Growth and development in Toxoplasma gondii
by Michael Nicholas Guerini
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy in Veterinary Molecular Biology
Montana State University
© Copyright by Michael Nicholas Guerini (2002)
Abstract:
The significance of Toxoplasma gondii to human and animal health, along with its value as a model for
other pathogenic protozoa, makes this microorganism an important model in the field of Apicomplexa
research. It is clear from numerous studies of the diseases caused by this family of microorganisms that
parasitaemia itself is key to pathogenesis, and thus, an understanding of the growth processes in these
pathogens could provide better treatments. In this work, the relationship/regulation between the cell
cycle and development was investigated. A G2 population arose during tachyzoite-to-bradyzoite
differentiation thus, providing a mechanism of linking the cell cycle with development. To gain further
insight into the regulation of the parasite cell cycle, both a reverse and forward genetic approach was
used. One approach used bioinformatics and cDNA library screening to identify cell cycle related
proteins. Two distinct proliferating cell nuclear antigen (PCNA) genes in T. gondii were identified and
this work demonstrated that these two genes were differentially expressed during development and the
tachyzoite cell cycle. Further work showed that each PCNA likely acts independently and provided
evidence that TgPCNA1 potentially serves as the major replisomal PCNA. The function of PCNA2 in
T. gondii remains unknown. Finally, a forward genetics approach focused on the complementation of
the tachyzoite temperature sensitive (ts) mutant 11C9, which arrests within 1 to 2 divisions at the
non-permissive temperature (40°C), and approximately half of the parasites arrest with a single
undivided nucleus (2N) placing the defect at some point in mitosis. Complementation of tel 11C9 with
a homolog of the eukaryotic XPMC2 suggests that the cyclinB/cdk or wee1/chk1 pathway in this
mutant is likely affected. In T. gondii, a proposed linkage between growth and development seem
consistent with a developmental timer system (“clock mechanism”), yet the presence of checkpoints
suggests that the domino model plays at least a partial role in regulating the tachyzoite cell cycle. GROWTH AND DEVELOPMENT
IN TOXOPLASMA GONDII
by
Michael Nicholas Guerini
A dissertation submitted in partial fulfillment
of the requirements for the degree
of
Doctor of Philosophy
in
Veterinary Molecular Biology
MONTANA STATE UNIVERSITY
Bozeman, Montana
April 2002
©COPYRIGHT
by
Michael Nicholas Guerini
2002
All Rights Reserved
APPROVAL
5
of a dissertation submitted by
Michael Nicholas Guerini
This dissertation has been read by each member of the dissertation committee and
has been found to be satisfactory regarding content, English usage, format, citations,
bibliographic style, and consistency, and is ready for submission to the College of
Graduate studies.
Michael W. White
(Signature)
Date
Approved for the Department of Veterinary Molecular Biology
Allen Harmsen
Approved for the College of Graduate Studies
/ - f " GTL
Bruce R. McLei
(Signature)
Date
iii
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I further agree that copying of this dissertation is allowable only for scholarly
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for extensive copying or reproduction of this thesis should be referred to Bell and Howell
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Signature
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//<9 Z
ACKNOWLEDGMENTS
The accomplishment of completing my Ph.D. resulted from the efforts of numerous
people. The most important contributions have come through encouragement, support
and friendship.
I thank Albert and Barbara Guerini for emotional and financial support. I also thank
Carla and Rick Wiesend, and Randy and Tammy Crone for being true friends and giving
me the support and encouragement during some of the difficult times. My final thanks go
to Dr. Rob Keene and Bart Goodman who fed me and kept me entertained.
Finally, I appreciate the help I received on the technical aspects of my dissertation from
all involved, especially:
Maria Jerome
Jay Radke
Chris Kvaal
Xuchu Que
Sharon Reed
V
TABLE OF CONTENTS
LIST OF TABLES.......................................................................................................... viii
LIST OF FIGURES........................................................................................................... ix
ABSTRACT............................ ' ........................................................................................ xi
1. TOXOPLASMA GOAD//AND APICOMPLEXANS.....................................................I
Toxoplasma gondii.....................................................
I
Life cycle..............................................................................
I
Disease and clinical relevance of Toxoplasma infections....................................... 4
Apicomplexan Biology................................................................................................... 8
Genetics.................................................................................................................. 8
Cell Biology of Growth..................................................................
