AN ABSTRACT OF THE DISSERTATION OF
Wasna Viratyosin for the degree of Doctor of Philosophy in Microbiology
presented on June 6, 2002 Title: Genetic Variation of Chlamydial Inc Proteins
Redacted for Privacy
Abstract approved:
Daniel D.
Genomic analysis is a new approach for the characterization and
investigation of novel genes, gene clusters, the function of uncharacterized
proteins, and genetic diversity in microorganisms. These approaches are important
for the study of chlamydiae, a system in which several genomes have been
sequenced but in which techniques for genetic manipulation are not available. The
objective of this thesis is to combine computer-based analysis of chiamydial
inclusion membrane proteins (Incs) with cellular and molecular biological analysis
of the bacteria. Three different experimental lines of investigation were examined,
focusing on Incs of C. trachomatis and C. pneumoniae.
Chlamydiae are obligate intracellular bacteria that develop within a nonacidified membrane bound vacuole termed an inclusion. Putative Inc proteins of C.
trachomatis and C. pneumoniae were identified from genomic analysis and a
unique structural motif. Selected putative Inc proteins are shown to localize to the
inclusion membrane.
Chiamydia trachomatis variants with unusual multiple-lobed, nonfusogenic, inclusion were identified from a large scale serotyping study.
Fluorescence microscopy showed that IncA, a chiamydial protein localized to the
inclusion membrane, was undetectable on non-fusogenic inclusions of these
variants. Sequence analysis of incA from non-fusogenic variant isolates revealed a
defective incA in most of the variants. Some variants lack not only IncA on the
inclusion membrane but also CT223p, an additional Inc protein. However, no
correlation between the absence of CT223p and distinctive inclusion phenotype
was identified. Nucleotide sequence analysis revealed sequence variations of C.
trachomatis incA and CT223 in some variant and wild type isolates.
Comparative analyses of the three recently published C. pneumoniae
genomes have led to the identification of a novel gene cluster named the CPn1O54
gene family. Each member of this family encodes a polypeptide with a hydrophobic
domain characteristic of proteins localized to the inclusion membrane. These
studies provided evidence that gene variation might occur within this single
collection of paralogous genes. Collectively, the variability within this gene family
may modulate either phase or antigenic variation, and subsequent physiologic
diversity, within a C. pneumoniae population.
These studies demonstrate the genetic diversity of Inc proteins and
candidate Inc proteins, within and among the different chiamydial species. This
work sets the stage for further investigations of the structure and function of this set
of proteins that are likely critical to chlamydial intracellular growth.
© Copyright by Wasna Viratyosin
June 6, 2002
All Rights Reserved
Genetic Variation of Chiamydial Inc Proteins
Wasna Viratyosin
A DISSERTATION
Submitted to
Oregon State University
In Partial Fulfillment
of the Requirements for the
Degree of
Doctor of Philosophy
Presented June 6, 2002
Commencement June 2003
Doctor of Philosophy dissertation of Wasna Viratyosin presented on June 6. 2002
APPROVED:
Redacted for Privacy
Major Professor, repreenting\Microbio1ogy
Redacted for Privacy
Head of the Department of Microbiology
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Dean of tFIdrrduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Redacted for Privacy
Wasna (Tiratyosin, Author
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health Award
# R29A142869 and R01A148769, and the Oregon State University Department of
Microbiology N. L. Tartar Award Program. I would like to thank the Thailand
National Center of Genetic Engineering and Biotechnology and the Royal Thai
Scholarship program for their funding and support.
On a professional level I have to first and foremost acknowledge my major
advisor, Dan Rockey, for mentoring my educational development as a scientist and a
professional, and for helping me fulfill my goals. Thanks for all the advice and
support.
I would like to thank my committee members Dr. Dennis Hruby, Dr. Lyle
Brown, Dr. Jerry Heidel, and Dr. George Bailey for their time and support.
Additionally, I would like to thank the rest of the faculty, especially Dr. Jo-Ann
Leong, for time, advice, and support.
Thank you to the past and present members of the Rockey lab and my other
friends I have made over the years at OSU for being there to talk, share enthusiasm,
and have fun. Thanks to everyone for their friendship, cheerful support, and laughs. I
would like to thank all my family: Mom, my sisters and brothers who always love
and support me.
TABLE OF CONTENTS
INTRODUCTION
1
LITERATURE REVIEW
3
Molecular phylogeny of Chiamydia
3
Variants of chlamydiae
4
Developmental cycle
6
Elementary bodies and reticulate bodies
8
Cell surface associated proteins of Chlamydiae
Major outer membrane protein (MOMP)
Cysteine-rich membrane proteins (CRPs)
PorB
Polymorphic membrane proteins (Pmps)
Lipopolysaccharide (LPS)
10
Inclusion membrane proteins (Incs)
20
Genetic variation in chlamydial cell surface proteins
29
Genetic and phenotypic variation in other bacterial pathogens
32
MATERIALS AND METHODS
11
14
15
16
19
35
Wild type and mutant C. trachomatis isolates
35
C. pneumoniae isolates, plasmids and genomes
37
Chlamydial cell cultures
39
Purification of elementary bodies
39
TABLE OF CONTENTS, continued
Identification of putative Inc proteins using hydrophilicity analysis
40
Production of putative Inc proteins
41
Purification of maltose fusion proteins
44
Antibody production
46
Antisera and reagents for non-fusogenic variant studies
46
Immunofluorescence microscopy
47
Preparation of Chlamydiae infected cells for protein
gel electrophoresis
49
Preparation of LCR lysates for amplification reaction
49
Amplification reaction and DNA sequence analysis of incA
and CT223
50
Electrophoresis and immunoblot
52
In silico analysis of the CPn1O54 gene family of C. pneumoniae
genomes
53
Phylogenetic analyses
54
DNA amplification, and sequence analysis of the CPn1O54
gene family
54
Recombinant clones and sequence analyses
55
TABLE OF CONTENTS, continued
RESULTS
58
Identification and bioinformatic analysis of putative
Inc proteins
58
Protein localization using immunofluorescence staining
63
Fluorescence microscopy of cells infected non-fusogenic
variant isolates
66
Immunoblot analysis of selected non-fusogenie variant isolates
71
Sequence analysis of incA of non-fusogenic variant isolates
73
DNA sequence analysis of ORF CT223
77
Upstream sequence analysis of incA and CT223
78
In silico analysis of the CPn1O54 gene family
81
The conserved hydrophobic domain of the CPn1O54 gene family
85
Homopolymeric cytosine (poly C) tract and the variation of the length
of poly C tract in the CPn1O54 gene family
93
The tandem repeat motif in CPn0007
94
Framshift mutations and gene conversion in CPnOO 10
97
DISCUSSION AND SUMMARY
104
BIBLIOGRAPHY
124
LIST OF FIGURES
Figure
1.
The Chiamydia developmental cycle of the infection and replication
2. Electron microscopy of a chiamydial inclusion of C. psittaci in
7
9
infected cell
3.
Hydropathy profiles for the secondary structure of known
inclusion membrane proteins
4. Schematic representation of the contig containing the selected inc
25
42
genes in C. trachomatis genome
5.
Comparison of predicted proteins encoded by C. trachomatis CT058
and C. pneumoniae CPnO 107, CPn0367, CPn0369 and CPnO37O
62
6.
Immunofluorescence microscopy of C. trachomatis infected cell
HeLa cells fixed with methanol and probed with specific antibody
against each tested putative Incs
64
7.
Double-labeled fluorescence microscopy of C. pneumoniae labeled
with anti-HSP6O (A) and anti- C. pneumoniae IncA (CPnOI 86)(B)
65
8. The distinct pattern of the localization of CT223 on the inclusion
membrane
9.
Fluorescence microscopy of McCoy cells infected with a serovar J(s)
variant of Chiamydia trachomatis probed with antibodies against
67
69
J-JSP6O, IncA, and CT223
10. Fluorescence microscopy of McCoy cells infected with serovar G(s)
459, an IncA positive variant, of C. trachomatis probed with
antibodies against IncA
70
11. Immunoblot analysis of non-fusogenic C. trachomatis isolates probed
with antibodies against HSP6O, IncA, and CT223p
72
12. A graphic representation of the results from the DNA sequencing of
incA encoded by 10 wild type and 25 non-fusogenic C. trachomatis
isolates
74
LIST OF FIGURES (continued)
Figure
Pg
13. Multiple sequence alignment analysis of CT223p of fusogenic and
non-fusogenic variant isolates of C. trachomatis isolates
79
14. A schematic representation of C. pneumoniae CWLO29 contigs
82
15. Phylogenetic tree analysis of the DNA sequence of the CPn1 054 gene
family obtained from C. pneumoniae CWLO29
84
16. The upstream and 5' end sequence of the CPn1O54 gene family
86
17. A schematic representation of the structure features and comparison
of paralogous genes of CPn1O54 gene family in C. pneumoniae CWLO29
88
18. Hydropathy plot analysis of the members of the CPn1O54 gene family
89
19. The conserved hydrophobic domain in the CPn1O54 family
91
20. Phylogenetic tree analysis of amino acid sequence of hydrophobic
phobic domain of the CPn1O54 gene family obtained from
C. pneumoniae CWLO29
92
21. Variation of the length of the poly C tract of the CPn1O54 gene family
95
22. Schematic representation of the open reading frame of CPnOO1O-00l0.1,
1054, 0045 identified in C. pneumoniae CWLO29, AR39 and J138
99
23. Frameshift mutation and gene conversion in CPnOO 10
100
24. Gene conversion ofCPnOOlO in C. pneumoniae isolates
102
LIST OF TABLES
Page
Description of the wild type and non-fusogenic variants of
C. trachomatis isolates used in this study
36
2. Description of the 12 independent C. pneumoniae isolates
38
1.
used in this study
3. Oligonucleotide primers used for preparation of putative inc/maleE
hybrid recombinant clones for production of fusion proteins
43
4. Oligonucleotide primers used for PCR and nucleotide sequencing
57
of the internal regions of the CPn1O54 gene family from
C. pneumoniae CWLO29
5.
Putative Inc genes identified by hydrophilicity profile within
each sequenced chiamydial genome
6. DNA sequence identity of the CPn1O54 gene family using
Pairwise of BLAST 2 sequence program
59
83
This thesis is dedicated to my mom and my family
for
love, encouragement and support
Genetic Variation of Chiamydial Inc Proteins
INTRODUCTION
Chlamydiae are obligate intracellular bacteria that cause a variety of
human diseases. Two main human pathogens are C. trachomatis and C.
pneumoniae. Chiamydia trachomatis is leading cause of preventable blindness
and sexually transmitted disease (STD) including pelvic inflammatory disease,
chronic pelvic pain, ectopic pregnancy, and epididymitis (Schachter et al., 1990)
in the developing and developed countries, respectively. Chlamydiapneumoniae
is an important cause of community-acquired pneumonia (Saikku et al., 1985)
and bronchitis, and has recently been implicated in the etiology of
atherosclerosis (Kuo et al., 1993; Mlot et al., 1996). Both pathogens represent a
major public health burden.
Among intracellular bacterial pathogens, chlamydiae possess a unique
biphasic development cycle. They reside and replicate in non-acidified
membrane bound vacuoles termed an inclusion. Proteins that localize to the
inclusion membrane are defined as inclusion membrane proteins (Incs). Little is
known about the development of chiamydial inclusions and the functions of Inc
proteins.
A major area of interest in chlamydial biology is to understand the
interaction between the chlamydial inclusion and the host intracellular
environment. The role of chlamydial inclusion membrane proteins in the
2
chiamydia development cycle and pathogenesis remain to be elucidated. The
lack of a method for conducting genetic manipulation in the chlamydiae is the
primary limiting factor to attempts to characterize the unknown proteins.
Recently, the availability of several complete Chiamydial genomes
provides an alternate approach to elucidate thechlamydial biology. The
identification of new potential or functional assignment is extensively facilitated
by genomic approaches. Novel gene clusters or families, transport mechanisms,
and biochemical pathways are defined and characterized based on the sequence
similarity, and structural domain obtained from the genome data.
The focus of this thesis is to identify the putative Inc proteins and/or a
new gene family in the chiamydial genome based on structural motifs and the
sequence similarity. We first use the unique bi-lobed hydrophobic domain
commonly present in Inc proteins as a primarily predictive marker to determine
the putative Inc (s) in the C. trachomatis and C. pneumoniae genomes. The
second area of investigation is to determine the association of the non-fusogenic
inclusion phenotype of C. trachomatis variant isolates and incA sequence.
Finally, the comparative genomic analysis was used to identify a new gene
family and study the possible role of genetic variations of paralogs encoded
within the new gene family named the CPn1O54 gene family in C. pneumoniae.
Collectively, these studies described variability within the Chlamydiae not
previously examined in the literature.
3
LITERATURE REVIEW
Molecular phylogeny of Chiamydia
All members of the order Chiamydiales are obligate intracellular bacteria
that have a unique biphasic developmental cycle. They all have greater than 80 %
rDNA sequence identity with chlamydial 16S rRNA genes and/or 23s rRNA genes.
The order Chiamydiales recently has been revised into four families:
Chlamydiaceae, Parachlamydiaceae, Simkaniaceae, and Waddilaceae (Everett et
al., 1999; Bush and Everett, 2001). The family Chlamydiaceae, which previously
consisted of only a single genus, Chiamydia, was recently divided into two major
genera, Chiamydia and Chiamydophila. Members of genus Chiamydia include
C. trachomatis, C. muridarum, and C. suis in which the 1 6S rRNA and 23 S rRNA
gene sequences are 97 % identity. The genus Chiamydophila consists of C.psittaci,
C. pnuemoniae, C. pecorum, C. abortus, C. felis, and C. caviae, which their
ribosomal genes are 95 % identical. The nine species in these genera were
decisively distinguished by analyses of phenotype, antigenicity, associated disease,
host range, biological data, genomic endonuclease restriction, DNA-DNA
hybridization information, and phylogenetic analysis using the ribosomal operon of
16S rRNA and 23S rRNA (Everett et al., 1999; Everett and Andersen, 1997;
Katlenboeck etal., 1993; Pudjiatmoko et al., 1997).
The new proposed reclassification of the order Chiamydiale, however,
remains a controversial issue (Schachter et al., 2001). The minor sequence
difference may not be sufficient to create a new genus or species. The
taxonomically significant biological properties for genus or species differentiation
in the proposed reclassification were not completely devised. Tanner et al. (1999)
showed that C. trachomatis, C. psittaci, C. pecorum, and C. pneumoniae strains
represent a taxonomically and phylogenetically coherent grouping of 16s rRNA
into one group. Only 16s rRNA analysis, therefore, may not be a best choice for
speciation. Recently, five other genes that encode for major outer membrane
protein (MOMP), GroEL chaperonin, KDO-transferase, small cysteine-rich
lipoprotein and 60 kDa cysteine-rich protein were used as key determinants for
phylogenetic reconstruction (Bush and Everett, 2001). Phylogenetic analysis of
those genes demonstrated all genes evolved in concert with the ribosomal genes.
However, there are no common biological markers for genus or species
differentiation between the organisms within both proposed genera.
Variants of chlamydiae
Chiamydia trachomatis is currently classified into 19 serovars and
categorized into three serogroups or classes, which are: B class (B, Ba, D, Da, E,
Li, L2 and L2a), C class (A, C, H, I, Ta, J, Ja, K and L3), and intermediate class (F
and G). These groups are determined based on amino acid similarity of variable
domains within the major outer membrane proteins (MOMP). The serovar
distribution of B, C, intermediate class and "mix serovars" are 49%, 27.9%, 20.5%,
and 2.6%, respectively, in clinical infection (Dean et al., 2000). The classification
is consistent with the serological determination of chiamydial strains using
polyclonal antisera or monoclonal antibodies against major outer membrane
proteins (MOMPs). Serovar A, B, Ba and C are responsible for endemic trachoma.
Serovar D- K especially D, E, and F are the major prevalent C. trachomatis strains
implicated in sexually transmitted disease (STD) worldwide while significant
persistent infections are associated with cervical infection by the C class (Dean et
al., 2000). The C class appears less virulent, and requires higher multiplicity of
infection and longer incubation period, for replication in the tissue culture cells. A
recent longitudinal study provided evidence that C. trachomatis serovar G is
associated to the developing cervical cancer, but remains controversial.
Chiamydia trachomatis can also be subdivided into three biovars: trachoma,
lymphogranuloma venereum (LGV), and mouse pneumonitis. The classification is
based on sources, distinct differences in clinical disease, and differences in their
in
vitro cell culture growth characteristics. The trachoma biovar consists of human
serovars A to K; plus Ba, Da, Ta. Serovar A-C are primarily associated with
endemic trachoma, and serovar D-K are involved in urogenital tract infection. The
LGV biovar contains human serovars Li, L2, L2a, and L3, which are invasive
lymphotrophic, sexually transmitted agents. This biovar is more invasive
in vivo
and more readily infects cultured cells in vitro (Moulder et al., 1988). A mouse
biovar causes pneumonitis in an experimental murine model but is not known to be
pathogenic for humans or other animals. Classification of C. trachomatis into
different biovars at the molecular level remains questionable. Genotyping of C.
trachomatis did not correspond to biovar designation. Sequence analysis of ompA,
which encodes for major outer membrane proteins (MOMPs), could not
differentiate among the serological complexes. Analysis showed that A-, C- and Htrachoma are closely related to the L3 serovar while B- and E- trachoma are related
to the Li and L2 serovar. However, the 1 6S rRNA analysis revealed that the
trachoma biovariants are more closely related to each other than they are to LGV
isolates.
Chlamydiapneumoniae is classified into three distinct biovars: TWAR,
Koala, and Equine (Everett et al., 1999) Different C. pneumoniae isolates have 94-
100% homology with each other but less thani0 % DNA homology with C. psittaci
and less than 5% DNA homology with C. trachomatis (Kuo et al., 1995).
Developmental cycle
Chlamydiae are obligate intracellular pathogens that possess a unique
developmental cycle. The cycle consists of two distinctive forms: elementary
bodies (EBs) and reticulate bodies (RBs) (Figure 1). Elementary bodies are
infectious, metabolically inactive forms while reticulate bodies are metabolically
active but noninfectious forms. The nature of the development cycle is the
conversion of EB to RB. This cycle begins when EBs come to attach to susceptible
7
.:>
Infection (EB1
HOST
S
o.:o.
' 0'
ÔLL
Transformation
. .5.:,..
.0.
\ of EB to RB
:°'
S
0
.0 5
.
I
.1511
.00 10.1
S
I
.0 I
Replication
.
'. .EBs and RBs
0
S...,
I
.0
S
I
.0
I\ 'Q
I
": .10
\\ SI..
Lysis
/
SI
\ .0
/1..
I
Figure 1. The Chiamydia development cycle of the infection
and replication. This cycle typically take 48-72 h although
some species have longer cycles.
ri]
eukaryotic cells leading to internalization. Within the first 2 h following
internalization, EBs, which entirely reside in the non-acidified membrane bound
vacuoles known as an inclusion, differentiate into RBs. RBs undergo multiple
round of binary fission. As bacterial multiplication, the inclusion expands. After
24-48 hours post infection (hpi), RBs differentiate back into infectious EBs which
are subsequently released through host cell lysis. In most chlamydiae, this cycle is
complete within 48-72 hpi.
Elementary bodies and reticulate bodies
Elementary bodies (EB5) and reticulate bodies (RBs) are primarily defined
by morphological criteria (Ward, 1 983)(Figure 2). EBs are small structures which
are from 0.2 to 0.4 im in diameter and possess an electron-dense nucleoid. RBs are
much larger pleomorphic forms (1.0 to 1.5 tm in diameter). They are less dense,
and possess an evenly dispersed, reticulate cytoplasm. Intermediate bodies are
present at the transitional point in the developmental cycle. Aberrant RB forms also
are present in either gamma interferon or ampillicin treatment and tryptophane
depletion condition. EBs are denser and more resistant to mechanical and osmotic
stress than are RBs. (Tamura et al. 1967 a, b; Caldwell et al., 1981). The DNA of
C. trachomatis EB is packed into a condensed nucleoid structure by two histone
like proteins, Hcl and Hc2 (Christiansen et al. 1993; Hackstadt et al., 1991).
-.
-
/
,-,,-
'
/
F
/
3*
Figure 2. Electron microscopy of a chlamydial inclusion of C. psittaci
in infected cell. Morphological differences between EBs and RBs
multiplying binary fission were evident.
10
Elementary bodies were previously described as dormant like bodies.
Recently, whether EBs really are metabolically inactive bodies became a question.
It has been shown that EBs carry a pooi of ATP. Several active transcripts were
identified in EBs (Douglas et al., 2000). The fact was further strengthened by
proteomic analysis of the C. pneumoniae EBs (Vandahi et al., 2001). The findings
suggested that EBs are capable of metabolizing energy and thereby providing fuel
for protein expression and active transport of proteins at the very beginning of the
developmental cycle. The study also showed that EBs carry a large number of
proteins involved in transcription and translation that were functionally active.
These findings suggest that EBs are nondividing but metabolically active, at least at
a minimal level.
Cell surface associated proteins of chlamydiae
Chlamydiae
are phylogenetically and structurally classified as Gram-
negative bacteria. The chiamydial envelope is similar to the outer envelope of
gram-negative bacteria, consisting of an outer membrane, an inner membrane and a
periplasmic space. However, unlike typical gram- negative bacteria, chlamydiae
possess a unique cell envelope containing a cysteine-rich outer membrane, an inner
membrane and a periplasmic space, and deficient or novel peptidoglycan structure.
Chlamydial outer membranes consist largely of disulfide cross- linked major outer
membrane proteins (MOMPs), two cysteine-rich proteins, polymorphic outer
membrane proteins (Pmp) and a unique truncated lipopolysaccharide (LPS).
11
Presumably, the cross- linked supra-macromolecular complex may be the
functional equivalent of peptidoglycan (Hatch and Everett, 1995).
Major outer membrane protein (MOMP)
MOMP is the predominant surface-exposed chiamydial protein. It is present
in both RB(s) and EB(s) (Stephen et al., 1986) and constitutes 60% of the total
outer membrane protein complex (Caidwell et al., 1981). Although MOMP is
present throughout the developmental cycle, it is structurally different in the two
developmental forms (Hatch et al., 1984, 1986; Hackstadt et al., 1985; Newhall et
al., 1987). In the RB, MOMP is predominately a monomer whereas in the EB,
MOMP is present as a dimer, trimer, and multimeric complex in association with
truncated LPS (Birkelund etal., 1988).
MOMP is a 4OkDa outer membrane protein with a p1 of 4.9 that is
synthesized most actively at 20 hpi. It is encoded by ompA (Stephen et al., 1986),
formerly referred as ompl, which is transcribed from early in development through
the entire cycle. It is a chiamydial outer membrane protein that exhibits structural
heterogeneity between different members of the chlamydiae species. Sequence
variation in MOMP is confined mainly to four hypervariable domains (VD5) that
are flanked and interspaced by five conserved domains (Yuan et al., 1989).
The antigenic determinants in the hypervariable domains of ompA elicit the
serovar- subspecies-, serogroup-, and species-specific antibodies (Caidwell and
Schachter, 1982; Newhall etal., 1987; Stephens etal., 1982). C. trachomatis
12
serovar variants were analyzed and defined as deduced amino acid sequences of
OmpA, that varied by one or more than 1 amino acid from the prototype serovars
for variable domains. Therefore, MOMP represents the primary chiamydial
serotyping antigen of C. trachomatis isolates (Yuan et al., 1989; Zhang et al.,
1989).
The significance of variable domains was studied and showed that VD2 and
VD4, which are surface-exposed epitopes of MOMP, play a role in attachment
since treatment of C. trachomatis with specific antibodies to these epitopes blocks
attachment and infectivity. VD3 is the smallest and least variable domain, and
responsible for potential immunogenicity. Studies of protective or neutralizing
antibody (Allen et al., 1991), and immune specificity of murine T- cell against
MOMP (Ishizaki et al., 1992) reveals that an important antigenic T -cell epitope of
C. trachomatis is located at the upstream and within VD3.
Several studies strongly suggest that MOMP plays an important role in
pathogenesis. Treatment of infectious EBs with trypsin is correlated with reduced
attachment of chlamydiae to HeLa cells (Hackstadt et al., 1985; Su et al., 1988,
1990). Monoclonal antibodies specific to conformation-dependent determinants of
C. pneumonia MOMP are also potent in the neutralization of infectivity in vitro
(Wolf et al., 2001). Collectively, MOMP is such an immunodominant antigen that
it can be potentially a candidate for a subunit vaccine against chlamydial infection.
13
Analyses of two C. psittaci strains, guinea pig inclusion conjunctivitis
(GPIC) and meningopneumonitis (Mn) strain, and C. trachomatis revealed the
structural conserved region of MOMP, which seven of eight cysteine residues and
the four variable domains are identified at precisely the same positions (Zhang et
al., 1989). In all C. pneumoniae isolates, protein profiles are identical, with a
prominent 39.5 kDa band homologue to the MOMPs of other chlamydiae
(Campbell et al., 1990; lijima et al., 1994). All seven cysteine residues involved in
disulfide-linked structure of other chiamydial MOMPs are similarly conserved in
C. pneumoniae (Melgosa et al., 1994). Comparative genomic analyses also
revealed that ompA of C. trachomatis and C. pneumoniae have striking sequential
and structural similarities (Stephen etal., 1998). Such conserved structure suggests
the importance of structure and conformation of the MOMP protein.
Sequence analyses of VD4, the largest hypervariable region of MOMP,
were shown to be identical in 13 different C. pneumoniae isolates (Keltenboeck et
al., 1993). It is likely that C. pneumoniae MOMP was conserved among different
isolates. In previous studies (Campbell et al., 1990; Christiansen et al., 1999)
immunoblot and immuofluorescence analysis of C. pneumoniae infected sera failed
to detect MOMP, which suggested that C. pneumoniae MOMP may be
immunorecessive and not surface accessible. However, recent work by Wolf et al.
(2001) demonstrated that the C. pneumoniae MOMP contains nonlinear and
surface-exposed epitopes. Their study also suggested that these epitopes are
conformation -dependent and immunodominant.
14
Several studies directly or indirectly suggest that the MOMP has a role in
the adhesion of C. trachomatis and C. psittaci to host cells. The attachment process
to the host ligand was demonstrated in recombinant E. coli expressed maltose
binding protein (MBP)MOMP fusion protein and visualized by scanning electron
microscopy (Su et al., 1996). MOMP has also been shown to bind heparin in a
concentration-dependent manner. Excess heparin or heparan sulfate inhibits
binding of MBP-MOMP to eukaryotic cells. Mutant eukaryotic cells defective in
glycosaminoglycan elicit the reduction of binding of MBP-MOMP and native EBs.
Cysteine-rich outer membrane proteins (CRPs)
Major cysteine-rich outer membrane proteins (CRPs) were the first outer
membrane complex proteins identified in the sarkosyl-insoluble fraction of purified
outer membrane protein of EBs. They are encoded by OmcA and OmcB (Lanmden
et al., 1990), and envA and envB, in C. trachomatis and C. psittaci i respectively
(Everett et al. 1994). OmcA is the smaller cysteine rich membrane proteins, which
is annotated as 9 Kda-CRP. In contrast to ompA, omcB is extremely conserved and
the gene product is present only in EB (Hatch et al., 1984, 1986) in the ratio of 5
MOMP: 2 OmcA: 1 OmcB (Everett et al., 1991) and does not appear to be surface
exposed (Stephens et al., 2002). These lipoproteins are structurally similar to
murein lipoproteins present in gram- negative bacteria (Everett et al., 1994).
15
The cysteine- rich outer membrane is considered to be primarily responsible
for maintaining the structural integrity of the outer membrane in the absence of
peptidoglycan (Newhall and Jones, 1983; Hatch et al., 1984; Hackstadt etal.,
1985). The cysteine residues within CRP are highly conserved in all chlamydia
species, suggesting function in formation of disulfide-liked complex structure
(Everett et al., 1991). Proteolytic treatment or heat denaturation blocked the
binding of OmcB to the host cells. It indirectly suggested OmcB may play a role in
the attachment process.
PorB
PorB is a relatively cysteine-rich protein predicted to be in the outer
membrane. Like CRPs, it is a highly conserved gene present in the Chiamydiales.
Its MW is 39 kDa and a predicted p1 is 4.9, which are similar to those of MOMP.
PorB possesses a predicted leader sequence with an amino acid sequence that ends
in phenylalanine, a typical characteristic of outer membrane proteins of Gramnegative bacteria. It is present in the outer membrane complex of EB and is surface
accessible in C. trachomatis (Kubo et al., 2000). PorB is present in the outer
membrane in much smaller amounts than MOMP. Its transcription and translation
were found throughout the chiamydial development cycle. Functional studies using
in vitro liposome-swelling assay revealed that PorB is a substrate-specific porin
that preferentially facilitates diffusion of dicarboxylates through the outer
membrane (Kubo et al., 2001). It may be responsible for the diffusion of some
16
metabolites required for chiamydial growth. Furthermore, immunological studies in
tissue culture showed that it is a target for antibody-mediated neutralization.
Polymorphic membrane proteins (Pmps)
The Pmp(s) were identified as a 90 kDa antigenic protein family and
initially named putative outer membrane proteins (POMP) in C. psittaci. POMPs
were renamed the polymorphic membrane proteins (Pmp) because they may or may
not all be in the outer membrane (Grimwood et al., 1999) At least six putative
Pmp(s) were identified in C. psittaci on the basis of their size (90-110 kDa)
(Longbottom et al., 1996). Four of these Pmp(s) are very similar to each other.
They are located in pairs at the two different loci on the chromosome. It is likely
that these Pmps resulted from duplication. The first locus encodes the omp9]A and
omp9OA gene, which are absolutely identical. Another locus encodes omp9lB and
omp9OB, which share 89 % similarity and are 86 % similar to Omp9O. The 90 kDa
proteins are major immunogens recognized by post-abortion antisera of sheep
infected with C. psittaci (Lombottom et al., 1998). Similarly, in C. pneumoniae, a
homologous 98 kDa protein was shown to be an immunogen in a natural infection
(Campbell etal., 1990). Immunoelectron microscopy and immunogold studies of
C. psittaci and C. pneumoniae showed that at least one member of the Pmp family
was exposed on the surface of both EBs and RBs (Longbottom et al., 1998;
Knudsen et al., 1998).
17
Genomic analysis of complete C. trachomatis and C. pneumoniae genomes
has also revealed the existence of a complex gene family named the polymorphic
membrane protein (Pmp) family, that consisted of predicted outer membrane
proteins (Kalman ez' al., 1999; Grimwood et al., 1998; Stephens and Lammel,
2001). The Pmp family shares similarity to the 90-kDa gene family (POMP) of C.
psittaci. Surprisingly, it is a large family of 9 related pmp genes in C. trachomatis,
identified and orderly named PmpA to PmpI. A larger homologous family present
in C. pneumoniae consists of2l relatedpmp genes (Pmpl to Pmp2l)(Kalman et
al., 1999). Unlike C. psittaci Pmps, which have high sequence similarity (Tanzer et
al., 2001), C. trachomatis and C. pneumoniae Pmps are remarkably polymorphic in
amino acid sequence, molecular weight and predicted p1. These differences lead to
difficulty in determining the accurate amino acid identities between proteins. Most
proteins show identities below 25%, which is not significant for implying structural
and functional similarity between proteins. Phylogenetic analysis of the Pmp family
of C. pneumoniae and C. trachomatis, however, revealed six related groups
containing at least one Pmp from each species (Grimwood et al., 1999). The six
related families also suggested the possibility of multiple specific roles for the Pmp
family.
All Pmps contain the common signature tetrapetpide repeat, glycine-
glycine-alanine-isoleucine/leucine/valine (GGAI/L/V). This motif is repeated
numerous times (2-12 times) throughout the NH3-terminal half of each protein.
Other repeat motif FXXN also occurs multiple times (4-23 times) within the NH3-
18
terminal half of all Pmps. The C-terminal half of all Pmps, except one truncated
protein, CPn452, are also characterized by conserved tryptophans and a carboxyl
terminal phenylalanine, a common feature of the outer membrane proteins of gramnegative bacteria (Struyve et al., 1991). Like many integral outer membrane
proteins in prokaiyotes, most Pmps possess predicted signal peptide leaders for
crossing the inner membrane, which suggests a localization of the expressed
proteins in the chlamydial outer membrane (Stephens et al., 1991). Christiansen et
al. (1999) demonstrated that at least three of C. pneumoniae Pmps are present on
the surface of EBs and differentially expressed.
Comparative genomic analysis of pmps of two strains, CWLO29 and J138,
revealed a nucleotide sequence identity of 89.6-100% and deduced amino acid
sequence identity of 71.1-100%. (Shirai et al., 2000). In addition, some pmps may
not be expressed as functional proteins, as five contain predicted frameshifts, and
one contains a nonsense mutation leading to stop codon. Mass spectrometry
analysis shows that major constituents of C. trachomatis L2 outer membrane
complex consist of PmpE, PmpG, and PmpH (Mygind et al., 2000; Tanzer et al.,
2001). All of the C. trachomatis and C. pneumoniae pmp however, are transcribed
during infection but differentially expressed at the surface of the EBs (Grimwood et
al., 2001). Expression of Pmps, in both C. trachomatis and C. psittaci, are
identified late (72 hpi) in the developmental cycle (Grimwood et al., 2001). By
two-dimension gel electrophoresis and mass spectrometry analysis, Pmp2, 6, 7, 8,
19
10,13, 14, 20 and 21 are found in EB, but there is no evidence of expression of
truncated genes responsible for Pmp5 and Pmpl2.
Recently, Pmps were predicted to be transported over the inner membrane
by the autotransport system based on the conserved amino acid stretch,
GG[AIL/V/I][J/L/V/Y] and FXXN repeat. Henderson etal. (2001) suggested that a
carboxyl terminal of the Pmp forms a predictive C-terminal ft-barrel traslocation
unit, which functions an anchor of the type IV autotransporters. The repeat motifs
in Pmp(s) resemble several autotransporter passenger motifs including rOmpA and
rOmpB of the Rickettsia. Collectively, homology search and structural motif
approaches suggested that the Pmp are predicted autotransporters.
Lipopolysaccharide (LPS)
The outer membrane of the chiamydial cell wall consists of a truncated
lipopolysaccharide, which harbors a groupspecific epitope. Unlike MOMP, LPS is
conserved among diverse Chlamydiae strains (Dhir etal., 1971). According to the
taxonomic reclassification of the order Chlamydiales, the formally described
genus-specific antigen of chlamydial LPS is now named a family-specific antigen
(Everett et al., 1999). Loosely associated with both EB and RB cell surface,
chlamydial LPS, is an immunodominant antigen that elicits humoral immune
response in all types of chlamydial infection The group specific epitope of LPS,
therefore, has been useful in diagnosis of chlamydial infection.
20
The surface exposure of LPS on RBs and EBs was characterized using
immunogold staining with two monoclonal antibodies. These findings also
suggested that the chemical structure of LPS may differ during the developmental
cycle (Kuo et al., 1987). Unlike other gram- negative bacteria, chlamydia harbors a
minimal LPS form containing lipid A and two Kdo residues that chemically
resemble the rough (Re type) LPS of Salmonella enterica serovar Minnesota Re
595. The predominant structure of chlamydial LPS is a trisaccahride of 3-deoxy-Dmanno-oct-2-ulosonic (Kdo) residues of the sequence alpha-Kdo-(2---8)-alpha-
Kdo-(2---4)-alpha kdo (Kosma et al., 1999; Rund et al., 2001). The chlamydiae
LPS exhibits low endotoxicity relative to LPS from other species. Antibodies
against LPS do not prevent infection of host cells. The low endotoxicity of
chiamydial LPS is related to the unique structural features of the lipid A, which is
highly hydrophobic due to the presence of unusual long fatty acids.
Inclusion membrane proteins (Incs)
Chlamydiae are obligate intracellular bacteria that have a unique
developmental cycle. Unlike other intracellular bacteria that reside in the host
cytoplasm, once endocytosed by host epithelial cells or non-professional
phagocytes, Chlamydiae replicate and sequester themselves within a nonacidified membrane bound vacuole, termed an inclusion, throughout their
intracellular developmental cycle. The inclusion rapidly dissociates from
endosomes or lysosomes and establishes interactions with exocytic vesicles by
21
fusion with trans-Golgi network (TGN)-derived sphingolipid at an early stage of
development. Both fusion of exocytic vesicles and avoidance of lysosomal
fusion require early chiamydial protein synthesis (Scidmore et
al.,
1996). Both
chiamydial proteins and host factors that govern chiamydial inclusion
trafficking, however, remain to be characterized.
The trafficking of Chlamydiae in the host cells is directly shown by the
absence or the presence of specific markers of endosomes and lysosomes on the
inclusion membrane. It is clearly demonstrated that neither early or late
endosome markers such as transferrin receptor and the cation-independent
marmose 6-phosphate receptor, or late endosomal/lysosomal markers including
the lysosomal glycoprotein LAMP 1, LAMP 2, cathepsin D and the vacuolar H-
ATPase, are present on the inclusion membrane (Heinzen et
etal.,
1996; Hackstadt et
al.,
1995; van Ooij
etal.,
al.,
1996; Scidmore
1997) In addition, fluid-phase
markers including fluorescein isothiocynate-conj ugated dextran and lucifer
yellow are not trafficked to the chlamydial inclusion (Heinzen et
al.,
1996).
The chiamydial inclusion has been described as an aberrant Golgiderived vesicle. Although it does not display any markers of the endocytic
pathway, it is labeled with a vital fluorescent dye analog of ceramide, 6-[N- (7nitrobenzo-2-oxa- 1, 3 -diazol-4y1) aminocaproyl sphingosine] (C6-NBD
ceramide), which has been used extensively to study sphingolipid trafficking in
viable cells. Incorporation of this vital dye into the inclusion was suggested as
the result of its fusion with a subset of exocytic vesicles (Hackstadt et
al.,
1995).
22
C6-NBD ceramide, like endogenous ceramide, is processed to sphingomyelin or
glucosylceramide within the Golgi apparatus at the cis or medial Golgi
compartment before transport to the plasma membrane via a vesicle-mediated
process (Lipsky and Pagano, 1985). It becomes clear that the acquired
sphingomyelin from C6 NBD ceramide is incorporated and can be detected in
the chiamydial inclusion within 2 hpi (Hackstadt et al., 1996). Approximately,
50% of the sphingomyelin endogenously synthesized from the added fluorescent
ceramide analog is diverted to the chlamydial inclusions.
The interaction of the inclusions with sphingomyelincontaining
exocytic vesicles was demonstrated as a common character of all Chiamydia
(Hackstadt et al., 1996; Rockey etal., 1996; Wolf et al., 2001). The transiently
direct trafficking of fluorescent sphingomyelin from the Golgi apparatus to the
inclusion is an active process that is energy-and temperature-dependent and is
sensitive to inhibitors of bacterial RNA and protein synthesis (Hackstadt et al.,
1996), and sensitive to BrefeldinA (Wylie et al., 1997). This trafficking leading
to the acquisition of sphingomyelin is initiated before differentiation of BBs to
RBs. Unlike the Toxoplasma gondii vacuole, vesicle interaction with the
exocytic pathway and the chlamydial inclusions is not dependent upon route of
entry of EBs but determined by the chlamydial early gene expression (Scidmore
et al., 1996). The interaction of inclusions with the exocytic pathway has been
proposed as a potential mechanism enabling chlamydiae to escape from
lysosomal fusion (Hackstadt etal., 1997).
23
The origin, development and function of the chiamydial inclusion remain
to be characterized. Only a set of chiamydial proteins termed inclusion
membrane protein (mc) has been identified on the inclusion membrane of all
Chlamydiae except C. pecorum. The development of the inclusion is presumably
accomplished by the integration of multiple inclusion membrane proteins into
the host vesicle membrane. During the study of differences in serological
response between infected animals and those immunized with formalin-treated
EBs, the three Inc proteins, IncA (Rockey and Rosquist, 1994; Rockey et a!,
1995; Bannantine et al., 1998a), IncB and IncC (Bannantine etal., 1998b), were
initially identified and characterized in C. psittaci. An alternative approach using
antisera against total purified membrane fractions of C. trachomatis infected
cells and directly staining the inclusion membrane has accomplished
identification of other Inc proteins. Immunofluore scent staining revealed a new
set of four chlamydial inclusion membrane proteins, Inc D-G (CT116-119)
localized to the C. trachomatis inclusions (Scidmore et al., 1998). The operon
containing incD-G was located downstream of C. trachomatis incA. Genome
data analysis revealed that IncD-G are C. trachomatis specific Inc proteins and
are absent in C. psittaci and C. pneumoniae genomes. Genes encoding for IncD-
G are expressed as an operon with transcription detectable in the first 2 h
following internalization while incA is expressed in the 18 hpi. The early
expression of the incD-G operon suggests that they are required for the
modification of the nascent inclusion.
24
Genomic analysis of the complete C. trachomatis serovar D and C.
pneumoniae
CWL029 genome led to the identification of homologous IncA,
IncB and IncC. IncA of C. trachomatis was demonstrated to localize in the
inclusion membrane (Bannantine etal., 1998). To date, there are 10 identified
Inc proteins: IncA, B, C of C. psittaci, IncA-G of C. trachomatis and C.
pneumoniae IncA. Surprisingly, these Inc proteins do not share significant
sequence similarity (less than 20 %), nor are they are similar to any genes within
the sequence database. However, each identified Inc protein has the unusually
large bi-lobed hydrophobic motif of 60-80 amino acid residues (Figure 3). It was
demonstrated the C- terminal hydrophobic domains of IncA, meG and IneF of
C. trachomatis are exposed to the cytoplasm (Hackstadt et al., 1999). IncA of C.
psittaci, is also exposed on the cytoplasmic side of vacuoles and phosphorylated
by host cell kinase on the serine and threonine residues (Rockey et al., 1997).
Therefore, Inc proteins may play a critical role as mediators of host-parasite
interaction via signal transduction pathways in host cells.
The function of the Inc proteins in the chiamydial developmental cycle and
hostpathogen interaction remain to be elucidated. Recently, the first eukaryotic
protein that interacts with chiamydial inclusion membranes was identified. The
specific association of C. trachomatis inclusion membrane and mammalian 14-3-313, a
phosphoserine binding protein, via its interaction with meG has been revealed using a
yeast two-hybrid system and immunofluorescent microscopy (Scidmore et al., 2001).
The specific localization of 14-3-313 to the inclusion membrane was identified only in
25
C. trachomatis IncA
50
250
200
150
100
C. Dsittaci IncA
>..
U
>
x -4
100
150
(.,. psniac, inca
40
80
120
160
Figure 3. Hydropathy profiles for the secondary structure of known inclusion
membrane proteins. Profiles were determined using the algorithm developed
by Kyte and Doolittle with a window size of seven amino acids. The vertical
axis displays relative hydrophilicity with negative scores indicating relative
hydrophobicity. The horizontal axis indicates amino acid number. Note that the
size of each graph is similar while the length of each protein is distinct.
Hydrophobic region is similar size and shapes are circle in each plot. Each of
these proteins has been shown to localize to the chlamydial inclusion membrane
using fluorescence microscopy with specific antibody.
26
C. trachomatis. This is consistent with the absence of an meG homologue, as
determined by genome data analysis, in either C. psittaci or C. pneumoniae. However,
the role of 14-3-3(3 in the C. trachomatis infections remains unclear. The speciesspecific interaction of 14-3-3(3 and the inclusion membrane may mediate the signal
transduction and/or other ligand proteins in infected cells. Several studies of
chlamydial development have shown the similarities and differences of the
development cycles of various Chiamydia spp. Each species and strain possesses
distinct characters associated with inclusion development. The significant differences
include inclusion morphology, content of the inclusions, kinetics of the inclusion
development in different eukaryotic tissue cells and interaction of cellular organelles.
In C. trachomatis, C. pneumoniae and some strains of C. psittaci and C. pecorum,
multiple lobed inclusions formed initially are eventually fused to generate a large
inclusion even when the cells are multiply infected (Campbell et al., 1989; Matsumoto
et al., 1991; Patton et al., 1990; Ridderhoffet al., 1989; Rockey et al., 1996). Unlike
C. trachomatis inclusions, inclusions of many strains of C. psittaci including strain
GPIC, do not fuse but appear to divide within infected cells.
Little is known about the function, regulation, and mechanism of the unique
homotypic fusion property of C. trachomatis. Ridderhof and Barnes (1989)
demonstrated that inclusions harboring different serovars of C. trachomatis can fuse
and eventually form a single large inclusion. Treatment of infected cells with
cytochalsin D, a microfilamentdisrupting drug, led to the delay of perinuclear
transport and inclusion fusion of C. trachomatis serovar L2 but not serovar E
27
(Schramm et al., 1995). A reduced effect of cytochalsin D on the fusion of inclusions
was also identified in HeLa cells co-infected with C. trachomatis serovar E and F
(Ridderhof and Barnes, 1989). Van Ooij etal. (1998) showed that fusion of inclusions
is also inhibited at temperature 32°C in several serovars of C. trachomatis. The
multiple inclusions formed at 32°C are derived from a high multiplicity of infection as
opposed to from the dividing of an inclusion. The inclusion fusion is temperature
dependent and required bacterial protein synthesis.
Recently, Hackstadt et al. (1999) demonstrated that the homotypic vesicle
fusion of C. trachomatis was inhibited by microinjection of antisera against IncA
whereas the fusion of inclusion membranes with sphingomyelin containing vesicles
was not disrupted. In addition, antibodies against other Inc proteins such as anti-IncF,
IncG were unable to inhibit the homotypic fusion of C. trachomatis inclusions. The
detection of the inclusion fusion of C. trachomatis at about 10-12 hpi is correlated to
the temporal expression of IncA. In addition, the yeast two-hybrid analysis showed
that carboxy-termini of IncA form homodimers interacting with the other IncA.
Collectively, these data showed that IncA plays a role in homotypic fusion of C.
trachomatis inclusions.