11
References..................................................................................................................... 14
2. TWO GENES ENCODING UNIQUE PROLIFERATING-CELL-NUCLEARANTIGENS ARE EXPRESSED IN TOXOPLASMA GONDII......................... 20
Introduction.................................................................................................................. 20
Materials and Methods................................................................................................. 22
Cell culture and parasite growth........................................................................... 22
PCNA cDNA cloning................... .............................................................. *....... 22
Phylogenetic analysis........................................
23
Southern and Northern Blot Analysis............................................................ ••••••24
Production of Recombinant Protein and Antibody Reagents............................... 25
SDS-PAGE and Western blot analysis................................................................. 26
Results.......................................................................................................................... 26
Cloning and sequence comparison of the PCNA genes in T gondii.................. ..26
Phylogenetic analysis of Apicomplexan PCNA sequences.................................. 29
TgPCNAl and 2 mRNA expression.....................
31
TgPCNAl and 2 protein expression..................................................................... 32
Discussion................................................................................................................... 35
References................................................................................................................... 38
3. CHANGE IN THE PREMITOTIC PERIOD OF THE CELL CYCLE IS
ASSOCIATED WITH BRADYZOITE DIFFERENTIATION IN
TOZORJMaua GOJVDJT.....................................................................................42
Introduction................................................................................................................... 42
Materials and Methods.................................................................................................. 44
Cell culture and parasite growth........................................................................... 44
Immunofluorescence analysis............................................................................... 45
Fluorescent-Acitvated-Cell-Sorting(FACS)......................................................... 46
Results........................................................................................................................... 47
Expression of TgPCNAl and 2 during
sporozoite-initiated development.......................................................................... 47
Analysis of DNA content changes during
sporozoite-initiated development.......................................................................... 48
Parasites expressing both SAGl and BAGl markers comprise
most of the G2 fraction............................................................
52
Bradyzoite de-differentiation is kinetically similar to development
initiated by sporozoites........................................................................................ 53
Discussion..................................................................................................................... 56
References...................................................................................
60
4. PROLIFERATING-CELL-NUCLEAR-ANTIGENS OF TOXOPLASMA
GONDII ARE DISSIMILAR IN EXPRESSION AND POTENTIAL
FUNCTION......................................................................................................... 64
Introduction................................................................................................................. 64
Materials and Methods................................................................................................. 66
Cell culture and parasite growth....................................................
66
Production of Antibody Reagents......................................................................... 66
Cell cycle specific expression of TgPCNAl and 2 ............................................... 66
TgPCNAl and 2 protein interaction..................................................................... 68
Transcomplementation of a cold-sensitive S.cerevisae POL30
PCNA mutant........................................................................-.............................. 69
Generation of TgPCNA gene “knockout” constructs........................................... 69
Analysis of TgPCNAl “knockout” parasites....................................................... 70
Results......................................................................................................................... 72
TgPCNAl is transported in and out of the nucleus during
parasite replication................................................................................................ 72
TgPCNAl and 2 form homotypic but not heterotypic complexes....................... 74
TgPCNAl but not TgPCNAl complements aPOL30 yeast mutant.................... 75
Generation of TgPCNA gene “knockouts”........................................................... 77
Phenotypic analysis of ATgPCNAl parasites...................................................... 80
Discussion.................................................................................................................... 81
References.................................................................................................................... 84
vii
5. GENETIC COMPLEMENTATION OF A TOXOPLASMA GONDII
CELL CYCLE MUTANT................................................................................... 88
Introduction................................................................................................................. 88
Materials and Methods............................
90
Cell culture and parasite growth........................................................................... 90
Construction of destination vector and complementation of tell C9.................... 91
Analysis of the cDNA for TgXPMCZ................................................................. 92
Phenotypic analysis of TgXPMCZ-complemented tellC 9.................................. 93
Results........................................................................................................................ 94
Complementation of cell cycle mutant tel 1C9.................................................... 94
Molecular Biology of Toxoplasma XPMC2............................................
96
Cell cycle analysis of TgXPMCZ transformants................................................. 97
Discussion................................................................................................................ 101
References................................................................................................................ 103
6. GROWTH AND DEVELOPMENT IN TOXOPLASMA.................................
108
Summary................................................................................................................... 108
References................................................................................................................. 113
LIST OF TABLES
Table
Page
1-1. Apicomplexan diseases of medical and veterinary significance............................... 5
3-1. Cell cycle and developmental distributions during sporozoite-tobradyzoite differentiation....................................................................
52
LIST OF FIGURES
Figure
Page
1- 1. Life cycle of Toxoplasma gondii............................................................................. 2
2- 1. Amino acid alignments of the putative functional domains of two
unique classes of PCNAs found in Toxoplasma gondii, Plasmodium
falciparum and Cryptosporidium parvumcompared to human PCNA................. 28
2-2. Southern analysis of T. gondii RH strain genomic DNA hybridized with
TgPCNAl- and 2-specific probes........................................................................ 29
2-3. Consensus tree of Apicomplexa and representative eukaryotic PCNAs.................30
2-4. Relative levels of TgPCNAl and 2 mRNAs from three different T. gondii
tachyzoite strains................................................................................................. 32
2-5. Cross reactivity of polyclonal antiserum to TgPCNAl and 2 recombinant
proteins..................................................
33
2- 6. Western blot analysis of T gondii tachyzoite lysates................................... *...... 35
3- 1. Developmental expression of TgPCNAs........................................................ *........ 49
3-2. Flow cytometric analysis of sporozoite, tachyzoite and bradyzoite
DNA content......................................................................................................... 50
3-3. Two color flow cytometric analysis of differentiating parasite populations............ 54
3-4. Analysis of the growth rate and BAGl expression during recrudescence............... 55
3- 5. Model of Toxoplasma gondii development in the intermediate host.................... 57
4- 1. The cellular distribution of TgPCNA changes during the tachyzoite cell cycle...73
4-2. TgPCNAl and 2 form homotypic but not heterotypic complexes........................... 76
4-3. TgPCNAl complements a POZ30 (PCNA) mutant yeast strain...............................77
4-4. Genetic structures of TgPCNAl and 2 and targeting construct designs..................79
4-5. Southern and Northern analysis of ATgPCNA2 parasites........................................ 80
5-1. Schematic representation of M1C9 complementation. ........................................95
5-2. Recovery of cDNA inserts from til 1C9 complements............................................96
5-3. Amino acid sequence of TgXPMC2.........................................................................97
5-4. Phenotypic rescue of til I C9 by TgXPMC2........................................................... 98
5-5. Temperature-sensitive, mitotic block of til 1C9 is rescued by TgXPMC2............100
ABSTRACT
The significance of Toxoplasma gondii to human and animal health, along with its value
as a model for other pathogenic protozoa, makes this microorganism an important model
in the field of Apicomplexa research. It is clear from numerous studies of the diseases
caused by this family of microorganisms that parasitaemia itself is key to pathogenesis,
and thus, an understanding of the growth processes in these pathogens could provide
better treatments. In this work, the relationship/regulation between the cell cycle and
development was investigated. A G2 population arose during tachyzoite-to-bradyzoite
differentiation thus, providing a mechanism of linlcing the cell cycle with development.
To gain further insight into the regulation of the parasite cell cycle, both a reverse and
forward genetic approach was used. One approach used bioinformatics and cDNA
library screening to identify cell cycle related proteins. Two distinct proliferating cell
nuclear antigen (PCNA) genes in T. gondii were identified and this work demonstrated
that these two genes were differentially expressed during development and the tachyzoite
cell cycle. Further work showed that each PCNA likely acts independently and provided
evidence that TgPCNAl potentially serves as the major replisomal PCNA. The function
of PCNA2 in T. gondii remains unknown. Finally, a forward genetics approach focused
on the complementation of the tachyzoite temperature sensitive (ts) mutant 11C9, which
arrests within I to 2 divisions at the non-permissive temperature (40°C), and
approximately half of the parasites arrest with a single undivided nucleus (ZN) placing
the defect at some point in mitosis. Complementation of tel 1C9 with a homolog of the
eukaryotic XPMC2 suggests that the cyclinB/cdk or weel/chkl pathway in this mutant is
likely affected. In T gondii, a proposed IinIcage between growth and development seem
consistent with a developmental timer system (“clock mechanism”), yet the presence of
checkpoints suggests that the domino model plays at least a partial role in regulating the
tachyzoite cell cycle.