The parasitophorous vacuole is likely a barrier that separates chlamydiae from
not only the hostile environment but also nutrient-rich environment of the host
cytoplasm. Chlamydiae obtain nutrients such as amino acids and nucleotides, from
host pools (Iliffe-Lee etal., 1999). Although the interaction of the inclusion
membrane and trans-Golgi-derived vesicles supplies the sphingomyelin for
28
chlamydiae, the delivery mechanism of other essential precursors remains unclear. It
was demonstrated by transfection and microinjection studies of fluorescent tracer that
the inclusion membranes do not contain channels or pores, which would allow passive
diffusion of nutrients and biosynthetic precursors as small as 520 Da (Heizen et al.,
1997). Neither specific transporter nor non-specific diffusion channels, however, have
been identified in the chiamydial inclusion membranes. It was suggested that
chlamydia may possess a novel mechanism to deliver the nutrient precursors from the
nutrient- rich environment of host cytoplasm across the inclusion membrane.
All known chlamydial inclusion membrane proteins have no apparent classical
signal sequences at the their amino termini (Rockey et al., 1995, 1997; Scidmore et
al., 1998). Studies by Masumoto et al. (1982) using electron microscopy
demonstrated that chlamydiae possess the surface projection on the cell surface that is
similar to the type III secretion machinery in gram-negative bacteria. The genome data
analysis of C. trachomatis and C.pneumoniae has revealed that chlamydiae possess
genes that may encode a putative type III secretion apparatus (Bayou et al., 1998).
This secretion system has been described in several bacterial pathogens including
Yersinia, Salmonella, Shi ge/la, and Pseudomonas. Unlike type III secretion system of
other pathogens, genes encoding the type III secretion apparatus in chlamydiae
genome are dispersed. The expression of the putative type III apparatus of C.
trachomatis was demonstrated using RT-PCR. With the absence of the classical
signal sequence in the peptide sequence and the presence of genes coding for type III
apparatus, Inc proteins are logically considered as a candidate proteins secreted by
29
type HI apparatus. According to BLAST search, CopN which shares sequence
similarity to the type III apparatus, is a chiamydial protein observed to be secreted by
chlamydiae to the inclusion membrane (Fields et al., 2000). Additionally, Subtil et al.
(2001) demonstrated that the IncA, IncB and IncC of both C. trachomatis and C.
pneumoniae
were secreted by the heterologous type III machinery of Shigellaflexneri.
These findings provided evidences suggesting that chlamydiae encode and use a type
III secretion system.
Genetic variation in chiamydial cell surface proteins
Antigenic polymorphism within C. trachomatis by allelic variation of the
single copy of ompA encoding MOMP was identified and considered the genetic
basis of antigenic change within C. trachomatis (Barnes et al., 1987; Brunham and
Peeling, 1994; Hayes etal., 1994; Lampe etal. 1993; Stephen etal., 1987; Yang et
al., 1993). Although the mechanism of genetic variation for ompA remains unclear,
comparative analysis of its sequences revealed that mutation and recombination
events between serovars may play a role in antigenic variation (Brunham and
Peeling, 1994; Hayes etal., 1994) Alteration of MOMP epitopes is considered to
play a critical role in the immune evasion in the host cells. Identification of new
variants of C. trachomatis isolates revealed a mosaic structure of ompA containing
the nucleotide sequence with different ancestries. These variants include Ia (I/H),
LGV (L1/L2). The polymorphism of ompA sequence suggested that the genetic
variation may result from the inter- or intra-chromosomal recombination within or
30
between different serovars. Recently, the intraspecies and interspecies
recombinations have been identified in C. trachomatis ompA. Comprehensive
statistical analysis of recombination of 40 ompA and omcB sequences from C.
trachomatis, C. psittaci, and C. pneumoniae support intragenic recombination of C.
trachomatis ompA (Millman et al., 2001). The higher degree of recombination was
in the downstream half of the gene. Investigations of mosaic sequences of ompA of
known recombinant strains including D/B 120, GIUW-57, E/Bour and LGV-98
revealed significant breakpoints for recombination just before and within VD3, a
region responsible for elicited T- helper cell responses. The high recombination at
the upstream region and within VD3, may be associated with the molecular
adaptation. Analyses of intragenic recombination demonstrated that the rate was
higher for C class than for B class isolates. Analyses also revealed interspecies
recombination of ompA between the rodent C. trachomatis mouse pneumonitis/
Niggli strain and the C. pneumoniae horse N16 strain. Unlike the C. trachomatis,
C. pneumoniae
isolates cannot be classified into different serovars because MOMP
is highly conserved. However, the strain diversity of C. pneumoniae remains
unknown.
Among the most interesting findings from the genome was the existence of
a large group of polymorphic outer membrane proteins (Pmps). Antigenic variation
of the Pmp family has been investigated. Although the biological role of Pmps
remains to be described, the unknown role of the Pmp family is of specific interest
focusing on their surface exposure and immunogenicity in natural infection.
31
Hatch et al. (1999) described the variation of Pmp expression in C. trachomatis L2
and in C. psittaci 6BC. Recently, Christiansen etal. (1999) have also proposed and
revealed the differential expression in vivo of Pmps in mice infected with C.
pneumoniae. Comparative analysis of the outer membrane protein genes ompA and
pmp of C. pneumoniae CWLO29 and J138 strain revealed the strain diversity of
pmp genes (Shirai etal., 2000).
Furthermore, Grimwood et al. (2001) revealed the evidence for interstrain
variation of some Pmps of C. pneumoniae CWLO29, TW-183 and AR39.
Interstrain variation of pmp gene expression was detected in pmpGl 0
(CPn0449/0450), which contains a homopolymeric guanine tracts (poly G) and a
predicted frameshift mutation. The variation of string of G residues in apmpGl0
nucleotide sequence can lead to a change in reading frame. Based upon the genome
sequence for strain CWLO29,pmpGlO contain a string of 13 guanine residues and
is out of reading frame. The PmpGlO expression was detected only in AR39 (14 G)
and TW- 183 (11 G) but not CWLO29 (13 G). Interestingly, CWLO29 with a
difference passage history containing 14 guanine residues, which are in frame,
produces a protein product (Knudsen et al., 1999). These findings revealed the
interstrain and intrastrain variation in C. pneumoniae PmpG 10 expression, which
are apparently modulated by slip-stranded mispairing mechanism. C. trachomatis
pmpG (CT871) also encoded the string of G which suggested the phase variation is
a common trait in PmpG. The differential expression of pmpl0 resulting from gene
switching has been identified and explained by the variation of the length of poly
32
guanine (poly G) tracts (Pedersen et al., 20001). In addition, Grimwood
et
al.
(2000) revealed the variation in size of PmpG6 in comparative studies of CWLO29
and AR39.Genomic analysis showed that PmpG 6 in CWLO29 contains the three
internal tandem repeats of 131 amino acids whereas of AR 39 contains only 2
repeats. It is likely that differentiate expression and gene activation or inactivation
of Pmps will contribute to the surface variability and altered phenotype These
findings also suggested the interstrain variation of Pmp family was modulated by a
novel molecular mechanism. The function of Pmps, however, in chlamydiae
growth and development remains uncharacterized.
Genetic and phenotypic variation in other bacterial pathogens
Mutation is considered as a mechanism of gene variation and adaptation,
often exploited in response to changes in the environment and to promote
evolutionary flexibilities. The mutation rate of each gene in the genome is likely
variable and it is found to be higher in some particular genes. Obviously, unlike the
housekeeping genes with universally conserved sequences, genes involved in the
interaction with the environment frequently have a high mutation rate in
unpredictable fashion. Such genes are referred to as contingency or mutable genes
(Moxon et al., 1994, 1998) Mostly, these hypermutable genes are involved in
production or modification of surface structures of microbial pathogens lipopolysaccharides, lipoproteins, capsular polysaccharides, flagella, pili, and
33
excreted enzymes, and the modification system enzymes (van Belkum
1999;
Deitsch etal.,
1997;
Moxon
etal., 1998,
et al., 1998; 2000)
In microbial pathogens, the variations of surface structures are roughly
classified into two types: phase variation and antigenic variation (Robertson and
Mayer,
1992;
Henderson etal.,
1999).
Phase variation is the reversible change in
the presence or absence of a particular surface structure. In contrast, antigenic
variation is the changes leading to different variants of a surface structure. Gene
conversion and recombination, which lead to gene cassette switching, intragenomic
and intergenomic gene transfer, are the major mechanisms of antigenic variation.
The variation of the surface structure can be mediated by either homologous or non
homologous recombination. Both phase and antigenic variations are reversible
genetic mechanisms facilitating microbial evasion of host immune response and
microenvironmental adaptation. Genetic variation of surface structures plays a
critical role in microbial pathogenesis adherence, colonization, invasion,
virulence, and persistent infection.
Phase variation involves the regulation of gene expression of surface
structures under different circumstances. The variation results from the reversible
mutation in the simple short DNA repeat sequence (van Belkum
et al., 1998).
The
short sequence repeat (SSR) or repetitive DNA, is a stretch of homopolymeric or
heteropolymeric DNA sequence. SSR can be found in either a noncoding region
including promotor or a coding or internal DNA sequence of the genome.
Therefore, the phase variable genes have been identified by the presence and the
length of DNA repeats interpreted on their sequence context. The variation of the
short DNA repeat is considered to be a result of a recA-independent slippedstranded mispairing mechanism (Levinson and Gutman, 1987). Variation of the
short DNA sequence repeat located within the open reading frame, which can alter
the translational reading frame thereby determining whether or not the protein is
translated or within the promotor regions where they affect the strength of
transcription.
Several microbial pathogens including Neisseria gonorrhoeae, Neisseria
meningitides, Haemophilus influenzae, Helicobacter pylon, etc. possess
mechanisms to ensure variability of their surface structures in particular
environments (van Belkun etal., 1997, 1999; Hood etal., 1996; Moxon et al.,
1994, 1996, 1998 ). Phase and antigenically variable switch systems are attributed
special status and distinguished from more conventional mutation events. (Dybvig
etal., 1993; Saunders etal., 1998, 2000; Henderson etal., 1999; Feguy, 2000).
35
MATERIALS AND METHODS
Wild type and mutant C. trachomatis isolates
Different serovar (B, D, if, E, F, G, H, I, Ta, J and K) of 13 wild type and
26 mutant C. trachomatis isolates collected over the course of 12 years were used
in this study (Table 1). These isolates were obtained from Dr. Walter E. Stamm,
Division of Allergy and Tnfectious Disease, School of Medicine, University of
Washington, Seattle. The mutant isolates with nonfusogenic inclusions of C.
trachomatis infected cells were identified from genital swabs of clinical patients by
observation of inclusion morphology during routine serotyping of C. trachomatis at
the Seattle-King County Department of Public Health Clinic between 1988 and
1999 (Suchland et al., 2000). Wildtype chlamydial reference strain of serovar D
(D/IJW-3/cx), serovar J (J/UW-36/cx) and serovar G (G/UW-57) were described by
Wang etal., 1985. The serotype of each C. trachomatis isolate was determined
using antibodies against MOMP (major outer membrane protein) using the low
passage microtiter plate format (Suchiand etal., 1991). The serovar and a
laboratory reference number were indicated for each clinical isolate. The mutant
isolates, possessing nonfusogenic inclusion phenotypes, were represented with a
"(s)" designation
36
Table 1. Description of wild type and non-fusogenic variants of C. trachomatis
isolates used in this study
serovar
Isolate
year isolated
Source of isolation
Wild types
MT9227
MT9291
MT9332
MT9334
MT9301
MT9309
MT2364
MT3464
MT9295
MT9325
MT9329
MT9336
MT9346
B
MT93 11
K
E
F
G
Ia
J
J
J
J
J
J
J
2000
2000
2000
2000
2000
2000
cervical infection
urethral infection
cervical infection
cervical infection
urethral infection
urethral infection
cervical infection
cervical infection
urethral infection
cervical infection
urethral infection
cervical infection
rectal infection
urethral infection
1987
1993
1994
1998
1996
1995
1996
1999
1986
1995
1995
1988
1996
1999
1994
1995
1997
1998
1988
1994
1996
1997
1998
1997
1994
1992
cervical infection
cervical infection
cervical infection
rectal infection
cervical infection
cervical infection
cervical infection
rectal infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
urethral infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
cervical infection
2000
2000
2000
2000
2000
2000
1994
1994
Non-fusogenic variants
MT1 129
MT1O12
MT2923
MT4013
MT5058
MT3991
MT5328
MT8323
MT1986
MT4 022
MT8039
MT459
MT5642
MT9005
MT2 197
MT3839
MT6565
MT7713
MT893
MT1980
MT5942
MT6276
MT6462
MT6686
MT8068
MT2481
B(s)
D(s)
D(s)
D(s)
D(s)
if(s)
if(s)
if(s)
E(s)
F(s)
F(s)
G(s)
H(s)
H(s)
Ia(s)
i(s)
J(s)
Ia(s)
J(s)
J(s)
J(s)
i(s)
i(s)
J(s)
J(s)
K(s)
37
C. pneumoniae isolates, plasmids and genomes
Purified genomic DNA used in this study (Table 2) was obtained from Dr.
Lee Ann Campbell, Department of Pathobiology, University of Washington,
Seattle. Chiamydia pneumoniae was grown in HEp 2 cells for 72 h. Elementary
bodies were harvested, and purified using Renografin gradient as described below
(CaIdwell et al., 1981). Genomic DNA of C. pneumoniae were isolated from
purified EBs using QiaAmp DNA mini kits (Qiagen Inc., Valencia, CA) according
to the manufacture's instructions. DTT (Dithiothretol) was added to lyse the
chlamydial membrane proteins. Extracted DNA was eluted in 100
tl
of Qiagen
elution buffer, and stored at 20°C.
Genomic data of C. pneumoniae CWLO29 (http://chlamydia-www.berkeley.
edu- 423 1/, GenBank accession number AE001363 and NC 000922), AR39
(http://www.tigr.org/, GenBank accession number AE002161), and J138
(http ://w3 . grt.kyushu-u.ac.jp/J 138/ GenBank accession number ABO3 6071-
ABO3 6089 and NC_002491) were analyzed. Open reading frames (ORFs) and
annotated were designated based upon the C. pneumoniae CWLO29 genome. The
TA and Zero
BluntTM
PCR cloning kits (Invitrogen, San Diego, CA) were used to
attain clones for genetic variation study.
Table 2. Description of 12 independent C. pneumoniae isolates used in this study
Strain
City/Year Isolated
Origin/clinical presentation
CWLO29 Atlanta, US/1987-88 oropharyngeal swab/pneumonia
AR32
Seattle, US/i 983
Reference
Campbell
1991
et al.,
throat swab/phaiyngitis
conjunctiva
TW183
Taiwan/i 965
AR458
1986
acute respiratory infection
AR388
1985
acute respiratory infection
AR23 1
1984
acute respiratory infection
-
AR277
1984
pneumonia
-
PS32
1995
carotid endarterectomy
-
AC43
Japan/i 989
nasopharynx/pneumonia
KA5C
Finland/1987
pneumonia
KA66
Finland/i 987
pneumonia
2023
Brooklyn, US/1987-89 acute respiratory infection
-
Yamazaki
et al.,
1990
Ekman etal., 1993
Chirgwin
et al.,
1991
Note: All of AR isolates were isolated during a period of endemic acute respiratory
disease in Seattle during 1983-86. KA isolates were isolated from military trainees
* isolated form the Providence Medical Center
39
Chiamydial cell cultures
Semiconfluent monolayer cultures of HeLa 229 epithelial cells, (CCL 2.1;
American Type Culture Collection) and McCoy murine fibroblast cell line (CRL1696; American Type Culture Collection) were routinely grown in Minimal
Essential Medium (MEM, Gilbo BRL) supplemented with 10% fetal bovine serum
(FBS) and 10 g/ml gentamicin (MEM-lO; Whittaker Bioproducts) at an
atmosphere of 5% CO2 in humidified air at 37°C before infection. For
immunofluorescence microscropy, cell cultures were grown on 12-mm-diameter
glass coverslips (no 1) in 24 well plates.
Purification of elementary bodies
Purification of EBs from selected C. trahomatis serovars and C.
pneumoniae isolates were conducted from infected HeLa cell monolayers and
HEp2 cell lines with serial Renografin gradients (Squibb Diagnostics, New
Brunswick, N.J.) as previously described (Caldwell et al., 1981). Infected HeLa
cells with 90% of cells containing inclusions were harvested at 48 hpi for C.
trachomatis and 72 hpi for C. pneumoniae. Chlamydiae were removed from the
monolayer using 4-mm glass beads and cold Hanks balanced salt solution (HBSS,
Gibco). The cell suspensions were pooled and ruptured using sonication
(Braunsonic model 1510). The cell lysates were centrifuged at SOOxg for 15 mm at
4°C.
The supernatants were then layered over 8 ml of a 35%(vol/vol) Renografin
solution (diatrizote meglumine and diatrizate sodium) in 0.O1M HEPES (N-2hydroxyethylpiperazine-N'-2 ethanesulfonic acid) and 0. 15M NaC1. The
Renografin was then centrifuged at 43,000 x g for 1 h at 4°C. The pellets were
suspended in SPG buffer (0.25 M Sucrose, 5mM L-glutamic acid, 10 mM sodium
phosphate, pH 7.2), and layered over discontinuous Renografin gradients (13m1 of
40%, 8 ml of 44%, and 5 ml of 52% Renografin, vol/vol). The gradients were
centrifuged at 43,000X g for 1 h at 4°C. The purified EB fraction located at the
44/52% Renografin interface was collected, diluted with 3 volumes of SPG and
then centrifuged at 30,000xg for 30 mm. The EB pellets were washed in SPG to
remove the residual Renografin. Chlamydiae were stored at 80°C in SPG. Prior to
inoculation, Chlamydiae were diluted in SPG and kept on ice.
Identification of putative Inc proteins using hydrophilicity analysis
Sequence analyses were performed from all ORFs identified from the C.
trachomatis and C. pneumoniae genome database. Hydropathy plots from Mac
VectorTM
6.0 (Oxford Molecular) using KYTE-DOLITTLE algorithms with a
window of seven were conducted to determine the hydrophobic domain in the
amino acid sequences (Kyte and Doolittle, 1982). Deduced amino acid sequence of
Chlamydiae genes in which secondary structures contain bi-lobed hydrophobic
domain were identified as putative Inc proteins. The sequence similarity of putative
Inc proteins was determined using the BLAST (Basic Local Alignment Search
41
Tool) search and BLAST 2 sequences program from http://www.ncbi.nlm.nih.gov/.
All putative Inc proteins of C. trachomatis and C. pneumoniae were subjected to
gap BLAST analysis to identify similarities with known proteins in the database.
Production of putative Inc proteins
Six putative inc genes of C. trachomatis: CT119, CT223, CT229, CT233,
CT288, CT442, CT484, and CPnO1 86, a candidate incA gene of C. pneumoniae,
were selected to identif' the protein localization. We selected two genes, CPO 186
and CT233 which gene products are similar to known Inc proteins. The gene
product of CPnO186 has 20-23% similarity to IncA of C. trachomatis and C.
psittaci while the gene product of CT233 has 44 % similarity to IncC of C. psittaci.
The other four putative inc genes were randomly selected with an attempt to
provide a cross-section of putative Inc. ORF CT223 and CT229 were selected to be
investigated because both are encoded in a cluster of seven putative inc genes
(Figure 4), Gene product of CT228 consists of two independent hydrophobic
domains whose structural feature of two tandem IncA-like protein joined tail to
head. ORF CT484 encodes a gene product that is significantly similar to CPnO6O2
and also structurally similar to IncA protein but the predicted protein possesses a
more hydrophobic profile over the entire sequence. Six putative inc genes of C.
trachomatis and one of C. pneumoniae, were amplified using specific
oligonucleotide primers (Table 3). In some case, the 5' end of the gene was deleted
due to the full-length constructs of genes apparently toxic to E. coil.
42
Cntig 2.3
4
113
114
115H116II 118J
I
119
oI 121 '122
,cA 1-I
123
I
-
--ø-
-,.
-0- -0-
- -0 -ø
.
ContIg 3.5
231
U
NT
230
60.T
-0-
-
I
I 229
I
I
-==-
T1
223 222 I
Iefldc
H226 1225r224i
-0- -0- -0- -0-0-
229 1227
-4- -0-
-0-
=--=
IWT
.
---
ik
-- --
I
w_I
50
100
200
150
250
=w
.
_,__...
C _,
._.---..--------
--
St £
. ----20
IU
a.
40
:1 8
.
P11 I l_
*886*
I89VV
!
30
'1
I"l '9881
w
m.t
60
60
WI0
100
120
140
*I
Figure 4. Schematic representation of the contig containing the selected inc
genes in C. trachomatis genome. (A) ORF maps of two 10 kb segments of the
C. trachomatis genome labeled contig 2.3 and contig 3.5. ORF encoding
putative Inc(s) including CT1 15 to CT1 18 from contig 2.3 and CT223 to CT229
from contig 3.5 Note the close proximity of IncA, B, and C to each of the
respectively putative Inc clusters. (B) Hydrophathy plots of C. trachomatis
putative Inc proteins used for production of specific antibodies. Note that each
protein contains a bi-lobed hydrophobic domain similar to that seen for the
known Inc proteins
43
Table 3. Oligonucleotide primers used for preparation of putative inc/malE hybrid
recombinant clones for production of fusion proteins
Target ORF
Forward and reverse oligonucleotides
Region
(5' -------- 3')
C. trachomatis
CT1 19
(incA)
Full length
AGCCATAGGATCTGGTTTCAGCGA
GCGCGGATCCTAGGGAGCTTTTTGTAGGGTGA
CT223
Full length
ATGGTGAGTTTAGCATTAGGGA
GCGCGGATCCTACACCCGAGAGCCGTAATTGAA
CT229
Full length
ATGAGCTGTTCTAATATTAAT
GCGCGGATCCTTATTTTTTACGACGGGATGCCTG
CT233
(inc C)
ATGACGTACTCTATATCCGAT
GCGCAAGCTTAGCTTACATATAAAGTTTG
CT288
316-1452
CGAAAACAAAAACAACTAGAAGAG
GCACAAGCTTAATGCACTCTCCACAAATCTTCTAA
CT442
Full length
ATGAGCACTGTACCCGTTGTT
GCGCGGATCCTCATTGGGTCTGATCCACCAG
CT484
433-1014
ATAGTGCCTCCCTACGACTCTATC
GCGCGCAAACTTCTATTTAGCTACTACCCCGTAGAA
Full length
C. pneumoniae
CPnO186
(incA)
GCGCGGAATTCATAAGTGGAGGAGAAG
GCGCGGATCCTTACTGACCTCCTG
Full length
Maltose binding protein (MBP) fusions of selected putative Inc proteins were
constructed using pMAL-c2 (New England Biolabs). The amplification products of
putative inc genes were produced using Pwo polymerase (Boebringer Mannheim)
and serovar D genomic DNA as the template and digested with restriction enzyme
Xmn land BamHI for CT119, 223, 229 and CPnO186 and XmnI and HindIll for
CT288, CT442 and 484. The vector was digested sequentially with those enzymes.
The digested insert was ligated into the pMAL-c2 vector and transformed into
competent E.coli strain DH5c. Positive recombinant clones were identified using
restriction enzyme analysis, amplification reaction, and sequence analysis and then
overexpressed for production and purification of fusion proteins.