I
CHAPTER I
TOXOPLASMA GONDII AND APICOMPLEXANS
Toxovlasma gondii
Life cycle
The phylum Apicomplexa is divided into four classes, the Gregarinea,
Haemosporidea, Piroplasmea and Coccidea, which together comprise over 5,000 named
species of protozoan parasites [I]. The coccidea are the largest class (-1,500 species)
and include various pathogens of man and animals [Eimeria, Sarcocystis, Neospora,
Cryptosporidium, Cyclospora and Toxoplasma gondii], T gondii, the subject of this
thesis, is capable of infecting an unusually broad range of hosts and many different cell
types. Toxoplasma is facultatively heteroxenous (two host life cycle) and causes one of
the most common parasitic zoonoses in the world (Fig. 1-1) [2-3].
There are three
infectious stages in the life cycle of T. gondii; sporozoites, tachyzoites and bradyzoites.
T gondii is unique among heteroxenous coccidians in that all three of these parasite
forms are infectious to both intermediate and definitive hosts.
Three routes are
responsible for Toxoplasma infection: I) horizontal transmission by oral ingestion of
oocysts (sporozoites) from the environment, 2) horizontal transmission of tissue cysts
(bradyzoites) by oral ingestion of raw or undercooked meat, 3) vertical transmission of
2
tachyzoites via the placenta of infected mothers [3-4]. It has also been reported that
tachyzoites may be transmitted through milk from mother to offspring [3],
,Bradyzoites <X “
Gametes
Tissue cysts x
(bradyzoites)
Intermediate Hos
Feline Host
oocysts
(sporozoites)
Merz,
Figure 1-1: Life cycle of Toxoplasma gondii.
As with other coccidians, the T gondii oocyst is the environmentally protective
housing for the sporozoite [2], When oocysts are ingested by an intermediate host,
sporozoites excyst and actively penetrate cells of the intestinal tract where they
differentiate into replicating tachyzoites.
Tachyzoites multiply rapidly by repeated
rounds of endodyogeny and will ultimately develop into bradyzoites contained within a
protective tissue cyst. Tissue cysts are the end-stage of the intermediate life-cycle and
3
are capable of persistence for the life of the host. It is postulated that tissue cysts break
down periodically and bradyzoites differentiate back into tachyzoites, which renews the
pathogenic
cycle
(recrudescence)
and
is
the
basis
of chronic
disease
in
immunocompromised individuals [3]. Alternatively, bradyzoites are released from tissue
cysts when ingested by a new animal host and differentiate into a tachyzoite to begin the
intermediate life cycle. The location and number of tissue cysts in animals differ with
respect to host and T. gondii strain [5], In mice and rats, tissue cysts were more prevalent
in the brain [5] whereas higher mammals present more cysts in muscular tissues [5],
Within the feline host, bradyzoite development follows the definitive life-cycle
[2], Bradyzoite invasion of feline enterocytes results in differentiation into merozoites
that replicate for several cycles prior to forming gametocytes. The ensuing processes of
gamogony and oocyst formation take place within the epithelium of the small intestine.
After fertilization of a macrogametocyte by a micro gamete, the zygote forms within an
oocyst that is passed into the intestinal lumen and shed in the feces. The zygotes (the
only diploid stage) in the oocyst undergo meiosis to form eight haploid sporozoites,
which are subcompartmentalized into two groups of four, each group is contained in a
sporocyst.
Sporozoites and tachyzoites ingested by a feline must first develop into
bradyzoites prior to initiation of the definitive life cycle. Thus, the incubation period
prior to oocyst shedding is characteristic of which developmental stage is present in the
inoculum. Oocysts are shed 3 to 10 days after ingesting tissue cysts, >13 days after
ingesting tachyzoites and >18 days after ingesting oocysts [3].
4
Disease and clinical relevance of Toxoplasma in.ff.r.tions
A relatively small number of Apicomplexa species are responsible for the
diseases of medical and veterinary significance (Table 1-1) [6-7]. Examples include
Plasmodium spp., which cause malaria, Eimeria spp., which cause coccidiosis in animals,
and Toxoplasma gondii which causes toxoplasmosis [2],
Toxoplasmosis in healthy,
immunocompetent humans is common yet clinical presentation is often inapparent or
misdiagnosed. Toxoplasmosis can be categorized into three states; acute, sub-acute and
chronic.
In the acute state (caused by tachyzoite parasitaemia), the most common
symptoms are swollen lymph glands, associated fever headaches, and anemia, all of
which are symptoms typically confused with influenza [2-3,8], The sub-acute stage is
characterized by formation of cysts in tissues such as liver, heart, brain and eyes [2-3,8].
The recrudescence of the tissue cyst (bradyzoite) back into the proliferative tachyzoite
leads to a chronic infection.
A number of factors influence the severity of disease
symptoms including pathogen strain, susceptibility and age of the host, and the state of
acquired immunity within the host.
5
O R G A N IS M
D ISEA SE
A FFLIC TS
Toxoplasm a
T o x o p la s m o s is
All w a rm -b lo o d e d a n im a ls ,
b ird s (3 0 + s p p .)
P lasm odium
M a la ria
H u m a n s a n d A n im a ls
S arcocystis
S a rc o c y s to s is
H u m a n s , M a m m a ls , B irds
a nd R e p tile s
Eim eria
C o c c id io s is
M a m m a ls a n d B ird s
C yclospora
C y c lo s p o ria s is
Hum ans
Isospora
s o s p o ro s is
H u m a n s a n d A n im a ls
C ryptosporidium
C ry p to s p o rid io s is
H u m a n s a n d A n im a ls
Babesia
B a b e s io s is (re d w a te r fe v e r)
C attle, o th e r a n im a ls , m an
(ra re )
Theileria
T h e ile rio s is (E a s t C o a s t F e ve r)
C a ttle
Table 1-1: Apicomplexan diseases of medical and veterinary significance [6,7].
T. gondii infection within healthy individuals in the United States is thought to
occur most commonly through the consumption of contaminated meat products, although
the relative contribution of contaminated food versus environmental sources remains an
open question [3-4]. Tissue cysts are present in edible tissues of approximately 30% of
sheep and swine in the United States [2,9-11]. Outside of Europe and North America, the
prevalence of infected animals increases to >50% (ex. in pigs) thereby leading to high
rates of infection in some countries [3]. A higher percentage of food source infections
have been found in populations of individuals within France, Panama and the continent of
Africa where the seropositive rate can exceed 65% [4]. These findings support the
hypothesis that crowded living conditions and cultural/eating habits contribute to the
wide range of seropositivity among various populations [3],
6
Documented epidemics of oocyst infection have occurred raising concerns that
environmental contact with oocysts is a threat to urban populations [12-14], Two cases
in North America have provided the foundation for this concern. At a riding stable in
Atlanta Georgia 43% of individuals exposed to an oocyst source became seropositive
[15]. In a case in Vancouver, Canada, 110 individuals presented with T. gondii infections
as a result of contaminated water from a municipal water source [12]. In this local
epidemic, it was estimated that several thousand individuals were exposed to
contaminated water. High seropositive rates are seen in farm workers, gardeners [4], and
a vegetarian life-style does not seem to minimize the risk of exposure [4] suggesting
environmental transmission is a significant cause of toxoplasmosis.
The unborn and the immunocompromised are at significant risk for acquiring a T.
gondii infection. The symptoms presented by these two groups include encephalitis,
sepsis syndrome/shock, myocarditis, hepatitis and retinochoroiditis [3],
Congenital
toxoplasmosis usually occurs when the mother acquires a primary T. gondii infection
during pregnancy and may result in an abortion, neonatal death or fetal abnormalities,
Vertical transmission occurs in mothers previously exposed to T. gondii who enter into an
immunocompromised state during pregnancy either through the use of steroids, systemic
lupus erythematosus or an HIV infection [3], It has been suggested that in these women,
the reactivation of a latent infection (recrudescence of the bradyzoite) resulted in
transplacental transmission [3].
Children who survive a prenatal infection may have a significantly reduced
quality of life with defects in vision, hearing, and central nervous system (CNS)
deficiencies [3], The frequency of prenatal infection varies from I to 100 per 10,000
7
births [3], The risk of an interuterine infection increases during pregnancy but the most
severe manifestations of toxoplasmosis occur when transmission comes at an early stage
of pregnancy. In some states of the U.S.A. and various European countries (Austria,
Italy, Denmark, Poland and France) programs have been initiated that are aimed at early
detection of prenatal infection in neonates [3]. The prophylactic use of drugs on women
of childbearing age is an unreasonable proposal.