Purification of maltose fusion protein
Maltose fusion proteins were purified as described in the protocol's
instruction (New England Biolab). Recombinant E. coli clones containing the
fusion plasmid were grown in LB medium supplemented with ampicillin at 3 7°C. A
single colony of each recombinant clone was inoculated into 10 ml LB broth
containing 100
tl
2 M glucose and 20 l ampicillin (50 mg/ml), and grown
overnight at 37°C with shaking. Ten ml of the overnight culture was inoculated into
one liter of LB broth containing 10 ml 2 M glucose and 2 ml ampicillin (50 mg/ml)
and grown at 37°C with shaking until cell culture was 2x1 08 cells/ml (optical
density (OD)0.4-0.6). Three ml of 0. 1M isopropyl-13-D-thiogalactoside (IPTG)
was added to the culture and then incubated 2 h at 37°C with good aeration. The
45
cell cultures were then centrifuged 20 mm at 4000 x g at 4°C. The supernatant was
removed and pellet was resuspended in 50 ml lysis buffer (10 mM sodium
phosphate buffer pH 7.2, 0.5 M NaC1, 0.25% Tween 20, 1mM sodium azide, 10
mM 2-mercaptoethonal and 1 mM EGTA [ethylene-glycol tetraacetic acid]). The
cell suspension was kept overnight at 20°C and then thawed in cold water. The
cell suspension kept cold on the ice container was sonicated using short bursts to
avoid heating the extract. The sonicated cells were centrifuged 14,000 x g for 20
mm at 4°C. Supernatant or the crude extract containing the fusion proteins was
pooled and stored at -20°C.
The fusion protein was subsequently purified using 12 g of amylose resin
(New England Biolabs), which was swollen in 50 ml column buffer (10 mM
sodium phosphate buffer pH 7.2, 0.5 M NaC!, 1mM sodium azide, 10 mM 2mercaptoethonal and 1 mM EGTA)) for 15 to 30 mm. Amylose resin was loaded
into a 2.5x10-cm column and washed with 3 column volumes of column buffer.
The crude extract was diluted 1: 5 with column buffer and then loaded onto the
column at a flow rate of 1 ml/min. The column was washed with 3 to 5 column
volumes of column buffer and added with 10 ml of 10 mM maltose diluted in
column buffer to elute the fusion protein. The elution was colleted into 10 x 1 ml of
each fraction.
Antibody production
The purified fusion proteins of putative Inc CT223, 229, 288, 442, 484
CPnO 186, were used as antigen for the production of monospecific antibody.
Antibody production was performed in rabbit, guinea pig, or mice as previously
described (Rockey et al., 1995). Monoclonal antibodies against putative mc, CT
were produced from the Monoclonal Antibody Facility of the University of
Oregon. Mice immunized with CT223p were sacrified and splenic lymphocytes
collected. These cells were fused to SP/2 myeloma cells, using a modification of
standard hybridoma product techniques.
Antisera and reagents for non-fusogenic mutant studies
Rabbit polyclonal antibodies against to C. trachomatis IncA was produced
using described methods (Bannantine et al., 1998). Monoclonal antibody A57B9
(IgGik) is against a 60 kDa heat shock protein (HSP6O), a genus common
determinant (Yuan et al., 1992). Monoclonal antibody 20F12 (IgGik) and 12E5
(IgG2a
k), obtained from the Monoclonal Antibody Facility of the University of
Oregon, are specific for the CT223p of C. trachomatis. Rabbit polyclonal
antibodies against IncA were determined with 35S-labeled staphylococcal protein A
(3700 Bq/ml, Amersham, Piscataway, NJ). Secondary antibodies were purchased
from Pierce Chemical Co. (Rockville, III).
47
Immunofluorescence microscopy
In putative Inc protein studies, indirect immunofluorescence staining was
used to determine protein localization of Incs on the inclusion membranes.
Chlamydiae infected cells were performed as described above. Semiconfluent HeLa
cells in 6 or 24 well culture trays grown on 12- mm-diameter sterile glass
coverslips were infected with Chiamydia trachomatis strain LGV 434 (serotype
L2) and C. pneumoniae strain TW-1 83 diluted in HBSS (Gibco). These cells were
grown in the MEM. 10, supplemented with 10% fetal bovine serum and 10 tg/ml
gentamicin, and incubated for 1 h at room temperature with gentle rocking.
Alternatively, cells were inoculated by centrifugation at 2000 rpm (900 x g) for 1 h.
A multiplicity of infection (MOl) of 0.1 and 1 were used for immunofluorescence
staining.
Inocula were removed, and cultured cells were incubated with MEM- 10
plus 0.7 jg/ml cycloheximide at 37°C for 30 and 40 h. Chiamydia-infected cells
were washed twice with 1% phosphate buffer saline (PBS), and fixed with absolute
methanol for 15 mm or 4 % para-formaldehyde in 25 mM NaPO4 and 150 mM
NaC1, pH 7.4 for 1 h. Para-formaldehyde treated cells were permeablized with
0.1% Triton X-100 (Pierce Chemical Co) and 0.05 % SDS in PBS for 3 mm and
then washed twice with PBS. The permeablized cells were then incubated in the
blocking solution (10% Tween 20 in 50 mM NaPO4, 150 mM NaC1, pH 7.4) for 30
mm and then immunostained with each specified polyclonal antibodies against to
putative Inc proteins. All polyclonal antibodies were diluted 1:100 with PBS
containing 2% (w/v) BSA. After primary antibody staining, cultures were washed 3
times in 1% PBS for 5 mm and stained with secondary antibody conjugated with
fluorescein or rhodamine for 1 h at room temperature. Coverslips were then
mounted onto glass slide using Vectashield (Vector Laboratories) mounting
medium.
In non-fusogenic mutant studies, C. trachomatis infected McCoy cells were
cultured and fixed as described above and probed with genus-specific monoclonal
antibody (mAb) CF-2 to the lipopolysaccharides (LPS) determinant of Chlamydiae.
(Washington Research Foundation, Seattle WA). Mutant isolates were propagated
through several cycles of growth in Mc Coy monolayers.
Double immunofluorescence staining was used to identify localization of
Inc proteins, IncA or CT223p and HSP6O in the study of mutant isolates with
nonfusogenic inclusions. Wildtype and mutant infected cells were probed with
antibodies against HSP6O (mAb A57B9) and Inc protein CT223 (mAb 20F12
orl2E5) or IncA (rabbit polyclonal antisera). Monoclonal antibodies (mAbs)
against CT223p and chlamydial heat shock protein 60 (HSP 60) were used as
controls for immunostaining of inclusion membrane proteins and intracellular
developmental forms respectively. Nucleus DNA was labeled with 4,6-Diamidino-
2-phenylindole (DAPI), which was diluted 2 ig/ml to the mounting medium.
Immnuostaining was visualized using fluorescence microscopy.
Fluorescence images were performed on a Leica DMLB microscrope using the 100
x objective, and digitally collected with SPOT camera (Diagnostic Instruments,
Sterling Heights, MI). Images were processed with Photoshop 6.0 (Adobe
Software, San Jose, CA) and Canvas 6 (Deneba Software, Miami, FL).
Preparation of chlamydiae infected cells for protein gel electrophoresis
Confluent monolayers of HeLa cells or McCoy cells in six well trays were
infected with lxi 06 wildtype and lxi 08 nonfusogenic mutant isolate of C.
trachomatis in HBSS for 30 mm on a rocker platform. The HBSS was removed and
replaced with RPMI- 1640 (Gilbo BRL) supplemented with i 0% fetal bovine serum
(FBS) and iO tg/m1 gentamicin. Monolayer cells were subsequently incubated at
37°C in 5% CO2. Chlamydia- infected cell and mock infected cells were harvested
after 30 h. Cultured cells were washed twice with phosphate buffered saline (PBS;
150 mM NaC1, 10 mM NaPO4pH 7.2) and lysed in 600
tl
of ix SDS-PAGE
sample loading dye (i% sodium dodecylsulfate, 50mM Tris, pH 6.8, i% 2mercaptoethanol, and 10 % glycerol). Lysates were mixed and boiled for 5 mm,
aliquoted, and stored at 20°C. These lysates were used for electrophoresis and
immunoblot analysis.
Preparation of LCR lysates for amplification reaction
Monolayer culture of McCoy murine fibroblast cells grown in 6 well tissue
culture tray were infected with different serovar of nonfusogenic mutant and
wildtype strains of C. trachomatis (Table 1) at multiplicities of infection of
approximately 3 and incubated at 37 °C for 30 h. Culture cells were washed with
50
HBSS, and lysed with a brief sonication. Sonicated cells were centrifuged at high
speed (13 000Xg) in a microcentrifuged tubes for mm and supernatants were
removed. Pellets were then resuspended in a commercial ligase chain reaction
sample collect buffer (LCR Resuspension Buffer, Abbott laboratories, Abbott Park,
IL) was lysed and consequently heated to 98°C for 15 mm. The LCR lysates were
used for DNA amplification of wild type and non-fusogenic mutants of C.
trachomatis isolates.
Amplification reaction and DNA sequence analysis of incA and CT223
In non-fusogenic mutant study, five microliters of the LCR lysates were
used as DNA template for amplification of target sequences. All amplification
products were generated with Taq polymerase in 100 p1 standard reaction, and then
purified using the Qiaquick Columns (Qiagen, Valencia, CA). In case of incA
sequence analysis, two independent region of incA were amplified with specific
oligonucleotide primers that cover the first 500 bp region and full length of the
gene. The first fragment consisted of the 5' half of incA and 429 nucleotide
upstream of the coding sequence which was amplified using specific
oligonucleotide GTTTTTTCATGGCCTCTTCTCT and GATCTGCTATG
ATTTCTTGCG. The second fragment contained the 45 nucleotides upstream of
the predictive start codon and the full length incA coding sequence which was
amplified using specific oligonucleotide AGCCATAGGATCTGGTTTCAGCGA
and GCGCGGATCCTAGGAGCTTTTTGTAGAGGGTGA. DNA sequence
51
analysis was performed on the 5 'half of IncA contained in both of two fragments.
The DNA sequence of the upstream region of incA was analyzed and sequenced
from the amplification products of the first fragment.
Additionally, the entire incA sequence was sequence, in case incA of C.
trachomatis
mutants isolates was not altered in the first 450 nucleotides. The
amplification product of full length incA gene were generated using Taq
polymerase. The PCR products was ligated into p CR2.1 vector, and transformed
into competent INVaF'E. coil cells. Transformation was performed using a
standard technique and plated on LB plates containing 50 tg/ml of ampicillin and
incubated overnight at 37°C. Positive recombinant clones were identified using
restriction enzyme digestion analysis and polymerase chain reaction. Plasmid DNA
of positive recombinant clones were isolated, purified by Qiagen column and
sequenced.
The amplification of the upstream and full length of CT223, encoded for
known Inc protein named CT223p was conducted using specific oligonucleotides.
The primer sequences for upstream region of CT223 and full length were
GCAGCGACAGCACTAATTTTATAA and CTCTGGACAGATTTGTATTG
GAAA and ATGGTGAGTTTAGCATTAGGGA and GCGCGGATCCTACA
CCCG AGAGCCGTAATTGAA respectively.
DNA sequence analysis was performed using an Applied Biosystem 373A
or 377 DNA sequencer (Foster City, CA). Sequence editing was performed using
assemblylign 1.07 (Oxford Molecular Group, Campbell, CA).
52
Electrophoresis and immunoblot
To determine the production of IncA in non-fusogenic mutant of Chiamydia
trachomatis isolates, protein profiles of chlamydial infected cell lysates at 18 and
30 hpi were determined using SDS-polyacrylamide gel electrophoresis. Five
microliters of each diluted lysate sample was loaded on to 12% polyacrylamide gel
and electrotransferred to nitrocellulose membrane. HSP6O was used as a
Chiamydial protein to demonstrate that equivalent amounts of total chiamydial
antigens were load for each sample. Polyacrylaminde gel electrophoresis was
conducted as previously described (Rockey and Rosquist, 1994) and immunoblot
was performed on a Bio-Rad Trans blot cell (BioRad Laboratories) using 25 mM
sodium phosphate blot buffer (pH 6.8). Proteins were transferred at 1.0 A for 1-2 h.
After transfer, the membrane was blocked with PBS plus 0.1% Tween 20
and 2% BSA in for 1 h. The membrane was then incubated with primary antibody
diluted in blocking solution for lh at room temperature. Before used, antisera were
incubated with uninfected HeLa cells and commercial E. coil extract (Promega) for
6 h at room temperature on a platform rocker to increase antibody specificity.
Pellets were removed from treated antisera by centrifugation at 1 ,200xg for 30 mm.
Absorbed antisera were used for immunofluorescence microscopy and
immunoblotting and were stored at 4°C in 0.02% sodium azide.
Immnuostaining with heat shock protein 60 (HSP6O) was used as a control
for a constitutively expressed gene product and for protein quantitation.
Monoclonal antibody 20F12 against to CT223 was used for protein localization on
53
the inclusion membranes. The unbound antibody was removed by three time of
washing in PBS with 0.1% Tween 20 for 5 mm. The membrane was
immunostained with 35S-anti- immunoglobulin G for IncA. HSP 60 and CT223p
stained with monoclonal antibodies were detected using the peroxidase-conjugated
chicken anti-mouse IgG for 1 h. The membrane was twice washed in PBS with
0.1% Tween 20 for 15 mm. The nitrocellulose membrane stained with radioactive
reagent was incubated at 37°C until completely dried. For the peroxidase reaction,
membrane was visualized by incubation in chemiluminescent solution (ECL
reagent, Pierce Chemical Co) for 5 mi
Autoradiographs were performed and
subsequently scanned into images and processed using Photoshop (Adobe system)
and Canvas (Deneba Software) graphic system.
In silico
analysis of the CPn1O54 gene family of C. pneumoniae genomes
The following sets of ORFs of C.pneumoniae CWL 029: CPn0007,
0008/0009, 0010/0010.1, 001 1/0012, 0041/0042, 0043/0044, 0045/0046,
0124/0125, 0126, 1054 and 1055/1056 were aligned using CLUSTALW analysis of
Mac VectorTM 6.0. Each gene and each predicted gene product was also subjected
to gap BLASTX and BLASTN respectively (http://www.ncbi.nlm.nih.gov/
BLAST!) (Altschul
et al.,
1997) to identify homology to known genes and proteins
in GenBank. The similarity between two different DNA sequences was determined
using BLAST 2 sequence program from http://www.ncbi. nlm.nih. gov/blast/bl2seq
54
/b12.html (Tatiana, 1999). The hydrophilicity profile of the gene product of each
ORF was determined using hydrophathy plot of Mac VectorTM 6.0.
Phylogenetic analyses
The homology within the selected genes mentioned above was
demonstrated using phylogenetic analysis. Multiple sequence alignment of the
paralog or gene family was initially performed using CLUSTALW (Mac VectorTM
6.0) and subsequent phylogenetic analyses. Amino acid sequences corresponding to
the hydrophobic domain of the selected genes were also aligned. Phylogenic
analyses of both DNA and amino acid sequences were performed using PAUP
(Swofford, 2001). In this study CPnO 186 (IncA), and additional four putative
inclusion membrane proteins, CPn0284, CPn0285, CPn0829 and CPnO83O, were
selected as outgroups. Phylogenic trees were inferred by neighborjoining to
estimate evolutionary distances. Bootstrap values were obtained from a consensus
of 1000 neighbor-joining trees.
DNA amplification, and sequence analysis of the CP1054 gene family
Purified genomic DNA of selected isolates (Table 2) was amplified using
specific oligonucleotide primers flanking the DNA region of interest in CPn0007,
0010/0010.1, 0041, 0043, 0045, 1054 and 1055 (Table 4). The amplification
products were purified using a Qiaquick PCR purification spin column kit (Qiagen,
55
Valencia CA). The purified PCR product was sequenced using the ABI PRISM 377
(Perkin-Elmer, Norwalk, CT).
Recombinant clones and sequence analyses
The variation of the length of the poiy C tract within a strain of the
CPnOO43, CPn1O54 and CPn1O55 was determined. Genomic DNA of C.
pneumoniae AR39 was amplified using Pwo polymerase and specific
oligonucleotide primer for CPnOO43, CPn1O54 and CPn1O55 (Table 4). The Pwo-
PCR products were directly ligated with vector using the Zeroblunt cloning system.
According to the manufacturer's instructions, the ligation with the pCR II vector
was conducted with a 1:3 molar ratio of vector to PCR insert and transformed into
competent INVaF'E. coli cells. Transformation was performed using a standard
technique and plated on LB plates containing 50 tg/ml of ampicillin and incubated
overnight at 37°C. Positive recombinant clones were identified using restriction
enzyme digestion analysis and polymerase chain reaction. Plasmid DNA of 8-10
different positive recombinant clones were isolated, purified by Qiagen column and
sequenced. Variations of the length of the poiy C tract of CPnOO43, CPn1 054 and
CPn 1055 within C. pneumoniae AR 39 were determined for each recombinant
clone.
Comparison analysis of the variation of the length of the poly C tract in the
CPn1O55 using two different DNA polymerases in the amplification reaction was
further studied. Genomic DNA of C. pneumoniae AR39, PS32 and AR458 were
56
amplified using Taq and Pwo DNA polymerases and specific oligonucleotide
primer for CPn1O55 (Table 4). The PCR product was then cloned into pCR 2.1 and
Zeroblunt (TOPO) vectors respectively and transformed into E. coli. Plasmid DNA
of positive clones were isolated, purified by Qiagen column and sequenced.
Additionally, the variation of the length of poly C tracts in PCR products
amplified from recombinant plasmids but not genomic DNA was determined.
Recombinant plasmid DNA extracted from positive recombinant clones of
CPnOO43, CPn1O55, and CPn1O54 derived from pCR2.1 and Zeroblunt vectors
were used as a template to generate a set of four amplification products using Pwo
polymerase in the amplification reaction. The PCR products were isolated, purified,
and sequenced as described above.
57
Table 4. Oilgonucleotide primers used for PCR and sequencing of the internal
regions of the CPn 1054 gene family from C. pmeumoniae CWLO29
ORF
Region Amplified
Forward Primers (5'----3')
Reverse Primers (5'----3')
CPn0007
7488-8694
AAGAGTTTTCGCTAT
TGACGACTCTTAGATGTTTCG
CPnOO1O
13152-13502
GAAAACACCTCTTGTGGCTAG
AGACATACCCACGGAATAATAAA
CPnOO 10
13175-14431
TACTCGCGAGGACCATACCTAGC
AACTCATGGAACGTTATCTCGTCC
CPnOO 10.1
15178-16324
GCTTGGATGATTTACGACGAG
CTAAGATTTTTTGGGTGGCACG
CPnOO4 1
55728-56987
GAAAATTCTTTTCGCAGAGATGGA
TCCTGTGCTCTTCATAACGCTCAC
CPnOO43
58069-59173
TGCTTTTCAGGCATTCCAGG
GCGAGCTACGACCTCTTTAGCGA
CPnOO45
60755-61856
ATTTGAATCGATGGAGATTCCAGA
TATCCTGCAGCCATTCTACATCTT
Cpn 1054
1206885-1208142
AAAGGAAGTTTTCTTGAAGATCTT
ACTCATGGAACGTTATCTCGTCC
CPn1 055
1209382-1210530
AGTTCCTGAGATTCCTGAGGC
TATCTCCTCAACTACTCGAGATAAC
RESULTS
Identification and bioinformatic analysis of putative Inc proteins
J4ydrophilicity profiles of all open reading frames (ORFs) present in the C.
trachomatis and C. pneumonae genomes were determined using hydropathy plot
analysis. A deduced amino acid sequence containing a bi-lobed hydrophobic motif
(60-80 amino acids) in the secondary structure was predicted as a putative Inc
protein. Based on the selection criterion, 45 of 894 ORFs of C. trachomatis, and 68
of 1073 ORFs of C. pneumoniae, were identified as putative Inc proteins (Table 5).
Putative inc genes occupy 2.7 % of chlamydia-specific coding capacity in
C. trachomatis, and 5.5 % in C. pneumoniae. Open reading frames corresponding
these putative Inc proteins appeared to be randomly distributed throughout each
chlamydial genome. There are, however, some exceptions. Two contigs encoding a
cluster of inc gene products were identified in the C. trachomatis genome. The first
cluster contained four inc genes: CT115-118 in the contig 2.3, and the second was
seven inc genes: CT222-229 in the contig 3.5. The four-inc gene cluster was
located downstream of the known inclusion membrane protein of C. trachomatis
incA (CT1 19). The gene products of CT1 15-118 were recently identified on the
inclusion membrane using immunofluorescence staining (Scidmore-Carison et
al. 1999) named and recognized as IncD-G of C. trachomatis. A seveninc gene
cluster, CT 222-229, is located close to genes encoding amino acidand
59
Table 5. Putative inc genes identified by hydrophilicity profile within each
sequenced chlamydial genome
Putative ORFs
unique to each species
Putative ORFs showing
conservation between the species
(Orthologs)
C. trachomatis C. pneumoniae
484a
728
195
565
005
383
288
058
006
850
0602
0869
0288
0822
0443
0480
0065
0369
0442
E- value
C. trachomatis
e85
036
1.8 e50
115
116
117
118
134
135
164
192
196
5.2
2.7e42
2.2
2.0
2.5
e30
&26
e20
1.5 e5
1.7 e13
1008
7.4 e'2
2.9 e12
101
0312
1.4e'2
232
058
618
483
233
442
058
440
449
345
119 (IncA)
0291
4.2e1°
0370
0753
26'°
2e'°
0601
4.1
0292
0556
0367
0554
0565
0026
0186
1.5e°7
e08
1.2e°7
2 e05
1.2e°6
0.00045
0.0021
0.002
214
222
223
224
225
226
227
228
229
300
357
358
789
813
a boldface identifies ORFs examined in this work.
C. pneumoniae
0007
0008
0010
0011
0041
0043
0045
0067
0124
0126
0130
0131
0132
0146
0147
0164
0166
0169
0174
0215
0216
0225
0240
0241
0266
0267
0277
0284
0285
0352
0354
0355
0357
0365
0366
0371
0372
0375
0431
0432
0439
0440
0524
0585
0829
0830
1054
1055
sodiumdependent transporter proteins. BLAST analysis revealed that gene
products of CT232 and CT233 have significant sequence similarity to known Inc
proteins of C. psittaci IncC and IncB. In C. trachomatis, clusters of putative inc
genes were tightly linked to both the incA (CT119) and the incB/incC
(CT223/CT222) homologues. Such gene order, however, is not present in the C.
pneumoniae genome. The predicted sequences of IncA of C. trachomatis and C.
pneumoniae were more similar to that of C. psittaci than to each other.
Additionally, like C. psittaci incB/incC, several putative inc genes in C.
trachomatis and C. pneumoniae genome, were arranged in tandem. These included
CT134/135, CT232/233, CT357/358, CT483/CT484, CPn013O/0131/0132,
CPnO146/0147, CPnO215/ 0216, CPn0266/0267, CPn0284/285, CPn0354/0355,
CPn0365/03 66, CPnO37 1/0372, CPnO43 1/0432, CPn0439/0440, CPn0829/0830,
CPnO6O1/0602, and CPn1O54/1055.