Through the implementation of
effective screening programs, mothers can be treated with pyrimethamine and
sulfadiazine providing an in utero therapy for the fetus. These drugs have been shown to
decrease the severity of symptoms in infected children. More encouraging news comes
from a study in which pregnant women who showed evidence of a recently acquired
infection exhibited an ~40% decrease in the number of prenatal infections when given
spiromycin during pregnancy [3], Additionally, spiromycin has been shown to decrease
the percentage of fetuses with severe disease or intrauterine death from 11 to 3% [3].
In immunocompromised individuals, either a primary infection or a previously
acquired latent infection can lead to severe complications [3,16]. Encephalitis is the
leading affliction in immunocompromised individuals infected with T. gondii [3,16]. In
AIDS patients, the unique ability of bradyzoites and tachyzoites to interconvert is
considered to be the underlying cause of Tbxqptoma-induced encephalitis [16].
Consequently, AIDS patients are treated prophylactically with a combination of
trimethoprin/sulfamethoxyzole which is often not well tolerated. Prophylactic treatment
for this pathogen along with recent advances in antiretroviral therapies and immune
reconstitution has reduced the incidence of CNS toxoplasmosis in AIDS patients [3,16].
8
Apicomplexan Biology
Genetics
Apicomplexan parasites have a wide range of genome sizes (8 Mb to >200 Mb).
The genome of Cryptosporidium parvum is 8 Mb, Plasmodium falciparum 30 Mb,
Eimeria spp. ~60 Mb and Sarcocystis cruzi may have the largest genome at 200 Mb [17].
T. gondii posseses two organellar genomes (apicoplast, ~35 kb and mitochondria, ~7 kb)
and a nuclear genome estimated to be ~80 Mb (haploid) partitioned into at least 11
chromosomes ranging in size from 1.9 Mb to greater than 10 Mb [18]. Based on genome
size and a rudimentary knowledge of intron/exon abundance, it is estimated that
Apicomplexan genomes may contain as many as 10-15,000 genes [J. Ajioka, personal
communication].
This is likely an overestimate, particularly for the small
Cryptosporidium genome, although there appear to be few introns in this unusual
member of the Apicomplexa [M. Abrahamsen personal communication],
T. gondii
nuclear DNA has a 55% GC content as compared to Plasmodium spp., which have a 20%
GC content [16]. Unlike other coccidian parasites, T. gondii codon usage is relatively
unbiased [17].
Phylogenetic analysis indicates that three genotypic lineages of T. gondii account
for most of the strains found in the world [21]. The RH strain (Type I) is the most
extensively used strain for experimental and genetic manipulations [22]. Type I strains
have an essentially identical genotype leading to the hypothesis that they are derived
from a single clonal lineage [23]. Type I strains have a reduced ability to undergo the
definitive life cycle in cats and are highly virulent in mice as opposed to Type II and III
9
strains which are comparatively avirulent but are capable of efficiently initiating the
definitive life cycle in the feline. The laboratory strain ME49-Plk represents the Type II
variant most often identified within HIV patients that present with acute Toxoplasmaencephalitis [21]. The Type III strain is most commonly found in livestock and is the
second most common strain found in HIV patients. Interestingly, karyotype comparisons
between the three major clonal lineages show very little chromosome size variation as
compared to other Apicomplexa. The karyotype of RH strain, for example, has remained
virtually unchanged in more than 50 years of culture [18,23-24], T. gondii isolates can be
divided into two virulence classes based on experiments in mice. The 50% lethal dose
(LD50) for most strains is dose dependent (requires IO2-IO5 parasites) [25]. The acutely
virulent Type I strains cause mortality at a very low parasite inoculum (LD50 <102) [25].
Three Apicomplexan genomes have been sequenced; P. falciparum, C. parvum
and Theileria parvum [26-28]. The combined efforts of an EST project and a recently
funded genome sequencing project have begun to provide resources for T. gondii [29-31].
The current Toxoplasma EST database includes -3200 ESTs derived from sporozoite
cDNA libraries (1262 unique), 17,000 tachyzoite ESTs representing -6600 unique
sequences and -3300 ESTs obtained from bradyzoite cDNA libraries which represent
1289 unique sequences.