Sequence similarity analyses were conducted to determine the similarity of
putative Incs to known proteins in GenBank. BLAST search analysis showed that
there was no significant similarity of these putative Inc proteins to any known
protein in the database. However, the similarity analysis of putative Inc proteins of
C. trachomatis and C. pneumoniae using the BLAST search and BLAST 2
sequences program demonstrated that some ORFs are orthogous or paralogous
genes which have significantly sequence homology between and /or within the
species (Table 5). For example, ORF CT484 and CPnO6O2 are orthologs
considerably derived from gene speciation, which share significantly sequence
61
similarity each other between species. The similarity data are consistent with
BLAST data present in the Chiamydia Genome Project (Stephens et al., 1998).
Genomic analysis also revealed that several putative inc(s) were defined as
paralogs likely derived by gene duplication in C. pneumoniae genome. These
included CPn0007, 0008, 0010, 0041, 0043, 0045, 1054 and 1056, and CPn0367,
0369, 0370 and 0524. CPn0367, 0369, 0370, and 0524 have sequence similarity to
CT058, a putative C. trachomatis inc. These putative inc(s) were quite similar each
other and to CPnO 107, which is smaller than the others and lacks a bibbed
hydrophobic motif, the predictive marker for localization to the inclusion
membrane. Except CPn0524, sequence similarity alignment of these genes,
especially at the carboxy termini was shown (Figure 5). The similarity of
paralogous genes, CPn0007, 0008, 0010, 0041, 0043, 0045, 1054 and 1056 will be
further studied using phybogenetic analysis and comparative genomic approaches
later in the this chapter.
Phylogenetic analysis of the primary sequence of putative inc using the
PAUP method failed to identify the genetic relationship within the Inc group. This
was true for analyses of either complete protein sequence, the predicted
hydrophobic domain, or analyses of the genes remaining after removal of the
bibbed hydrophobic region. Collectively, among putative Inc proteins, a bibbed
hydrophobic domain is the only common structural motif present in the secondary
structure of the chlamydial Inc protein.
-____
ma
I.
=
5.00
4.00
3.00
2.00
Q
0.00
.
a'
- im
100
-200
> -300
50
-C.p. 369
C. p. 370
C. t. 058
C. p. 107
C.p. 367
C.p. 369
w
..
IINA MNNIIM*
-APIIIIIIIIIIIIIIIk*k,* AM *IIINIIMISi*:IllIIIIIIIIIIIIII.iIIIIIIIIIIIIIIIIIII
II...I..
11
BC. t. 058
C. p. 107
C. p. 367
i_I_IIIIIIIIIIIIIIIIF
-
is
ma ii
&&
mai
A1lNu*lnmmtcunnttmmmmnt.nhpIuuuupu*4NuIINI* *
w
o -100
I
62
1.-I'
.1.1'
-.-5I
NI PSTFITIFVST GQFSSLR
SARFR
N KIFA ALNQSKLIFVSTSGNIAQPR
N RYYVWEHQGADIVATTGDIAKPR
QQPCFI KLKDSKLIFISTSGDIAVPR
159031148183157-
N TGYA.%HLKNSNLTLISTLGPIEKPR
186- IMQSNLP- AA IANATLQSAAFAKRGQ
058- ILVTDSMSM I NAANIRTM]R175- IL
=
TS
210- [i K T
R
CR
V M I V N A A N S N MIS
V NI I V N A A N E N I
R
L
VN
GAGTN
GACTN
CGCTN
C. p. 370
184-V1KTQGI-VMIVNAATPNMAIN----NVKGTS
C. t. 058
215- D F P IXL L[T1DIIC %% E[1
o84QVILSAA1VSVID1SWG1LSQRPLNPE
C. p. 370
209- LALAKATSVRC%%fNSKKSPDLRSKQFL L
C.t.058
239-CECS ATWEDKNHLV
114-GECRAGM RNADGSN
230- CECRS P INRD TN
265-GECRSAKWENSDHTS
239-CECRSAKWENLNGT
C.p. 107
C.p. 367
C.p. 369
C.p.107
C.p.367
C.p.369
C.p.370
FElL
GTPL
E
GKGL V
200- AFLSAATHPTICWNINTRTSGG
235- KALS ATSLQCWJASRLPRAHS SGSQL P
I
1'
I
Figure 5. Comparison of predicted proteins encoded by C. trachomatis CT058
and C. pneumoniae CPnO1O7, CPn0367, CPn0369 and CPnO37O.
A)Hydrophilicity profiles for three of these predicted proteins.(Note the scale
differences for each plot). B) Similarity of these predicted proteins. Sequence
alignment of the carboxy termini of each protein using CLUSTALW (Mac
VectorTM 6.0). Amino acid similarity and position are indicated to the left of
each line.
63
Protein localization using immunofluorescence staining
Immunofluorescence staining was used to test the hypothesis that a hi-lobed
hydrophobic domain can be used as predictive marker for chiamydial inclusion
membrane proteins. Six putative inc genes of C. trachomatis: CT119, CT223,
CT229, CT233, CT288, CT442, CT484, and CPnO 186, a candidate incA gene of
C. pneumoniae, were selected to identify the protein localization (Table 5)
Fluorescence staining revealed that all selected putative Inc proteins were
localized onto the inclusion membrane except CT484 p (Figure 6). The
localization of gene product of CPn0186, the C. pneumoniae IncA homology, was
also shown on the inclusion membrane protein of C. pneumoniae infected cells
(Figure 7). Although this ORF encodes the sequence that is about 20 % similar to
C. psittaci and C. trachomatis incA, the secondary structure of the gene product
contains the common hi-lobed hydrophobic domain identified in known Inc
proteins. Five of six selected putative Incs were localized onto the inclusion
membrane, including the CT233 homologue, C. psittaci IncC, CT442 (a cysteine
rich protein), and three previously unexamined proteins encoded by open reading
frames CT223, CT229 and CT288. The localization of CT233 and CT288 cannot
be detected on the chlamydial inclusion after methanol fixation. Fixation with
paraformiadehyde and subsequent permeabilization with Triton X- 100 was used to
demonstrate the localization of these proteins on the inclusion the C. trachomatis
infected cells. Both proteins were shown to localize to discrete patches within the
inclusion (Figure 6). Anti- CT223p antibodies also labeled C. trachomatis L2.
64
Figure 6. Immunofluorescence microscopy of C. trachomatis infected HeLa
cells fixed with methanol and probed with specific antibody against each
tested putative Incs. Panels A and B represent a cell doubly labeled with antiIncA (A) and HSP6O (B) and demonstrate the characteristic labeling evident
for known Inc proteins. Panel C-H each represents cells singly labeled with
each specific antisera against CT229(C), CT223(D), CT228(E), CT233(F),
CT442(G) and CT484(H).Arrows indicate the inclusion membrane in
E,F and G. Bar in H represents 10 microns for each image.
65
Figure 7. Double-labeled fluorescence microscopy of C. pneumoniae
labeled with antiHSP6O (A) and anti-C. pneumoniae IncA (CPnO186)(B).
Infected cells were fixed with methanol at 44 h. and labelled with anti
HSP6O (A) and anti- C. pneumoniae IncA (CPnO 1 86)(B). Note the IncA is
distributed the margins of the chlamydial inclusion, similanly to as seen with
C. psittaci and C. trachomatis (Figure 6A).
inclusions with a unique pattern (Figure 8). The distinctive pattern between IncA
and CT223p was shown using double immunostaining with anti-IncA and CT223p.
A gene product of ORF CT484, one of selected putative inc genes was not
found on the inclusion membrane. The localization of gene product CT484 was
absolutely undetectable from either early or late inclusions fixed with either
methanol or paraformaldehdye. The gene product of this putative inc has extensive
sequence similarity with a corresponding gene product of CPnO6O2 in C.
pneumoniae
genome. Cross reactivity of antisera against CT484p, therefore, also
was determined. A weak immunostaining of C. pneumoniae-infected cells was
observed with anti-CT484p antibodies (data not shown).
Fluorescence microscopy of cells infected non-fusogenic variant isolates
A previous study by Suchland et al. (2000) revealed that 1.5 % (176 of
11,440) of independent clinical isolates of C. trachomatis infected patients
collected over the 12 years showed a variant inclusion phenotype. Variant isolates
form multiple-lobed inclusions within single cells infected at MOl greater than 1
whereas wild type isolates form a typical single inclusion, following infection of
cells at high multiplicities (Figure 6). In this study, 13 wild type and 26 nonfusogenic variant isolates were subjected to examine the localization of IncA
protein on the C. trachomatis infected cells using immunofluorescence staining.
67
Figure 8. The distinct pattern of the localizaion of CT223 on the inclusion
membrane. Chiamydia trachomatis serovar L2 infected HeLa cells fixed
with methanol and doubly labeled with anti-IncA (A) and anti-CT223p
antisera
The localization of IncA to the inclusion membrane is always demonstrated
in wild type clinical isolates. In contrast, multiple-lobed inclusions form in infected
cells under similar condition by variant isolates. Two distinct groups of
nonfusogenic variant isolates were identified with respect to the presence or
absence of IncA in fluorescence profile. Most non-fusogenic variant isolates lack
IncA protein localized on the inclusion membrane. Twenty four of 26 non-
fusogenic mutant isolates were negative for immunofluorescence staining of IncA
protein (Figure 9). Two non-fusogenic variant isolates {G(s)459 and J(s)5942}
were positive for IncA protein immunofluorescence staining (Figure 9,10). The
IncA-positive variants were morphologically distinct from the IncA-negative
nonfusogenic variants. A high degree of variability in the number, size and shape of
individual vacuoles was observed within the infected cells. The fluorescence
staining of IncA and CT223p in these isolates was very strong, suggesting possibly
high levels of each protein.
Additionally, the localization of CT223p on the inclusion membrane was
determined. Initially, identification of CT223p was used as the positive control of
the localization of Inc of C. trachomatis infected cells. In these clinical isolates,
four variant isolates showed the absence of the CT223p on the multiple-lobed
inclusion. One of the 13 wild type isolate (J3464) and 3 of the 26 variants
{J(s)1980, J(s)6276, J(s)6686} were shown to lack CT223 as determined by
fluorescence staining. All identified CT223-negative isolates were of serovar J or
J(s). There was no correlation between fusogenicity and non-fusogenicity, or any
Anti-IncA
Anti-HSP6O/CT223p
J(s)
5942
J(s)
893
J(s)
1980
Figure 9. Fluorescence microscopy of McCoy cells infected with serovar J(s)
variant of Chiamydia trachomais probed with antibodies against HSP6O, IncA
and CT223. Cells were infected and incubated 30 h prior to methanol fixation
and labeling with antibodies specific for IncA (left column), or with a combination of and anti-HSP6O (green) and anti-CT223 (red). The isolates number is
indicated to the left of each pair of pictures. Note that the images in the left column do not show the same cells presented in the right column. All images were
magnified and photographed under identical conditions.
70
Figure 10. Fluorescence microscopy of McCoy cells infected with serovar
G(s) 459, an Inc A positive variant, of C. trachomatis probed with antibodies against IncA. Cells were infected and incubated 30 h prior to methanol fixation and labeling with antibodies specific for IncA (A) Wildtype
serovar G; (B) servar G(s)459. In these cells, the varied size and shape of
the IncA-positive inclusion are evident. As with figure 8, note that these
images do not represent the exact same cells.
71
other observable aspect of inclusion development, and the presence of absence of
CT223p.
Immunoblotting analysis of selected non-fusogenic variant isolates
The protein profile of IncA and CT223p of wild type and variant infected
cells was determined by immunoblotting. It was used to confirm the result of
immunofluorescence staining of IncA and CT223p and to verify that those proteins
were not present on the inclusions at detectable levels within infected cells.
Chlamydial HSP6O was included as a control to demonstrate that similar amount of
total chlamydial antigen was loaded for each sample. The immunoblots of
representatives of serovar D, J, H, of wild type and variant isolates was shown
(Figure 11). The results of immunoblot were consistent with those of
immunofluorescence microscopy. Isolates that lacked detectable IncA and CT223p
in the inclusion membrane were also negative by immunoblot.
According to the presence of CT223p in immunofluorescence microscopy
and immunoblotting determined by monoclonal antibody, the possibility exists that
CT223p negative variants are actually producing this protein, but the alteration in
sequence eliminated the epitopes recognized by mAb. To address this, the variant
CT223 sequence was cloned into E. coil using a similar approach to that used for
production of the wild type CT223p. The mutant protein was recognized by the
anti-CT223p-mAb in immunoblot analyses (data not shown).
72
IncA
CT223p
HSP6O
123456789
t
-
-.. i
-_ -
Figure 11. Immunoblot analysis of non-fusogenic C. trachomatis isolates
probed with antibodies against HSP6O, IncA, and CT223p. Lysates of HeLa
cells infected with selected non-fusogenic and wild type C. trachomatis
isolates were eletrophored, transferred to nitrocellulose membrane, and
probed with either anti-IncA, anti-CT223p or anti-HSP6O.
Lanel+1O, Mock-infected lysate; 2+11, wild type serovar D; 3, D(s) 2923;
4, D(s) 8093; 5+ 12, wild type serovar J; 6, J(s) 893; 7, J(s) 1980;
8, B(s) 1129; 9,H(s) 5942; 13, J(s) 1980; 14, G(s) 459
73
Sequence analysis of incA of non-fusogenic variant isolates
The incA sequence from 26 independent nonfusogenic variants and 13 wild
type isolates representing ten serovars or sub-serovars were examined (Table 1).
We studied a single isolate for serovar B(s), G(s), H(s), E(s) and K(s), two isolates
of serovars F(s), and Ta(s) and three or more isolates for serovars D(s), D-(s) and
J(s). Wild type isolates used for control were of serovar B, D-, F, G, Ta, and E and
seven for serovar J. The variation of incA sequence in the population of wild type
C. trachomatis isolates was also examined. Sequence data and Genbank accession
number for variants are presented (Figure 12)
In these studies, 5 of 13 (3 8%) wild type isolates (E9332, J2364, J3464,
J93 11 and J 9325) contained incA identical to prototype incA sequence, and 8 of 13
(62%) wild type isolates showed nucleotide variation of incA sequence (Figure 12).
Three of 13 (39%) wild type isolates (D-9291, F9334, and J9336) were incA
variants with the substitution of isoleucine with threonine (147T) at codon 47 and
glutamic acid with lysine (Eli 6K) at codon 116 in the gene product. Such variant
sequences were also identified in nonfusogenic variant isolates. These sequences
were slightly different from the prototype incA sequence of C. trachomatis serovar
D{(D/UW-3/Cx), Genbank Accession #:AE001273} present in the genome project
However, there was no change resulting in alteration of a reading frame or
introduction of a stop codon of incA identified in those wild type isolates.
74
Figure 12. A graphic representation of the results from the DNA sequencing of
incA encoded by 10 wild type and 25 non-fusogenic C. trachomatis isolates. To the
left of the isolate designation are the accession numbers and the result from
fluorescence microscopy of the isolates with antibodies t o Inc. The complete IncA
sequence was determined for the isolates shown in bold while the first45O-550
nucleotides was analyzed for all other isolates. The scale of the amino acids in the
hydrophobic domain is used to position in the symbols representing the mutations.
In the legend the symbols are defined-note that a variety of distinct lesions within
the incA coding sequence are associated with the lack of IncA within the inclusion
membrane. Isolates G(s) and J(s)5942 are nonfusers encoding IncA that is properly
localized to the inclusion membrane., silent base changes in incA; *, a base
change that leads to an amino- acid substitution; v, a single base deletions or
insertions that lead to premature truncation of the protein; , single base changes
that leads directly to a stop codon. Solid bars indicate larger deletions and are
drawn to scale.
75
Nucleotide_ppst!on
Wild type
450
300
150
Acessioü
B9227
+
D-9291
+
*
F9334
+
*
G9301
+
AF327326
AF327328
-.".-i-*
+
J9329
J9336
J9346
+
+
I
I
*
*
r
U
*
*
I
I
+
I
Variant
B(s)1129
D(s)2923
D(s)4013
D(s)8039
D(s)1012
I
U
D-(s)5328
D-(s)3991
D
*
*
*
*
*
*
*
S
E(s)1986
I
H
*
*
F(s)8068
F(s)4022
AF279360
AF279343
I
*
V*
I
I
v
Ia(s)2197
la(s)7713
J(s)893
J(s)6462
-.
1
*
I
I
*1
+
U
AF327330
AF327331
AF327332
AF327333
AF279342
AF279359
+
H(s)9005
AF279344
AF279345
AF279346
AF327334
AF279347
AF279348
AF279349
AF279350
AF279351
AF279352
AF279353
AF279358
AF279354
AF279355
J(s)6686
Figure 12.
AF327329
AF276503
AF279341
I
H(s)5642
K(s)2481
ri
AF279340
AF276502
I
D-(s)8323
J(s)6276
AF327327
AF276501
AF1 63773
I
D(s)5058
J(s)1980
J(s)3839
J(s)6565
J(s)5942
i
I
1a9309
G(s)459
750
600
IncMf
V
AF279356
V
AF279357
76
On the other hand, these analyses identified a wide variety of distinct incA
sequence associated with the non-fusogenic phenotype (Figure 12). The sequence
analysis of 18 of 26 nonfusogenic mutant isolates revealed the alteration of the
5 'half of incA, which led to the truncated products. The incA sequence of most non-
fusogenic variant isolates (12 of 26) contained a frameshift mutation resulting from
single base change, which either altered the reading frame or caused directly to a
stop codon. Most of these are within the S'half of the gene, with the two exceptions
being D(s)1012 and F(s)8068. Seven of 26 mutant isolates: B(s)1 129, D(s)1012,
D(s)8323, D(-s)5328, D(-s)3991, E(s)1986 and J(s)1980 contained nonsense
mutations leading to the truncated gene product of incA.
Large deletions in incA were also identified from 4 non-fusogenic variant
isolates: J(s)893, J(s)6462, J(s)6686, and H(s)5642. An identical 51 nucleotide
(position 152-202) in-frame deletion of incA which resulted in the internally
truncated protein product was identified in J(s)893 and J(s) 6462 isolates. In
addition, J(s) 6686 and H(s)5642 have a14 nucleotide (position 18-3 1) and a 671
nucleotide out-of- frame deletion respectively, which leads to a change in reading
frame and a short, truncated, predicted products. A large deletion (671 nucleotides)
identified from H(s)5642 isolate extended from position 366 of incA into the 3'
untranslated region. The sequence analysis of several selected isolates of serovar
D(s), D(-s) and J(s) were performed. Three different isolates of serovar D(-s): 8323,
5328 and 3991 have an identical nucleotide change that leads to the introduction of
a stop codon.
77
The complete incA sequence was also determined from an IncAnegative
non-fusogenic isolates, D(s) 2923, and two IncA-positive non-fusogenic isolates
G(s)459 and J(s)5942. In D(s)2923, incA was altered at 3 nucleotides, leading to
the amino acid substitutions with the substitution of isoleucine with threonine
(147T) at codon 47 and glutamic acid with lysine (El 16K) at codon 116. Such these
changes in amino acid sequence were also identified in wild type isolates. In IncApositive non-fusogenic isolates, G(s)459 and J(s)5942 showed the variation of incA
but they still encode intact, complete full-length incA sequences. Therefore, there
were no mutations in incA that were uniquely associated with the IncA-positive
nonfusogenic isolates.
DNA sequence analysis of ORF CT223
The complete sequence of ORE CT223 was conducted from six wild type
and six non-fusogenic variant isolates. Included in these analyses were 3 serovar J
or J(s) isolates that were CT223p-negative by fluorescence microscopy and
immunoblot. Each DNA sequence were translated into predicted amino acid
sequence and compared to those of C. trachomatis serovar D and L2 provided
through the Chlamydia Genome Project. In contrast to incA, there is considerable
variation in the CT223 sequence of the serovar D and L2. The predicted sequences
of CT223 for serovar D and L2 have shared 91 % similarity. While incA varies at
only a few nucleotides, CT223 present in the database vary at 34 nucleotides
leading to 26 differences in amino acid sequence. The CT223p of the variant
78
sequences consisted of mosaic of the serovar D and L2 genomic sequence.
Sequence variation resulting from nucleotide substitution in CT223 was identified
in both wild type and variant isolates. In the three CT223p- negative isolates,
J3464, J(s)1980, J(s) 6686, the deduced amino acid sequences of CT223 were
identical to each other but distinct from the either prototype CT223 sequence
serovar D or L2 {Genbank accession number's for J3464 (AF279363); J(s)1980
(AF279362); J(s)6686 (AF279364)}. Each sequence had two unique nucleotide
substitutions that resulted in serine at position 22 changed to leucine, and glutamine
at position 120 changed to arginine (Figure 13). Neither of these mutations was
identified in any of six CT223 positive isolates or within the CT223 published
sequence from serovar D or L2. There is no association of changes in amino acid
and the absence of CT223p determined by immunofluorescence and immunoblot
studies.
Upstream sequence analysis of incA and CT223
The upstream region of incA of 6 of 13 wild type and 18 of 26 variant
isolates were amplified and sequenced in order to determine the variation of
predicted promotor regions of each isolate. These included D(s)2923 and J(s)893
isolates with intact coding sequences. Changes in the promotor sequence might be
responsible for the lack of the IncA in these isolates. However, the incA upstream
region of both of wild type and variant isolates was identical to that of the C.
responsible for the lack of the IncA in these isolates. However, the incA upstream
79
Figure 13. Multiple sequence alignment analysis of CT223p of fusogenic and
non-fusogenic variants of C. trachomatis isolates. Deduce amino acidsequence
from 6 wild type and 6 non-fusogenic variants were aligned and analyzed and
compared to those of C. trachomatis serovar D and L2 provided through the
Chiamydia Genome Project. The variant sequences consisted of mosaic of the
serovar D and L2 genomic sequence. In the three CT223 negative isolates,
J 3464, J(s)1980, J(s) 6686, the deduced amino acid sequences of CT223 were
identical but distinct from the either prototype CT223 sequence (serovar D or L2)
{accession #s for J3464(AF279363); J(s)(AF279362); J(s)6686(AF279364)}. Each
sequence had two unique nucleotide substitutions, which resulted in serine at
position 22 changed to leucine and glutamine at position 120 changed to arginine.
80
20 *
10
1419301(G)
1419346(J)
M19309(Is)
MT9334(F)
1419332(1)
CT223(servarL2)
CT223(serooaiO)
1413464(J)
MT2923(Ds)
MT4013(Ds)
M11012(Ds)
94r893(Js)
1416686(Js)
14r1980(Jo)
141401 3(Os)
141101 2(Ds)
14T893(Js)
MT6686(Js)
1411 980(Js)
MT9309(Ia)
MT9334(F)
1419332(E)
V C 1 FA F P
LFF
Y
A6V
A I V P.
UT9334(F)
MT9332(E)
CT223(oslv9r L2)
CT223(se,ovar 0)
1473464(J)
MT2923(Ds)
147401 3(Ds)
1411012(119)
M1893(Js)
1416686(b)
1411 980(b)
14193090.)
1419334(1)
1419332(1)
CT223(s.IvaI 12)
C1223(serov.r D)
1413464(J)
M12923(Os)
141401 3(Ds)
141101 2(Ds)
UT893(Js)
M16686(Js)
1411 980(Js)
Figure 13.
0
V
V
V
V
I
P.
GL
01
GI
GL
VI'I
V
(2
G
150
NLKP.W
WT
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EKQIEELKP.M
EKQIEEIKP.M
EKQIESLKP.M
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210
200
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INIL
INIL
CHE
CHE
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IVLL
INLL
180
170
150
.CCLN1EEKVRDLITKL0GYQERLKVLPA
OCCLNLEEEVRDLLTKLGG0060LKVLPA
LNLEKEvRDLLTKLGGYQERLKVLPA
A
LNLE!EVRDLLTKIOGYQERLKVIPA
A
LNLE[V)EVRDLLTKLOGYQERLKVLPA
S
LN gTov LqTKLrnoyQE9LKvLpA
A
Y LGGYQKRLKVLPA
-LV EflEV
L
LV EEV L I LIT1GYQERLKVLPA
S
LV 66EV L I LG0V ESLKVLPA
L
I LOGY EELKV LIP.
LV 68EV
A
L
I IGGY ERLKVIFA
LV 8.8EV
LOGY ERLKVIPA
LV ETEV
L
1
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LV ETEV
I I L .GY EELKVLPA
SCCLN 66EV
1
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LFGYVERLKVLPP.
220
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696
606
658
230
tIN
LFN
LFN
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240
flED
0
NED
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NED
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6
KY
'EJ
EKQIEELKP.M
EKQIEEI.KP.M1
EKQIEELKAML NY
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:
S
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C
I
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EKQI EELKAM
EKOILELKAM
200
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1419346(J)
VIOL
VIOl.
V
V
50
0GrVL
L
GGAVVL
Yroll
LVI.GL
V
A V I I G V 0 V S N I C S C C I P. 8
140
ISO
1419301(G)
1419346(J)
1419309(19)
I. 0
Q
Q
0
(2
MT1980(Js)
T
90
r
CT223(selov2rD)
14T1012(Ds)
MT893(Js)
1416686(Js)
V
FCTFAPPLPFYAGVAIVALGAVILGVGVSNTCSCCLRSP.KIEAQLILQQKLEISQLK
FCTFAPPLFFYAGVALVALGAVILGVGYSNICSCCLRSKKIEAREQLILQQKEEISQLE
FCTFAPPIFFVAGVALVALGAVILOVGVSNTCSCCLP.SKKIEARKQLILQQKKEISQLE
PCTFAPPLFFYAGVAIVALGAVILGVGVSNTC8CCLRSKKIEAREQLIIQQKKKISQIE
PCTFAPPLFFVAGVAIVALOAVILGVGVSNTCSCCLRS9KIEAHF1(IQLILQQKEEISQLK
FCTFAPPLFFYAGVALVALGAVILGVGV9NTCSCCLRSRKIEARTQLIIQQKEEISQIE
FCIFAPPLFFYAGVALVALGAVILGVGVSNTCSCCLRSI1KIEAREQLIIQQKKEISQLE
C1223(servarl2)
MI4013(Ds)
V
I
FCTFAPPLFPYP.GVP.LVALGAVILGVGVSNTC$CCLRSRKIF.AREQLIIQQKKEISQLE
FCTFAPPLFFYAGVP.LVALGP.VILGVOVSNTC8CCIRSI(KIEAREQLILQQKEEISQLE
PcTFApPIFFYAGVAIvAIGAVILGVGVSNTCSCcLRSRKIEP.HrnQLIIQQKKEISQIE
FCTFApPLFFVAGVALVAIGAVILGVGVONTCSCCLRSKRIEARrQLLIQQKEEISQLE
Q
Q
1413464(J)
14T2923(Ds)
I
V
V
V
CTPAP PLFFYAGVALVALGAV I LGVOV SNTC SCCLR SKK I EARQL
130
1419301(G)
1419346(J)
V
V
V
rfl
50
70
MT9301 (0)
1419346(J)
M19309Qa)
1419334(1)
1419332(E)
CT223(servac L2)
CT223(oerovar D)
MT3464(J)
M12923(Ds)
91
90
IVSLALGT9NGVEANNGIN1)L8PAPEA
OVSLALGT8NGVEANNGJNDLSPAFEA
(V8IALGTSNGVKANNGINDLSPP.PEA
OVSLAIGT9NGVEANVGINOL8PAFKA
4VSLALGT9NGVEP.NNGINDLSPAPEP.
6VSLALGTSNGVEP.NNGINDLSPAPFP.
4VSLALGTSNGVEP.NNOINDL8PAPKA
OV9IALGISNGVEANNGINDLIflPAPEA
IVSIALGT8NGVEANVGINDLTPP.PEA
4VSIALGTSNGVEANNGINOISPAPKA
409LALGT8NGVEANNGINOISFAPOA
4VSLALGTSNQVEAVNGINDLSPAPEP.
(VSLALGTSNGVKP.NNGIVDL PAPEA
IVSLP.IGTSNGVEANNGINDLIPAPEA
L
L
280
L
L
L
L
L
P.
A
P.
A
I
P.
L
A
270
VK9VNL IDLDPKSDS 000D0000FVYG9RVN
VK9VNL IDLDPKSDS SDGD0000FNYOSI1VN
VKSVNLIDLDPKSDS SDGDDDDGFVYGSEVN
VKSVNLIDLDPKSDS SDGD0000FNYG9RVN
FNYGSEVN
AKSVNIIOLD KSDS S0000FNYGSRVN
VK8VNLIDLD KSDS5060D
VKSVNIIDLDPX0DS S0000000FNYOSRVN
VK9VNL IDLDPK9DS906DD000FNYGSRVN
VKOVNL IDLDPKSIIS SDGDDDDOFNYGSRVN
VKSVNIIDLDPKSDS SDGDD000FVVG0RVN
VKSVNL IDLDPKSD$ SDGII0000FNYGSRVN
VKSVNL IDLDPKSD0S060D000FNYG8RVN
VKSVNIIDLDPKSDS 00600DDGFNYGSEVN
P.
L
L
L
I
L
1
L
0
6
1
1
L
59
(3
G
I
1
(3
1
1.
HR
V
V
0
G
V
V
..V
SlY
KV
KY
KY
81
region of both of wild type and variant isolates was identical to that of the C.
trachomatis prototype strain. There is no difference in the predicted promotor
region of incA in any selected isolates. On the other hand, the upstream sequence of
CT223 of variant isolates that lack the protein CT223p showed sequence variation.
The nucleotide substitution of thymine (T) with cytosine (C) of the upstream region
(at 111 from the start site) was identified in all CT223-negative variants but not
any wild type and CT223-positive variant isolates (data not shown) {Genbank
accession number's for J3464 (AF279363); J(s)1980 (AF279362); J(s)6686
(AF279364) }.
In silico analysis of the CPn1OS4 gene family
A novel gene family sharing extensive sequence similarity within the C.
pneumoniae genome was identified using Gap BLASTX and BLASTN search and
multiple sequence alignment analysis of CLUSTALW (VectorTM 6.0 ,Oxford
Molecular). This gene family, designated as the CPn1 054 gene family, consisted of
19 C. pneumoniae-specific genes: CPn0007, 0008, 0009, 0010, 0010.1, 0011, 0012,
0041, 0042, 0043, 0044, 0045, 0046, 0124, 0125, 0126,1054, 1055, and 1056.
These genes are located in four separate regions on the chromosome: contig 1.0-1.1
(CPnOO7-CPnOO12), contig 1.5-1.6 (CPnOO41-CPnOO46), contig 2.5 (CPnO124-
126) and contig 13.0-13.1 (CPn1O54-CPn1O56) (Figure 14). Similarity of DNA
sequences of paralogous genes using BLAST 2 Sequence program ranged between
82
Chiamydia pneumoniae CWLO29 genome
Contig 1.1
*
11000 12100 11300 14500 15400 I4
I*
1700 19600
17400
I
Contigs 1.5-1.6
-
54400 40000 (1400 (2700 (5400 (.4500 (4000 (7100 (*200 (*500
54100 57200
II
104
I*
Contig2.5
151200 152400 152400 154000
I54
t57300
56*20 159600
II
Contigs 13.0-13.1
1201
I2OO 1204015) 1200400 1304*00 l30O 1309600 1211000
I *S
øl*ø
w
9*9 9 9999
9
99
t
Figure 14. A schematic representation of C. pneumoniae CWLO29 contigs.
The CPn1O54 gene family is encoded in the contig 1.0-1.1 (CPn0007-0012),
contig 1.5-1.6 (CPnOO4 1-0046), contig2.5 (CPnO 124-0126), and contig 13.013.1 (CPn1OS4-1056).
83
Table 6. DNA sequence identity of paralogous genes in the CPn 1054 family
using Pairwise of BLAST 2 sequence program
% identity for paralogous genes of the DNA sequence
ORF
0008/9
0008/9
0010/10.1
81
0041/42
0043/44 0045/46
1054
a
1055/56
78
76
85
81
82
82
76
81
99
89
76
81
84
76
81
84
81
85
0010/10.1
81
0041/42
78
82
0043/44
76
76
77
0045/46
85
81
76
88
1054
81
99
81
76
1055/56
82
89
84
86
77
85
81
85
85
Pairwise of BLAST 2 sequence program was performed to determine the identity
between two different DNA sequences (Tatiana A, 1999). Each value was rounded to
the nearest integer, and values of >85% are presented in boldface. No analysis was
possible for CPnOO1 1/0012, CPnO124/0125, and CPnO126 because there was no
significantly specified sequence due to alignment of paralogs less than 20%.
a
CPnOO1 24-125
CPn0007
CPnO126
p10- 10.1
ci:
CPn1O55- 1056
0.1 substitutions/site
Figure 15. Phylogenetic tree analysis of DNA sequences of the CPn 1054
gene family obtained from C. pneunzoniae CWLO29. Multiple sequence
alignments were performed and analyzed using CLUSTALW and PAUP
program. Distance matrices were inferred by neighborjoining to estimate evolutionary distances. Bootstrap values (based on 1000 replicates)
are represented at each node.
85
20-99 % (Table 6). The existence of the paralogous genes and a gene family was
further strengthened using phylogenetic analysis performed by PAUP (Figure 15).
This also suggested that this gene family has extensive genetic relationship distinct
from the other putative Inc proteins. A classical signal peptide and conserved
Shine-Dalgarno sequence were not identified in the predicted products of this gene
family. In CPn0008, 0010, 0041, 0045 and 1055 contained the unusual putative
start codon GTG at the different site in different strains (Figure 16). Genomics
analyses revealed that the DNA sequence of CPn0008, 0010, 0011, 0041, 0043,
0045, 0124 and 1055 were similar to the 5' end of the CP1054 DNA sequence;
while CP0009, 0010.1, 0012, 0042, 0044, 0046, 0125 and 1056 were similar to the
3' end of the CPn1O54 (Figure 17).
The conserved hydrophobic domain of the CPn1O54 gene family
In a previous study, we demonstrated that a bi-lobed (5 0-60 amino acid)
hydrophobic domain is a predictive marker for localization into the inclusion
membrane. Consequently, 68 C. pneumoniae proteins, including several members
of the CPn1O54 gene family, have been reported as putative inclusion membrane-
associated proteins (Inc proteins; Figure 18). In this study, high amino acid
sequence similarity was identified in the bi-lobed hydrophobic domains of the
CPn1O54 gene family. Such high similarity of the bi-lobed hydrophobic domain in
the secondary structure in this gene family was unique which was not present in
any known Inc proteins (Figure 19). Additionally, the phylogenetic tree analyses
L'74
seJ
Figurel6. The upstream and 5' end sequence of the CPn1O54 gene family. Multiple
sequence alignments of the upstream region of the CPn1O54 gene family obtained
from the CWLO29 genome. Stop codon of CPn00009, 0040, 0042, 0044, 1054 (7)
were indicated. The unusual start codon GTG () was identified in CPnOO45,
0043,1055. Comparative genomics revealed that the start site of CPn0008,
0010,1054 (*) varied in different strains. Homopolymeric cytosine (poly C) tracts
encoded in either the predicted upstream or at the 5' end of coding region of the
CPn1O54 gene family.
V
V
80
0
CPnOO44-45(712-1017)
CPnOO42-43(8l84-8509)
CPn1O54-lO558326-9642)
CPn0009-IO(3073-3382)
CPnOO1O53-10546792-7O92)
CPnOO4O-41(5644-5845)
CPn0007-8(532-838)
ICA
C
AAIC
rAC* CCC
ACTAAC
AC A A
C CC
I CA
A TA C I
C
A TA C C
A
-
ACCAAC
C
TTAA
1 C
ITA
C T A I 0 _C CCAAC
ICGI
10
T
C
40
30
60
50
70
C
lIT
C AJC C C A CC A CCC AC III C C A CC A I I T
IC C A TAG A A C I T C C I GAG AT I C C T GAG CCCI
CAGCCCAACACCGACIICAAACCCITC AACCTCAACACSTTTAAAAGATI ---------
TACAA CAT ---------------
TTACTTAAAAACTGAJATCGTTTTC1
ACAATCTCIATACAAACCTCATTTTCI(ATITAACAGATTIITI
-------------- CT
TTccccAIATCTCCTTrnCCcICATIIAo*ACcTCC CTCAICCACAIX1A ------------ C
V
100
CPIOO44-45(712-1Ol7)
CPnOO42-43(8184-8509)
CPn1OS4-1055(9326-9642)
CPnOOO9-IO3O73-3382)
CPnOO1O53-1054(6792-7092)
CPnOO4O-41(5844-5945)
CPnOOO7-53238
CPnOOO9-13O73-3382)
C
_
A C A C TLC C C T I C I I I A T T I A C T C C C C A C C
C C
180
200
190
-
-
-
-
-
-
ACAACCAAAAICAA
-
230
220
240
-
I C I C I T I C A A I A A A A I A C T A I A I A I I A C I A C C I I A C T C C C I I I A A C I I A IC I C T I
_
260
AAAA
CATCACT
A C A CC TA CC C C C CCC CCC CJ C A C C A C I
CA C C AC I
IC A CC AC A ICC COC CA C A C A I IC A TA A IC - A A C TA CICIT C TA T CA C C ---- A C A CC TA C C C CCC C C CCC CL
AACACT
TO A IC C[C A 10CC A C C * CA CA I IC A IA A IC A A C IA chiC C IA T C ICC ----- C CA CC TA C C C C C C C C C C C C CC
TGATCCICCAIGCCACCACAGAI1CATAAIG AACTACITIICIATCTCC
IGATCC[cCAICCCACCACAGATTCATAAIC AACIAJC.IAICACC
ICAC ATICAICf1CAJT1CACA"ATICAT*ATC AACTJT1i1TAICTCC 330
540
350
360
-
-
GCAGCTACCCCCCCCCCCCC - -AACACT
-AACACT
ACAGCTACCCCCCOCCCC
TCAACIACCCCCCCCCCCCCC - - GACCCT
370
90
380
400
IA C I IC I TIC C A A C C TA A CC I ICC T{JT T I TA A C C A I IA C I I I I I IA C I IC T ICC C IC C I I I IA C TO A I I
C I I C I I T I I C I C I C I C I A A A I I I C C I C T I I I A C C A A I C A C I I I I I I A (1 1 1 C I I C C I C I C C 101 1 A C I C A I I
C IC IA 00CC C 1 1 1 1 1 1 1 IC IC I C IC TA A A I T IC C TO I T I IA CC A A I C A C T I I T I IA C I IC T I C C TO ICC I I I T!I CA I I
CTCIACCCTCCATIICTTCTCCATCIAAACTICGCGIIIIAGCCAITACTI1TTTACTTTTTCCTAICCT(IIACICAII
AICCICIIACICAII
C I C I ACCCICCAITICIICTCCAICTAAA C I I C C C C I I I I A C C C A 1 1 A C I I I T T I A C I I I T I C C
C C G IA GCCCC
C 1 0 1 A C C C C
CICIACCGTCCATTTCITCTCGATAIAAACTICCCCIITIACCCATIACITTTTTACTICTTCCTCTCCTTTTACTCATT
CICIACCGCTIICITCII ------410
Figure 16.
210
TTCCACCCTTTTCAAATGAGAAAAAGACCITCCAATAAA*TARI1ATAIflACIACCITACT1flflTIIAAC1IATGI1iI
*
______________
280
290
300
310
320
250
CPnl054-1O55(9326-9642
CPn0009-lO(3073-3382)
CPnOO1O5I-1054(8782-7092)
CPnQO4O-41(5644-5945)
CPn0007-8(532-838)
CAIAAAACICCACC
ACCAIACC
-
ICCICAICGATCCCAWCACACAITCAIAATG
IC A 1 1 CIc C A TJC C A C C A C A C A I IC A IA A IC A AC IA CIC{fI A IC A CC
CPn0O42-48184-85OQ)
160
-
CPnOO44-45(712-1017)
CPnOO42-43(8184-8509)
CPn1054-1O55(9326-9642
CPn9009-lO(3073-3382)
CPnU0lO53-lO548782-7O92)
CP0004O-41(5844-5945)
CPn0007-8(532-838)
CPnOO44-45(712-1017)
150
TA IC 301 ILG 1011CC I I TA A A A A I I IWT I TO A A IA A A A IJC TA TjJT A I TAG I A CC T TA CT ICC I IJA AC TI A T C
I IC I I T GA A TA A A A IA C TA IA TA I TA C IC C TA CI TA CI I IA A C I TA IC IC T I
I IC 1 1 10* A I A A A A TA C TA IA TOT TA C IA C C I TA C 10CC 1 I IA AC I I A IC I C I I
IICIIICAAIAAAAIACTAIAIAIT ACTACCIIACICCCIIIAAIIIAIC ICII
- I C I C I I I C A A I A A A A I A C T A I A T A I I A C T A C C I I A C I C C C 1 1 1 A A I T T A T C I C I I
C I I C I T T A C A ACCAACAICAA
IC IC IA CCC C C IC T I I I I I TA A A
I T C IC
- 101 C I I TA A A A A
A C C I C
I C I I I C I I I AAAA-
I I
I I C I I C 1 1 1
CPn0007-8(532-838)
V
140
ACCIICACAAACCAACTIITCII AACAICITITICCC11111AIAACA AAA
C
IC A AflA C C T I I IC 1 CAT TI IC A A A GA A A A TIC T I T ICC C A GA C A ICC A A IC A I TI T Cc TIC TA A A A TAG A A 1 1mG IC A A
C I A A A A CA r C A A A GA - I T IC T T C AC A A A C C A CCC I C IC AC CmTT C TA A C A A A T A --- C A A AT T C C T C C A I C C
CPnOO1O53-1051(6792-7092)
CPoOO4O-41(564#5945)
CP60007.8(532-838)
CPnOO44-45712-1O17)
CPnOO42-43(8184-8509)
CPn1O54-1O55(9328-9842
CPn0009-1O(3073-3382)
CPnOOIO53-1054(6792-7092)
CPnOO4O-4l5644-5945)
130
TAAAGCIGCCIICITTATITACTCGAGAACATCGITCCTACCACAACTCTACAAA
AACCACICIACGAC
ATAAACCICCG1ICT1TATTIACCGAGAACATCCTTCT
ATAAACCICCGTTCITIATTTACTCCC AGCACCATACCTACCATAACTCCAACAG
A A A GA C A A AOC A C C I C I I C
-AAAAAIACTCACCAAIAIACTC
170
CPnOO44-45(712-1017)
CPnOD42-43(8184-8509)
CPn1OS4-1055(9326-9842)
120
110
--AC.AATAGAAACTAACTCTTA
C(ACAA
AAACCCAGTITGC
CCAGAGGAGAAACCCAGITIGC
C T C
80
IICCTCGGCAAIACTIT(AACAATIICAATCGATCCACA11CC
--------------C C CC A C; C A G C CA C
CC A CC A T T 10* A T C C A TA C A A C I IC C T GA CA I IC C 1 GA C CCCI
420
440
430
CA CC A C C T C IC I I IC IA A C C I TJ GAG I T CC A
I C
A C C A C C I C T C I I I C I A A C C T I A C C C A I TrnC A
I C
ACCACCICICITICTCACCIIACCC AI1CCA-
IC A CC A CC T C IC II IC IC A C C I TOC CC A I 1WC A
-
450
470
460
- GO A C I ICC ICC A CCCC IC IC I I T ICC A I TAG CCI
- SCA TIC A C ICC A CC A A - .1 C I I I TOGA I IA
C I I I I C C A I T A C CA]
-CCAITCACICCACCAA
-- CC A C I C T C IC C A C I I C C I 1 IOC CT
-
-
TCACCACCTCICIITCTCACCIIACCCATTCCA --CCAITCACICCAC-A
I C A C C A C C I C T C I I I I T C A C C I TflC C C A I A C C A --- C C A C I C A C I C C
CC
I
TJ1CGA9lTCG(;CACICACTCCiACCCATCICTIIICGACCCCCITI0
CIT TTGGTTAGCCA
C
CC
TI
0
CIA
-CACICC
480
1209469
1207034
CPn1O54 (2436 bp)
13122
1i815
11666
10975
CPn0009 (1308 bp)
CPn0008 (714 bp)
15
1432U 1437w
3435
CPn0O1 0.1 (1371 bp)
CPnOO1O (894 bp)
16617
15692
19215
16644
CPnOO12 (1572 bp)
CPnOO11(726 bp)
5735
55996
56165
57.03
CPnOO42 (783 bp)
CPnOO41 (1350 bp)
60375 50419
5Uf
CPnOO43 (1929 bp)
62793 62790
51059
CPnOO45(1 725 bp)
1209694
1210524 121052/
CPn1O55 (831 bp)
325S
CPnOO46
1211231
Cpn1056(705 bp)
= 99
LIII
= 40-70%
11111
deletion
=20-30%
Figure 17. A schematic representation of the structure features and comparison of
paralogous genes of the CPn 1054 gene family in C. pneumoniae CWLO29. The 5
end of CPn1O54 was similar to CPn0008, 0010, 0011, 0043,0045, and 1055 while
the3' end was similar to the CPn0009, 0010.1, 0012, 0042, 0044, 0046, and 1056.
Range of similarity is 20-99 % Positions of the DNA sequence in the contig
included in the alignment were indicated with numbers on the top of each allele.
deletion
89
Figure 18. Hydropathy plot analysis of the members of the CPn 1054 gene family.
The analysis was performed using the Kyte-Doolittle method with a sliding
window of 7. These proteins are classified as putative inclusion membrane proteins
containing the bibbed hydrophobic domain (boxed), a predictive marker for
localization to the inclusion membrane (mc). Note that scale on the horizontal axis
is different for each protein.
, -rn.
..
I
4 *Ia 4 l
4 *4
[.1rn.i.it.j
IUk*
____
iI
....
-
- I
t.4
nmmm
==
.
:
-I
*1. .
4
'''
I II?II!1
III II: Itlilli! !!
4
* 1 4
..