T gondii has proven to be an important Apicomplexan model due to the relative
ease of in vitro genetic manipulations in the tachyzoite stage. More than 50% of parasites
express transiently after electroporation and non-homologous integration of DNA can
occur in 1/20 viable parasites (RH strain) transfected using one of several selectable
markers;
hypoxanthine-xanthine-guanine
phosphoribosyl
transferase
(HXGPRT),
10
chloramphenicol, pyrimethamine, or phleomycin [32-35],
Toxoplasma expression
vectors with a variety of promoter strengths, fluorescent markers (CFP, YFP, GFP, RFP),
epitope tags, and targeting sequences are now available [36], Using fluorescent markers,
proteins associated with the cytoskeleton [36], the nucleus [37], or targeted to specialized
organelles including the plastid [36,38-39] have all been characterized.
Antisense
protocols have been used to study the functions of nucleoside triphosphate hydrolase [40]
and HXGPRT [41], Recently a rudimentary tetracycline-inducible system was described
[42],
The high level of non-homologous integration in Toxoplasma allows for a large
portion of the parasite genome to be marked [43],
Through genomic tagging by
insertional mutagenesis, a gene can be identified whose inactivation is not lethal and for
which a suitable selection screen is available [43-45], Initial studies of gene function and
expression rely on the use of cDNA sequences (typically <2kb) that typically incorporate
into the genome by non-homologous recombination. Larger fragments of genomic DNA
can switch the targeting from a non-homologous to homologous type of integration.
Using genomic sequences >8kb, of which ~ 4 kb flank the selectable marker, has resulted
in ~50% of integrations occurring by homologous recombination.
Examples of
homologous integrations generating gene replacements include: dihydrofolate-reductase
(DHFR) [46], HXGPRT [47], bradyzoite antigen I [48], adenosine kinase [49]^ uracil
phosporibosyl transferase [50], rhoptry protein I [33] and dense granule protein two
(GRA2) [51]. Until recently, the major deficiency in the Toxoplasma genetic “tool-kit”
was a robust forward genetic model for complementation.
An episomal library
containing T. gondii genomic fragments [52] was unsuccessful due to the high integration,
11
rate. A new strategy has now been developed that combines phage recombination [53]
with the high level of stable integration in the tachyzoite [See Chapter #5 and 54].
Cell Biology of Growth
Apicomplexan parasites undergo three types of nuclear/cellular division.
Schizogony results when multiple nuclei are formed by alternating rounds of DNA
synthesis (S) and mitosis (M) ((S-M-ND)n-C-Gl) (representative organisms: Eimeria
callospermophilli and T. gondii merozoites ) [55, Speer unpublished observations].
Budding of daughter parasites (cytokinesis - C) is delayed until the last round of nuclear
division. In endopolygeny ((S-M)n-NDZC-GI) (representative organisms: Sarcocystis
tenella and Plasmodium berghei), multiple rounds of DNA replication occur in the
absence of mitosis, thereby generating a single polyploid nucleus that subsequently
undergoes multiple mitotic events in conjunction with concerted parasite budding [5657]. Endodyogeny, the mode of division of the T. gondii tachyzoite, has long been
considered a binary form of schizogony [58-59]. In endodyogeny (S-M/C-Gl), DNA
synthesis is followed by mitosis and parasite budding, which results in a process that is
indistinguishable from the final cycle of schizogony.
Interestingly, the scale, if not the number, of schizogonous nuclear divisions
appears to be predetermined in many of the Apicomplexa. For example, Eimeria bovis
sporozoites invariantly produce very large schizonts (with >100,000 first-generation
merozoites) [60], while sporozoites of Cryptosporidium parvum only form small
schizonts that produce 6-8 merozoites [61]. In each case the relative size of the schizont
12
is predetermined and obligatory to further development. Thus, it is evident that growth
control can directly affect the course of parasite development. The idea that growth
control and development are linlced was recently explored in T. gondii, and it was
established that tachyzoites emerging from sporozoite-initiated infections undergo a rapid
growth for 20 divisions, followed by a spontaneous shift to a slower growth rate that
precedes differentiation into the bradyzoite [62]. This growth shift occurs at least 24 h
prior to the expression of bradyzoite markers.
The structural features of parasite budding in T. gondii have been examined in
detail [58] by electron microscopy. Budding is first recognized by division of the Golgi
and by the appearance of conoid structures (apical complexes) in the tachyzoite
cytoplasm.
These structures appear prior to nuclear division and are almost always
anterior to the nuclear envelope [63].
As budding continues, two inner membrane
complexes emerge and extend posteriorly from each of the central apical complex
structures in order to enclose their half of the mother cell organelles. Into each immature
daughter parasite a divided nucleus migrates while the inner membrane complexes
continue to expand. Ultimately, the daughter cells consume most of the mother cell,
although they may remain joined posteriorly (rozetta) for several divisions.