-.
w **wvw4,_
IW I'4 *'W
S4
.......i
r''I
111111
a
Figure 18.
i
I
WI
40
/0
CPnO126
- LLUFSYYCM !FFFSGA I SSCGLLUSL
CPnO124 ------------
TLLLGILLIAS I IFLAVAIPGLSSAL)ALGL
CPn0007 --------- UIIi.IALSI ILIAG VVLLTUAIPGLSSVISSPA
CPn0008 ------------ -- ----- LSITLLUL ULLLTLOIPGLTAGISFGA
CPnOO41 ---------- JLATFLULGLLLIS ALFLTLGIPGLTAGVSFGL
ILl TFLVLGULLLIS ALFLTLGVPGLAAGLSFGL
CPnOO45
)LA TFLVFt1LLLISALFLTLGIPGLSAAISFGL
CPnOO1O
CPn1O54 ---------- ULA .TFLUFGMLLLISGALFLTLGIPGLSAAISFGL
CPnOO43 -----------VLA TFLVLGVLLLISGALFLTLGIS6VSLGV
CPn1O55
----------
ULFI
;LF$LGISGLSA.
ô0
70
!
C
I
t1TATVL
----------
ACACVt1ttDTVt
--
FSA
GVLUISGLLFL
LSA
GULI.JVSGLLCLLU--
I
LSA
GVLUVSGLLFFLI--
I
LSA
GULtIISGLLCLLV--
I
LSA
GVLMISOLLCLLU--
LSA
SVLVISGFLLL
L
I
GUI1
LCL
Figure 19. The conserved hydrophobic domain in the CPn 1054 gene family. Multiple sequence alignments of
the hydrophobic domains of the CPn 1054 gene family using CLUSTALW analysis. This analysis revealed the
remarkably high amino acid sequence similarity of the hydrophobic domain, which was found only in this
putative Inc protein family.
--
92
HCPnO284
HCPnO829
HCPnO285
HCPnO83O
100
77
HCPn0126
HCPnO1 86
84
HCPn1O55
I-ICPn0007
56
/
HCPnO1 24,,,,,,
/
I
I
HCPnOO43
HCPnO1O
HCPn04
HCPn0008
HCPnOO45
HCPnOO1 1
0.1 changes
Figure 20. Phylogenetic tree analysis of amino acid sequence of hydrophobic
domains of the CPn 1054 gene family obtained from C. pneunioniae CWLO29.
Multiple sequence alignments were performed and analyzed using CLUSTALW
and PAUP program. Distance matrices were inferred by neighborjoining to
estimate evolutionary distances. Bootstrap values (based on 1000 replicates) are
represented at each node.
also demonstrated the homology of the hydrophobic domain of the CPn1O54 gene
family (Figure 20).
Homopolymeric cytosine (poly C) tract and the variations of the length of poiy
C tracts in the CPn1O54 gene family
Genomic analysis of the CPn1O54 gene family revealed a short DNA repeat
of 6-15 homopolymeric cytosine (poly C) residues, positioned either upstream or at
the predicted S'end of CPn0008, 0010, 0041, 0043, 0045, 1054, and 1055 (Figure
16). Comparative genomic analysis of CWLO29, AR3 9 and J 138 also revealed
variation of the length of poly C tracts between strains in CPn0008, 0010, 1054 and
1055, but not in CPnOO41 (CCCCCCTCCCC), 0043(CCCCCCCCCCCC), and
0045(CCCCCC) (Figure 21 A). We further investigated the variation between
strains of the length of poly C tract for these genes from the 12 different C.
pneumoniae isolates. The variation of the length of poly C tracts between strains
was identified in CPnOO1O, CPnOO43, 1054, and 1055. No variation between strains
of the length of poly C tracts of CPnOO4 1 and CPnOO45 was identified in any
isolate.
The variation within a strain of the length of the poly C tracts of CPnOO43,
CPn1O54, and CPn1O55 was determined from 8-10 recombinant clones generated
by either Taq or Pwo amplified PCR products. Within C. pneumoniae AR 39, the
length of poly C tract of these genes in each recombinant clone was shown (Figure
21 B). Additionally, the variation within a strain and between strains of the length
of poly C tract of CPn1O55 was identified in C. pneumoniae AR 39, AR 458 and
PS 32 isolates (Figure 21 C). Whether or not the amplification process using either
Taq
or Pwo polymerase had an effect on the variation of the length of poiy C tracts
was determined. The intrastrain variation of the length of the poly C tract of
CPn1O55 in C. pneumoniae PS 32 was identified using either Taq or Pwo
polymerase in amplification reaction. (Figure 21 D). In contrast, no variation of the
length of poly C tract of CPnOO43, 1054, and 1055 was identified in PCR products
amplified from plasmid DNA of recombinant clones derived from either TA or
Zeroblunt vector.
The tandem repeat motif in CPn0007
Hydropathy plot and multiple sequence alignment analysis revealed that
CPn0007 contained three identical copies of a hydrophobic domain (Figure 18)
which corresponded to "tandem repeat motif' while the others genes of the interest
have only a single hydrophobic domain. Comparative genomics of the complete
genome sequence of CWLO29, AR39, and Ji 83 indicated that the three tandem
repeat motif in CPn0007 was not identical in all three strains (Shirai
et al.,
2000).
The CPn0007 of J138 was a truncated variant with the deletion of 110 nucleotides
that led to lack of the second domain in the tandem repeats.
Figure 21. Variation of the length of the poiy C tract in the CPn1O54 gene family.
(A) Interstrain genetic variation of the length of poly C tract in the CPn1O54
family identified using comparative genome analysis of the complete C.
pneumoniae CWLO29, AR 39 and J 138 obtained from the genomes
(B) Intrastrain variation of the length of poly C tract of the CPnOO43,
1054,1055 in C. pneumoniae AR 39.
(C) Intrastrain variation of the length of poiy C tracts of CPn 1055 identified in
C. pneumoniae AR 39, AR458, and PS32
(D) Comparison of two different DNA polymerase enzymes. Taq and Pwo,
were used for polymerase chain reactions. CPn1O55 of C. pneumoniae
PS32 was amplified by Taq and Pwo and directly cloned into pCR2. 1, TA
kit and Zeroblunt respectively.
20
10
ltttcrstrain variation of the Iottgth of the poly C
tract to the CP 1054 gene fatttily
A_j
B_j
lntrastratn variatton of the
length of thc poly C tract to AR 39
15
:..IFI
[
nFR
0
::
CWLO29
El AR39
iin I
AR39CPoOO43
5
.
:
J138
:
cJ
7.5
2:
JJ1
:
o
n
The length of the polyC tract
lntrastrrin vartattort of the
length of the poiy C tract of CPn 1055
DJ
4-
-
El
v
AR39CPttIOSS
3
C. Puoto,tjo PS32 CPtt 1055
-
-
-
El AR458 CPnIOSS
0
PS32CPnIOS5
2-
2:
HH
The length of the poly C tract
Figure 21.
The length ot the poly C tract
El
Paro
El
T
97
In this study the size variation of CPn0007 of 12 different C. pneumoniae
isolates generated using polymerase chain reaction was further determined. It
revealed that the PCR product corresponding to CPn0007 was identified in all C.
pneumoniae isolates tested. There was no evidence of deletion or insertion of a
repeat motif of CPn0007 in any isolates (data not shown).
Frameshift and Gene conversion in the CPn1O54 gene family
Comparative genomics analysis of the three genomes showed that the
sequence variation in CPnOO1O, CPnOO43, and CPn1O54 was caused by deletion,
nucleotide substitution and gene conversion. Frameshift mutations were found
which lead to a truncated sequence of CPnOO 10-0010.1 and CPnOO43 -0044 in
CWLO29 but not in AR 39 and J138. Frameshift mutations and nucleotide
substitution were also identified that led to a truncated sequence of CPn1 054 in
AR39 and J138 (Figure 22). In CWLO29, CPnOO1O-0010.1 and CPn1O54 shared
99% sequence similarity with a frameshift mutation and small difference identified
at the 3' end of each gene, while in AR39 and J138, CPnOO1O were identical to
CPn1O54 with one gap and gene conversion (Figure 23).
In this study, the frameshift mutation and gene conversion of CPnOO1O
were further investigated from 12 different C. pneumoniae isolates. The frameshift
mutation leading to the truncated sequence of CPnOO 10 was identified in CWLO29
and 4 other isolates, AR388, AR231, KA5C and KA66. Evidence for gene
conversion, in which CPnOO1O was converted to CPn1O54, was identified in 2 of 12
isolates, AR 39 and AC43 (Figure 24). The nucleotide sequences of the frameshift
sequence and the gene conversion of CPnOO 10 at the 3' end of the gene identified
from 12 selected samples were deposited in GenBank under following accession
numbers: AF4740 17-474026, and AF46 1543-461552 respectively.
99
CPnOOIO(894bp)
CWLO29I
15749
14328 14379
13435
H
CPnOO1O.1(1371 bp)
825448
827769
AR39
1
CPnOO1O (0764) (2325bp)
J
15428
13104
J138
CPnOOIO(2325bp)
I
1209469
1207034
CWLO29
CPn1O54 (2436 bp)
AR39
CPn1O54 (0797) (1566 bp)
CP0796 (723 bp)
I
f
1203437
J138
861969
862709 862691
864274
1203730
ICP1054I
(294 bp)
61069
CWLO29f
62794)
CPnOO45 (1725 bp)
Pn0046 (477 I)
777928
780132
AR39
63266
CPnOO45(728)(2208bp)
f
62947
J138
CPnOO4S(2208 bp)
I
Figure 22. Schematic representation of the open reading frame ofCPnOOlO0010.1, 1054, 0045 identified inC. pneumoniae CWLO29, AR39 and J138.
Comparative genomics anlysis demonstrated genefusion or gene fission found
in the CPnOO1O I in CWL-029 strain, CPn1O54 in AR 39 and CPNOO45 in
AR39 and J138.Truncated genes caused by frameshift mutation were
identified as shown.
100
Figure23. Frameshift mutation and gene conversion in CPnOO1O.
A) Comparative genomics revealed the frameshift mutation (*), which lead
to the truncated CPnOO1O (894 bp) in CWLO29 but not in AR39 and
J138 (2322 bp). Stop codons (V) in truncated CPnOO1O (CWLO29)
resulting from the frameshift were shown.
B) Gene conversion in CPnOO1O identified in AR39 and J138. Multiple
sequence alignment of the 3' end of CPnOO1O-0010.1(CWLO29),
CPnOO1O (AR 39, J138) and CPn1O54 (CWLO29) were constructed
using CLUSTALW analysis. In CWLO29, CPnOO1O-0010.1 is similar to
CPn1O54 but not identical. The difference between CPOO1O and CP1054
is found at the 3' end of the gene. In contrast, in J138 and AR39,
CPnOO1O is converted to be identical to CPn1O54 explained by gene
conversion.
101
A
CA
CPIO(CWLO29)
CPIO(JT38)
CPIO(AR39)
CP01054(CWLO29)
AT&T C TAA Aid UACGCAGAGT TA TA GACATAAATTTTTGTAGA 6
I G TAT C G GAG! CT GA CC CA GAG! TA TA GA GA TA A AT 11116 GA GAG uGh G
Ta! A IC C GAG! CT GA C C C A CA Gil A TA GA GA IA A AT TIll G GA GAG CGA
TGTATCGGAGTCTGACGCAGAGTIATAGAGAIAAATTTTTGGAGAGTI......G
9)1
931
9111
cAl
6CC C GA A GA I C CC AT A GA AT C AG C A TA TA A CCI C G TI A A GA GA I GA IC
AG G C GA A GA ICC CAT G C AA I G G C CAT AT AA CCI G Ti! A A GA GA! GAIT
CC GA A GA T C C CAT G C AA ICC G C A TAT AA CCITT Ti A A GA CAT GA!
G C GA A GA ICC CA IC GAA I GG GCAT AT AA CC! G G I IAA GA GA I GA!
CPIO(CWLO29)
CPIO(J 138)
CPIO(AR39)
GPO 1054(CWLO29)
*
911
A C TI A UT
CPIO(CWLO29)
GA A A A AT A A AT
CPIO(JT38)
CPIO(AR39)
GA A GA G I G CT I G T OCT GA A A A GA A C C Ti C C G GA I G C C CA G GA A C CI I AG
CP01054(CWLO29)
AT ACT GA
A A A A A A A CT
C A A A AT A C C A A
A A
GAA GA GTGG CT 61CC! GAAAAGAA GCT CGGGAT GC CGA GGAA CGTT GG
GAAGAGTCGGIGIGCIGAAAAGAAGCTTCGGGATGCCGAGGAACGITGG
V
111(1
CPI O(CWLO29)
931
(1)1
IlIAc
11801
CPIO(i738)
A GA AA TI! AG GA hA G CAT! Ciii! G G TI AG
CPIO(AR3G)
A A A A A I IT A G C A A A G C A C T C IT TI C C G I A G A A G A A G A C C C C G C C ill G A
CPOTO54(CWLO29)
AGAAATTTAGGAAAGCAGTCITTIGGGTAGAAGAAGACGGGCGC!IIGA
Ii!
2300
CP1054(CWLO29)
CPIO(AR39)
CPOIO-01O1(CWL029
2980)
CPIO(J138)
CPIO(AR39)
2395
A A AG A GA A A C Gill G CT I GOT ACT A A GA I AG TA C CA A CCC AGCA GC 0 AG
A A A GAG A A A C CT IT CC TI COT ACT A A C AT A G I AG CA A CC CA C CA CCC AG
A A A GAG AA A CCIII C OTT GG TACT A AG AT AG TAG C A A CCC AC CA CC GA
CP7O(J138)
CP1054(CWLO29)
234
232
IAAAGACAAACCTTTGGTTCGTACTAACATAGTACCAACCCAGCA CCC AC
237
238
239
24
TT0CAGCATTTGAATCCAAGAAGT1CCfGAGATTCCTAGGCCC CAG
TTCCAGCATTI ChAT CCATAGAAGTTCCTGACATT CCI GACGCCC CAG A
I CC A CC A ITT GA AT C CAT A GA A CIT C CT GAG AT I CCI GAG C C C C CA C A
CPO1O-01O. 1(CWLO29TI7IA CflA I I TflA AflC C A
Ir
2415
CP1O54(CVtO29)
CPIO(J138)
CPIO(AR39)
242
245
244
245
ACAAACCGAGTTTCCTGATAAACCGCGTICTTTATTACTCGCCAGbI
IC
A GA A A CCC ACT IT CCI CC AT A A A CCC CCI T CIT I AT I I ACT C C C GAG CI
A GA A A C C GAG I I I C CT COAT A A A CCC C G I I CIII Al I TACT CCC GAG Gd
CPOlO-OlO 1(C WLO29
A GA A Ar1C A GfqTJr!T CC AflA A A CCC C GflTflT I I AlT I AflT C GflGA C Cd
CP1054(CWLO29)
CATACCTAC
CPIO(i138)
CAT CCTAC
CATTCCIAG
2480
CPIO(AR39)
CPO1O-OlO.l(CIAtO293 C AT IC CT AG
Figure 23.
102
Figure 24. Gene conversion of the CPnOO1O in C. pneumoniae isolates.
The 3' end of the CPnOO 10 identified from 12 C. pneumoniae isolates were
aligned and compared with that of CWLO29. Two of 12 C.pneumoniae
isolates, AR 39 and AC 43, CPnOO1O are converted to be identical to CPn1O54
3CPIO(J138)
3CPI 0AR39
CPI 0-2023
CPI 0-KA66
CPIO-AR231
CPI 0-KA5C
CPI 0-PS32
CPI 0-AR277
CPI 0-AC43
CP1 0-AR388
CPI 0-AR458
CPI 0-TW183
CPI 0-AR39
30
20
10
3CPI 054(CWL)
-40
50
60
TAGCAACGCAGCAGCGAGTTGC* CA TTG ATCCATAGAAGTTCCTGAGATT(CTGAGG CCC
TAGCAACGCAGCAGCGAGTTGCAG'ATTTGAATCCATAGAAGTTCCTGAGATTCCTGAGGCCCC
T A G C A A C G C A G C A Ge A C, T T G C A G U A T I I G A A I C C A T A G A A G T T C C I G A (1 A I T C C T 0 A 0 13 C C C C
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT
CTGATATCGTTGAGTCTTC
CTGATATCGTTGAGTCTTC
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT
CTGATATCOTTGAGTCTTC
TAGCAACOCAGCAGCGGATCCAAGAATTTCAA - CCAT
CTGATATCGTIGAGTCTTC
CTGATATCGTTGAGTCTTC
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT
TAGCAACGCAGCAGCGGATCCAAGAATTICAA - CCAT - CTGATATCGTTGAGTCTTC
I A 0 C A A C G C A 0 C A GC GJTIf 13_C}A GJA I I TflA A T C C A T A 0 A A 01 1 C C I 0 AA TT_C_CIT G A GCE7JC
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT
CTGATATCGTTGAGTCTTC
CTGATATCGTTGAGTCTTC
TAGCAACGCAGCAGCGGATCCAAGAATTTCAA-CCAT - CTGATATCGTTGAGTCTTC
T A 0 C A A C GC A GC A G C GIlT_13_;A 0A T I TA A I C C A T A 0 A A G I T C C T 0 AA Fir_C_CIT G A GCC
CPI 0-CWL
TAGCAACGCAGCAGCGGATCCAAGAATTTCAAflCCATI -------- ICTGATATCGTTGAGTCTTC
3'CP7054(CWL)
GAGGAGAAACCGAGTTTGCTGGATAAAGCGCGT TCITTAITTAcTCGCGAGGACCATJCCTAG
70
3CPIO(i138)
3CPIOAR39
CPI 0-2023
CPI 0-KA66
CPI 0-AR23 1
CPI 0-KA5C
CPJ 0-PS32
CPI 0-AR277
CPIO-AC43
CPI 0-AR388
CPI 0-AR458
CPI 0-TWI 83
CPIO-AR39
CP1 0-CWL
13 A 13
0 AGAAA
80
C C GA 0 1 T T
90
13
C T 0 0 A
1
100
AAAG C 0 C 0 T I C T T T
110
A
120
T I T A C I C 0 C 0 AGGA C C A T T C C T
130
A0
GAGGAGAAA('UGAGT T TGC TOGAT AAAGCGCGT TCTTTATTT AC TCGCGAGGACCAT TCCTAO
AATGAGAAAGTGAGCCTT ATGGACAAAGCGCGATTT TTATIT AATCGTGAGGACCAT TCCTAG
AATGAGAAAGTGAGCCTTATGGACAAAGCGCGATTTTTATTT AATCGTGAGGACCATTCCTAG
AATGAGAAAGTGAGCCTTATGGACAAAGCGCGATTTTTATTT AATCGTGAGGACCATTCCTAO
AATGAGAAAGTGAGCCTT ATGGACAAAGCGCGATTTTTATTT AATCGTGAGGACCATTCCTAG
AATGAGAAAGTGAGCCTT ATGGACAAAGCGCGAT TTTTATTT AATCOIOAGGACCATTCCTAG
AATGAGAAAGTGAGCCTT ATGGACAAAGCGCGATTTTT ATTT AATCGTGAGGACCATICCTAG
JAjG A 0 A A AEJG A GTErT G S AA A A GC GC GflTET I I A I T I AJT C Sf0 A S G A C C A T T C C I A 0
AATGAGAAAGIGAGCCTT ATGGACAAAGCOCGATTTTT AlIT AATCGTGAGGACCATTCCTAG
AATOAGAAAGTGAGCCTTATGOACAAAGCGCGAT TTTTATTT AATCGTGAGSACC ATTCCTAG
AATGAGAAAGTGAGCCTTATGSACAAAGCGCGATTTTTATTT AATCSTGAGGACCATTCCTAG
EJAfS A G A A AEJG A GftT3JT 5 0 AfA A A SC SC GfTfT T I A I T T AfT C OfG A G G A C C A T T C C T A G
AATSAGAAAGTSAGCCTT ATGGACAAAGCOCGAT TTTTATTT AATCGTGAGGACCATTCCT AG
Figure 24.
U)
104
Discussion and Summary
A major area of interest in the chlamydial biology is to understand the
interaction between the chiamydial inclusion and the host intracellular
environment. The role of chlamydial proteins in the inclusion membrane in the
development cycle and pathogenesis remain to be elucidated. However, the lack of
genetic manipulation techniques in the chlamydiae is the primary limiting factor to
blocking characterization of unknown functional proteins, including inclusion
membrane proteins. A new approach is to analyze the microbial genome and
determine the function of proteins predicted from the sequence. This includes
similarity searching, structural motif analysis, and comparative genomics.
Attributes of function and structure are commonly inherited in a protein family.
The structural motif of putative chlamydiae Inc proteins
A protein family is commonly defined based on high sequence similarity.
However, a protein family can also be classified using a common structural motif
as a signature. The motif, small sequence units of protein families, is identified as a
highly similar region in alignments of protein sequence. Motifs are widely used to
predict functional regions of proteins (Henikoff et al., 1997). Motifs may reflect
either common ancestry or convergence from independent origins. Identification of
a signature motif can be important for the exploration of structural and functional
inferences within unknown genes or gene products.
105
The putative Inc family is mainly classified by a unique structural motif
rather than sequence similarity. Although they share less sequence similarity
between each other, they retain a common structural motif. This study
demonstrated that the structural motif, a bi-lobed hydrophobic domain in the
secondary structure of Inc proteins, is a motif for targeting protein to the inclusion.
The mechanisms exploited by chlamydiae to target the Inc proteins to the inclusion
are unknown. It appears that the bi-lobed hydrophobic motif is not the only
structural domain required for protein localization to the inclusion membrane.
Recently, some chiamydial proteins lacking the signature hydrophobicity domain
have also been identified on the inclusion membrane. These include Cap 1, a
protective antigen recognized by cytotoxic T cells (Fling et al., 2001) and CopN, a
predicted target of the type III secretion apparatus (Fields et al., 2000). It is
suggested that there are other, uncharacterized, structural motifs required for
protein localization to inclusion membrane.
Genomic analysis is a valuable approach for chlamydial researchers. Two
C. trachomatis and three C. pneumoniae genomes have been completely
sequenced, yielding many otherwise uninvestigated avenues of research. Gene
families and novel functional genes were also identified, including the polymorphic
membrane proteins family (Pmp) (Grimwood et al., 1999) and a chlamydiae
protein that interacts with an apoptotic death receptor (Stenner-Liewen et al.,
2002). The Pmp family was identified using the structural domain containing the
common signature tetrapetpide repeat, GGAI/L/V followed by the FXXN motif.
106
Recently, the predicted function of the Pmp family has also been determined using
a structural motif by Henderson and Lam (2001). They showed that the Pmp family
resembles other gram-negative bacterial proteins secreted via an autotransporter
system. The Pmp proteins harbor the structural motif for autotransporters including
a predicted signal sequence, a passenger domain {GG [A/I/V/I] [I/L/V/Y] and
FXXN}, and a 13-barrel structure at the carboxyl terminal region within the outer
membrane.
Recently, Stenner-Liewen et al (2002) has identified a hypothetical
chlamydial protein that shared significant identity and predicted structural
homology to the death domains (DDs) of members of the mammalian TNF-
receptor family. The chlamydial protein, which has homology to human DDs of
human DR4 and DR5 are referred to as chlamydia protein Associating with
eath
omains (CADD). In vitro experiment showed that CADD is capable of inducing
apoptosis and interacting with Fas domain.
Gene homology and Gene order
Gene arrangement of putative inc(s) is mostly dispersed but some are
clustered. Clusters of inc(s) in C. trachomatis were tightly linked to both the incA
and the incB/incC homologs, but no such gene arrangement was seen in
C. pneumoniae genome. These findings suggest that gene order of inc group is not
generally conserved among the Chiamydia.
107
There are some examples of conserved gene order and gene cluster. The
cluster of incB/incC and genes encoding amino acid-and sodiumdependent
transporter gene are conserved in not only C. trachomatis and C. psittaci
(Bannantine etal., 1998) but also C. pneumoniae. Typically, such a conserved gene
cluster conveys functional coupling between the genes product present in them
(Overbeek et al., 1999). Whether functional significance is related to gene
arrangement on the microbial chromosome remains to be elucidated. However,
these analyses demonstrated that the family of putative Inc protein is randomly
expanded in both C. trachomatis and C. pneumoniae.
Genomic comparison revealed that about 80% of all predicted coding
sequences in C.trachomatis and C. pneumoniae are orthologous. The average of
identity between orthologs is 62% at the amino acid level (Kalman etal., 1999).
Orthologs are defined as related genes in different genomes that have been created
by the splitting of a taxonomic lineage, while paralogs are related genes in the same
genome created by gene duplication events (Fitch, 1970). In putative inc analysis,
20 of 60 ORFs were defined as orthologs. The range of identity is about 20-70%
for the inc orthologs. The presence of both orthologs and paralogs in putative Inc(s)
suggested that some Inc proteins are conserved and required to function in
biological development in both C. trachomatis and C. pneumoniae, while other Inc
groups are species specific determinants that play a role in specific functions.
108
Possible functions of the putative Inc protein
Evidence of a large number of putative Inc proteins suggested that possibly,
the chlamydiae have an extraordinary repertoire of unique proteins in contact with
the cytosol of infected host cells although their functions remain uncharacterized.
They may be important in some aspects of communication with the host cell
cytoplasmic environment. This may include aspects of intracellular vesicular
trafficking, establishment or maintenance of the inclusion, and/or nutrient
acquisition.
The structural motif of a bi-lobed hydrophobic domain in all Inc proteins
leads to the speculation that one lobe of the motif may be embedded in the
chlamydial cell wall whereas the other may be in the inclusion membrane
(Hackstadt et al., 1999). These proteins may then serve to anchor the pathogen to
the inclusion, facilitating direct contact between the RB and the inclusion as well as
providing molecular contact between the RB and the cytosol. Microinjection
studies demonstrated that three C. trachomatis Inc proteins, including IncA, IncD
and IncG, are cytosol- exposed at the carboxyl terminus of the protein (I-Iackstadt et
al., 1999).
Non-fusogenic variant of C. trachomatis
Chiamydia trachomatis typically produces a single large inclusion during
development. Infection of an epithelial cell with the multiplicity of infection greater
than one of C. trachomatis results in multiple inclusions within the cell, which
109
eventually fuse to form a single large inclusion. Previous studies conducted by
Matsumoto eta! (1991) and van Ooij eta! (1998) suggested that the fusion of
C. trachomatis inclusion is a relatively late event in the developmental cycle. These
authors showed that the fusion of the C. trachomatis inclusion did not appear to be
initiated before 10 hpi. The developing inclusion was not uniformly completed
until between 18 and 24 hpi. Additionally, C. trachomatis can form atypical
nonfusogenic inclusions in low temperature culture (van Ooij et al., 1998). The
mechanism of homotypic fusion of C. trachomatis, however, remains to be
characterized
In this detailed study, we showed that the non-fusogenic variants were
apparently associated with the absence of the IncA in the nonfusogenic inclusion
membrane. Previously, Suchland et al. (2000) have identified the variant isolates
with the nonfusogenic inclusion phenotype that was cell line- and temperatureindependent in the clinical studies. They demonstrated that the variants are stable
upon repeated passages, and lack IncA on the inclusion. In our subsequent incA
sequence analyses, we demonstrated diversity in incA within the nonfusogenic
clinical C. trachomatis isolates. A variety of mutations of incA were identified,
including nucleotide substitutions that directly introduce stop codons, single base
frameshift, and large deletions. In most cases (24/26 IncA-negative isolates),
undetectable IncA in the inclusion membrane was correlated to the interruption of
the proper reading frame. In the remaining two isolates, the reason for the absence
of detectable IncA remains unclear.
110
The role of IncA in homotypic fusion of C. trachomatis inclusion was
supported by electron microscopy and microinjection studies of Hackstadt et a!
(1999). They demonstrated that the fusion of C. trachomatis inclusions can be
detected by electron microscopy by about 10 hpi. This is consistent with the
temporal expression of C. trachomatis IncA and detection of IncA on the
developing inclusion. Microinjection of antibody specific to IncA can block the
fusion of inclusions. Through two-hybrid system analysis, they also showed that
the IncA can interact with the homologous IncA via the carboxyl- terminus of
protein. These data suggested the model in which the interactions between C.
trachomatis IncA monomer may modulate inclusion structure, which possibly
facilitate the association of opposing membrane faces to favor membrane fusion.
In this study, sequence analysis lead to the detection of incA and CT223
variant isolates in the clinical samples. Three of 13 (23%) wild type and one of
nonfusogenic variant isolates, D(s)2923, possess IncA variants with the substitution
of isoleucine with threonine at codon 47 and glutamic acid with lysine at codon 116
(IncA 147T, El 16K) while 5 of 13 (3 8%) wild type isolates encoded prototype
IncA presented in the C. trachomatis serovar D genome. These results were similar
to the studies of clinical C. trachomatis isolates in Netherlands reported recently by
Pannekoek etal. (2001). They demonstrated that 9 of 25 (36%) wild type isolates
of clinical C. trachomatis encoded prototype
incA
and 7 of 25 (28%) were IncA
variants (IncA 147T and El 16K). However, there is no association with the
Ill
nonfusogenic inclusion phenotype of C. trachomatis and variant IncA (147T,
El 16K).
Though the non-fusogenic D(s)2923 isolate encodes an intact incA
sequence, expression remain unclear. Sequence analysis of the 300 nucleotide
upstream of incA in the isolates {D(s)2923, J (s)893, and J(s)6462} encoding
apparently intact polypeptides were identical to that of the serovar D genomic
sequence. Analysis of upstream region of incA for selected wild type and variant
isolates showed no variation. There are, therefore, no differences in the putative
regulatory region to lead to the absence of IncA in these isolates that have
apparently intact coding sequence. Therefore, the mechanism used by these
isolates to block the synthesis or accumulation of the IncA remains to be
characterized.
In contrast to incA, there is considerable variation in the serovar D and L2
CT223 sequence. While incA varies at only a few nucleotides, CT223 varies at 34
nucleotides leading to 26 differences in amino acid sequence. We also identified
the variation of CT223 sequence which contains identical two-amino acid
substitution in one wild type (J3464) and two nonfusogenic variant isolates
{J(s)1980; J(s)6686}. In these CT223-negative isolates, serine changes to leucine at
position 22; and glutamine changes to arginine at position 120. The CT223
sequence variant, however, was not directly associated with the absence of this
protein in either immunofluorescence or immunoblot analysis. Sequence analyses
of the upstream region of the CT223 for CT223-negative isolates reveal a single
112
nucleotide substitution of thymine for the cytosine. Combination of the evidence of
variant sequence within coding sequence and the putative regulatory region of
CT223p will lead to future studies that will focus on gene expression at the
transciption level. Serine is a candidate target for phosphorylation site in signal
transduction. Therefore, the effect of changes in serine to leucine at position 22 and
glutamine to arginine at position 120 on gene expression and protein product
remains to be characterized. The absence of CT223 in the inclusion membrane may
be associated with the requirement of the posttranslational modification or another
uncharacterized mechanism.
These findings have also allowed a phenotypic comparison study of strains
that produce IncA or do not produce IncA, which may lead to differences in the
biology of chlamydial infection and disease. Giesler etal. (2001) demonstrated that
the difference patterns of disease are associated with the non-fusogenic phenotypes.
The non-fusogenic phenotype might offer selective advantage to the variant strains
to cause persistent infections.
The CPn1O54 gene family and genetic variation
Comparative genomics analysis is an invaluable approach for the
investigations of novel gene families, species or strain specific pathogenesis, and
genetic diversity in the population. Recently, genomic comparison studies
demonstrated that the overall genome organization of C.
pneumoniae is
highly
conserved among various isolates. From detailed pairwise comparison of both
113
genome and proteome sequences, C. pneumoniae genome AR39, and Ji 83 are
greater than 99.9% identical to CWLO29 (Read et al., 2000; Shirai et al., 2000).
Although genomic organization and gene order of these independent strains are
very similar, there is evidence for genetic variation, which may contribute to strainspecific phenotypes.
Relatively little is known about molecular pathogenesis, genetic diversity
and the adaptive strategy of C. pneumoniae. A key adaptation strategy used in
several microbial pathogens is to change biological characteristics to gain "fitness".
A repertoire of variations is required for pathogens to generate the advantageous
variants or heterogeneous progeny within an isolate. Variability enables a single
bacterial clone to adapt without the acquisition of additional genes. Such
phenotypic variation can be achieved by regulating gene expression or through
genetic change mechanisms. Point mutations, homologous recombination, and the
action of transposon-like elements are key mechanisms by which gene flexibility is
achieved. Those mobile elements enable the DNA rearrangement and mutations,
which contribute to the new variants in the population. Except the bacteriophage
present in C. pneumoniae AR39, there are no functional inserted sequence (IS)
elements, transposons, retrons or prophages in the whole genome (Read et al.
2000). This unusual feature suggests a novel mechanism for genetic variation in C.
pneumoniae.
In genomic analysis, it is remarkably clear that coding regions of the
genome are organized hierarchically into gene families and superfamilies. A group
114
of genes in which all of the members have> 50% pairwise amino acid similarity is
defined as a gene family and an alignable groups of genes with similarity below
this threshold is a superfamily (Gruar and Li, 1999; Thronton and DeSalle, 2000).
In this study, we have identified a new cluster genes designated as the CPn1O54
gene family based on the single quantitative measure of pairwise similarity. The
significant similarity of these genes suggested that they are paralogous genes that
can be classified in a new gene family. Our previous study has identified some gene
products of these genes as putative inclusion membrane proteins (Incs). Seven
genes in the gene family, CPn0007, 0008, 0009, 0010, 0010.1, 0011 and 0012 are
encoded between the polymorphic membrane protein genes, pmpl and pmp2,
which encode cell surface proteins of C. pneumoniae. Members of the CPn1O54
gene family were categorized as paralogs, which apparently arose through gene
duplication, followed by diversification. Their protein products may thus serve
similar but not identical functions. The unusual start codon GTG was identified in
the CPnOO43, CPnOO45 and CPn1O55. The members of this family appeared to be
variably interrupted by frameshifts. These may be mediated by gene fusion or gene
fission, which involves the combination of two genes into one or the spitting of one
gene into two proteins. Such events could occur by deletion or insertion mutation
leading to removal of a start codon. These analyses also suggested that there were
substantial 5' and 3' conservation of these paralogs, with the greatest variation in
the midregion. These genes are found only in C. pneumoniae and have yet to be
ftinctionally characterized. They, therefore, are determined as unknown,
115
hypothetical, and C. pneumoniae specific genes, which may be required for specific
attributes.
Comparative analysis within and among the complete genome sequence has
provided evidence that genetic variation within this gene family may be modulated
through multiple mechanisms. Several paralogous genes, including CPn0008, 0010,
0043, 1054, and 1055 in the CPn1O54 gene family, contain poly C repeats either
upstream or at the predicted 5'end of the coding region. The length of the poly C
stretches in the CPn1 054 gene family varied between paralogous genes in each
genome, and within single genes in the three genomes. The length of the poly C
tract also varied between different isolates and within an isolate, which suggested
the presence of the heterogeneous population in each C. pneumoniae isolate. The
variation of the length of this repeat appeared to be involved in the determination of
the predictive start site of these genes. The variation in the length of the poly C
tract moved upstream-located ATG/GTG codon in or out of frame, which may
affect either transcription or translation. The repertoire of variants in the family and
the presence of variability of the length of the short sequence repeat suggests that
this family may also play a possible role in either functional or antigenic variation,
which can contribute to the genetic diversity or unknown virulence traits of C.
pneumoniae.
Short sequence repeats such as homopolymeric nucleotides, which usually
are identified in contingency or hypermutable loci, are involved in a molecular
mechanism of genetic variation in numerous pathogenic bacteria (Brian et al. 1992;
116
Moxon etal. 1998; Deitsch et al. 1997). The presence of contingency loci provides
a repertoire of variation, and therefore genomic flexibility for the bacterium. In
several pathogens, the deletion or insertion of a homopolymeric repeat can alter the
level of gene expression or the translational frame of gene encoding cell surface
molecules, thereby leading to the new variants in the population. In C. pneumoniae,
variation of the short repeat of homopolymeric nucleotides was first identified in
the Pmp family (Christen etal., 1999; Stephens and Lammel, 2001). Comparative
genomic analysis and cloning expression showed that the length of the poiy G tract
of pmpG 10 varies between strains and within an isolate (Pedersen etal., 2001).
Furthermore, variation of the length of poly G has been demonstrated that it plays a
role in the differential expression of PmpG 10 (Pedersen et al., 2001)
Variation in size via recombination within the tandem repeats was identified
in the Pmp family and CPn0007, a member of the CPn1O54 gene family (Shirai et
al. 2000 a, b). Comparative genomics analysis reveals the shorter sequence of
PmpG 6 in AR 39 and J138. In CWLO29, PmpG 6 contains three tandem repeats of
131 amino acids, whereas that of AR39 and J138 contains only two repeats
(Grimwood et al., 2001; Shirai et al., 2000a, b). Like PmpG6, the size variation of
CPn0007 is caused by the deletion of a tandem repeat. In J138 genome, CPn0007
encodes a truncated sequence with a gene product that lacks 110 amino acid
residues in the middle region (Shirai et al., 2000a) leading to the absence of the
second tandem repeat in this protein. This variation mechanism may play a function
in genetic variation in both families. We have further studied the variation of
117
CPn0007 in 12 clinical isolates. However, there was no evidence for insertion or
deletion of the repeat motif of CPn0007 in any isolates.
In the CPn1O54 gene family, each gene is derived from gene duplication.
About 50% of all gene duplication will lead to functional divergence (Wagner et al.,
1998). Each duplicated gene can elicit a differential expression pattern. They might
also be selected for subfunctionalization (Massingham etal., 2001). On the other
hand, not every gene duplication result in a new function. Some duplicate genes
have lost function resulting from mutation and become pseudogenes (Wagner,
1998). Comparative genomic analysis revealed frameshift mutations and nucleotide
substitution in CPnOO1O, 0045, and 1054, which resulted in prematurely terminated
gene products in the genome. These genes might be unfunctional or pseudogenes
that were transitionally inert in some isolates.
Recently, in the comparative analysis of C.pneumoniae strain CWLO29 and
AR 39 Jordan et
al.
(2001) identified and described an independent conversion
between paralogs Cp0764 (CPn 10540) and Cp0797 (CPnOO1O-0010.1) in AR-39
but not CWL-029. The nucleotide sequence of CP764 and CP797 are 100%
identical except for one deletion. In contrast, there is no gene conversion in
CWLO29 strain. It encodes the two paralogs , CPnOO1O and CPn1O54, which lack
100 % identity, but contains the 28 nucleotide difference at the 3' end of the coding
region. Gene conversion is an intragenomic, nonreciprocal recombination event
that results in identical (homogenized) gene sequence. It is likely that gene
conversion between related genes or paralogs may result in antigenic variation.
118
Comparative genomics reveal the genetic variation within this family that
may be mediated by gene conversion between paralogs (CPnOO1O and CPn1O54)
(Jordan etal., 2001). Apparently, there are two copies of either CPnOO1O or
CPn 1054 in the genome. In CWLO29, CPnOO1O, is very similar but not identical to
CPn1O54. The significant difference between these two paralogs is present at the 3'
end of the gene in CWLO29. In contrast, CPnOO1O ofAR39 and J138 are identical
to CPn1O54 of CWLO29 but different from CPnOO1O of CWLO29 (Figure23). In
AR 39 and J138, CPnOO1O sequence is identical to the CPn1O54 sequence, which
can presumably be explained by gene conversion. In this present study, we have
further investigated the 3' end of CPn1001O sequence in selected C. pneumonia
isolates. Two of 12 isolates (AR39, AC43) were identified in which CPnOO1O was
converted to CPn 1054. Mutations leading to the pseudogene of CPnOO 10 are
identified in some isolates. The result also showed that gene conversion of
CPnOO1O of AR39 was consistent to that found in the AR39 genome data. With the
difference of the 3' end sequence the two copies of CPn 10 or CPn 1054 in
chlamydiae genome may have altered function, lose function, or gain totally new
function.
Gene conversion is a mechanism contributing to genetic and, subsequently,
antigenic variation of a repetitive gene family. The mechanism of gene conversion
is plausibly explained by a nonreciprocal recombination of two separate
chromosomes within a cell or two branches of replication intermediates. This
recombination results in the excision of all or part of the nucleotide sequence of
119
one gene and its replacement by a replica of the nucleotide sequence from the other
gene. In pathogenic Neisseriae, pilin variation is caused by not only reciprocal but
also nonreciprocal recombination. The asymmetric recombination of pilE
(expressed loci) and puS (silent loci) leads to the transfer of the variable sequence
of unexpressed genes to the expression locus (Haas etal., 1986). In the Hop-family
member of H pylon, the conversion mechanism generates the chimeric sequence
consisting of the divergence of 5'region and identical 3' region (Jordan et al.,
2001).
Recently, the coding region of the CPn1O54 gene family has been proposed
to be the hypervariable region in the genome of C. pneumoniae (Daugaard et al.,
2001). The extensive variation of this DNA region between isolates was identified
using restriction fragment length polymorphisms analysis. The frameshift mutation
in CPnOO 10 resulting in gene fusion or gene fission of CWLO29 and AR39
identified in these studies was consistent with those of the genome sequences. The
CPnOO1O sequence of TW183, 2023 and two Finnish isolates (KA5C and KA66)
identified in these studies were consistent with those of C. pneumoniae isolates
studied by Daugaard et al. (2001). In addition, the importance of the poly C tracts
as sites of variation for C. pneumoniae has also been discussed. The difference in
the length of the poly C tract within a strain and between strains was identified in
CPn0008 and CPn1 054 respectively. These data supported the event of intrastrain
and interstrain variation of these genes.
120
The analysis presented here supports multiple mechanisms including;
slipped strand mispairing within the repeats, frameshift mutations, homologous
recombination and gene conversion for antigenic variation, and adaptive evolution
in C. pneumoniae biology. Although the proteins in the CPn1O54 gene family have
previously been classified as putative inclusion membrane proteins, their
subcellular locations and functions remain to be identified. It is also not yet known
whether the members of CPn 1054 gene family are expressed individually or
coordinately, or to what extent each gene is expressed during the course of an
infection. However, the variation, both within and between strains, is a potential
requisite for this gene family that may contribute to the unique biology of C.
pneumonzae.
Significance and summary of the research
These studies have used genomic analysis as an approach for identification
of uncharacterized gene clusters and variants from the Chiamydia genome projects.
The information derived from the genome sequences has provided an approach to
identify putative Inc proteins, a novel gene family and genetic variation.
A unique structural motif of a bi-lobed hydrophobic domain was shown to
be a common character for chlamydial inclusion membrane proteins Incs. Virtually
all Inc proteins lack significant similarity to known proteins within the database. A
large group of putative Inc proteins encoded in the C. trachomatis and
121
C. pneumoniae genomes can be defined in two groups: orthologs and paralogs.
These findings suggested that some Inc proteins are conserved and required for
fundamental development and growth and some are species-specific determinants
that may have specific attributes.
Chlamydiae develop and multiply in non-acidified vacuoles, which can be
thought of as safe niches to separate them from the hostile intracellular
environment (Sinai and Joiner, 1997). The mechanism of chlamydial inclusion
development, however, remains unknown. Without the available genetic
manipulation technique to conduct variant strains in chlamydiae research, a
discovery of clinical collection of C. trachomatis variants with the multiple lobed
inclusions become a research project of interest. Studies of these variants may
elucidate a mechanism of the inclusion development and new insight of chiamydial
biology. In our study, most of these non-fusogenic variants uniformly lacked IncA,
a chlamydial Inc protein, on the inclusion membrane. Sequence analyses of incA
with a variety of mutations are consistent with the absence of IncA in most of nonfusogenic variants. Our findings suggested that IncA is a chlamydial protein
involving in homotypic vesicle fusion. However, a few nonfusogenic variants have
IncA localized on the inclusion membrane. Therefore, in some isolates, additional,
yet unidentified factor, also play a role in the efficiency or rate of fusion of C.
trachomatis inclusion. Additionally, the absence of IncA and CT223p in the
inclusion suggested that these proteins may be not critically required for
pathogenesis and survival in the eukaiyotic environment. Sequence analyses of
122
incA and CT223 from clinical isolates revealed the first evidence of genetic
variation of C. trachomatis Inc proteins.
Analysis of the three recently published C. pneumoniae genomes has led to
the identification of a new gene family named the CPn 1054 gene family which
consists of 19 predicted genes and gene fragments. In this family, paralogous genes
share not only high sequence similarity but also common structural motifs. They
contain a bi-lobed hydrophobic domain in the secondary structure, which is a
common signature for protein localization in the inclusion. This family is a gene
cluster present in only C. pneumoniae. The CPn1 054 gene family, therefore, is
defined as a species-specific cluster that will provide significantly biological
attributes, particularly genetic and phenotypic variation in C. pneumoniae.
Comparative analysis of this gene family within and among the published
genome sequences has provided evidence that gene variation through multiple
mechanisms might occur within this single collection of paralogous genes.
Frameshift mutations are found that result in both truncated gene products and
pseudogenes that vary both within and among the different genomes. Variation via
recombination of tandem repeats is also observed in a single distantly related
member of the CPn1O54 gene family. Finally, several genes in this family contain
poly C tracts either upstream or within the predicted 5' end of the coding region.
The length of the poly C tracts varies between paralogous genes in each genome,
and within single genes in the three genomes. Sequence analysis of genomic DNA
from a collection of 12 C. pneumoniae clinical isolates was used to analyze the
123
extent of the variation in the CPn1O54 gene family. These studies demonstrated that
frameshifts, gene conversions, and changes in the length of the poiy C sequences
are evident both between strains and within strains at several of the different loci.
In particular, changes in the length of the poly C tract associated with different the
CPn1O54 gene family members are common in each tested C. pneumoniae isolate.
Collectively, the variability identified within this newly described gene family may
modulate either phase or antigenic variation and subsequent physiologic diversity
within a C. pneumoniae population.
These studies have opened new questions of whether variation of inclusion
membrane proteins may involve in pathogenesis and genetic diversity. These remain
challenges and much further investigation will be required to uncover the chiamydial
infection and host-pathogens interaction.
124
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