Recently, the features of tachyzoite endodyogeny have been placed within the
framework of the eukaryotic cell cycle. The tachyzoite cell cycle is unusual in that it is
comprised primarily of a Gl and a bimodal S-phase [37,64]. Tachyzoite division can be
partitioned into -60% Gl, -30% S phase and -10% G2+M phases [37,64], A significant
fraction of tachyzoites possess a 1.7-1.8 N DNA content, indicating that DNA synthesis
may be unequal with -20% of the genome replicating just prior to entering mitosis. This
13
cell cycle feature remains to be confirmed; however, it is noteworthy that gametocytes of
Toxoplasma and Plasmodium spp. appear to pause or slow chromosome replication (1.7
N) prior to invasion of the next host cell where chromosome replication is completed [17,
65-66]. In this regard, the Plasmodium microgametocyte is remarkable, as chromosome
replication is interrupted indefinitely until the gametocytes move into the insect host at
the time of a blood meal. Whether this pause in DNA synthesis represents a novel cell
cycle checkpoint remains to be determined.
The subsequent chapters describe work In T. gondii which has provided insight
into the mechanisms involved in the linlcage between growth and development. The
hypothesis for chapters 2 and 4 is as follows: Proliferating cell nuclear antigen (PCNA) is
an essential co-factor of DNA replication and repair in eukaryotes, and is present as a
single gene. Apicomplexa have two PCNAs and we hypothesize that one PCNA is
essential for DNA replication and the second PCNA functions in repair. The initial
characterization of these two genes was reported in Guerini et al. [67] and appears as
chapter 2 in this thesis. The work presented in chapter 3 begins with experiments that use
TgPCNAl and 2 as a marker for proliferation and this work is based on the hypothesis
that a cell cycle switching mechanism is involved in differentiation. Further work in this
chapter characterizes the recrudescent pathway.
Chapter 5 describes work using a
forward genetics approach [54] to complement a loss-of-function mutant in Toxoplasma.
This work was performed to prove that a forward genetics approach to complementing
temperature sensitive (ts) tachyzoite mutants was functional and that the Cyclin B
pathway might play a role in the defect associated with Al 1C9 [68].
14
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20
CHAPTER 2
TWO GENES ENCODING UNIQUE PROLIFERATING-CELL-NUCLEARANTIGENS ARE EXPRESSED IN TOXOPLASMA GONDII.
Introduction
The haploid genome of Toxoplasma gondii consists of 80 mb partitioned between
11 chromosomes [I]. Little is understood about T gondii chromosome structure or the
mechanisms of chromosome replication, although a low-resolution genetic map [2-3]
based, in part, on expressed-sequence-tags has been generated for this parasite [4], What
is Icnown about T. gondii division derives primarily from decades old ultrastructural
observations [5], Members of the phylum Apicomplexa, to which T gondii belongs,
replicate by simple binary division (endodyogeny) or by multinuclear processes
(schizogony) that either involve multiple rounds of S and M phase prior to cytokinesis or
the synthesis of polyploid chromatin followed by a single, multifocal mitosis and
cytokinesis [6], Schizogonous mechanisms appear to govern T. gondii multiplication in
its definitive feline host, whereas in the intermediate host, asexual replication of the
tachyzoite is exclusively by endodyogeny [6].
Rapid tachyzoite division is closely
associated with virulence, which parallels reports that virulent isolates have elevated
DNA polymerase activity [7], This observation is potentially important to understanding
acute disease caused by Type-I virulent strains [3]; however, further work is needed in
21
order to identify specific components of the DNA replication machinery that may be
linked to virulence.
Proliferating Cell Nuclear Antigen (PCNA) is a ubiquitous co-factor of eukaryotic
DNA synthesis and repair [8-9]. Attempts to generate APCNA diploids in yeast have
been unsuccessful, demonstrating this protein is essential for cell survival and implying a
critical role in DNA replication [10-11]. PCNA is thought to specifically function by
increasing the processivity of DNA polymerases S and e and in this way possesses a
similar function to the [3-subunit of the E. coli DNA polymerase III holoenzyme and the
product of gene-45 of bacteriophage-T4 [12]. Animal and yeast PCNAs are relatively
small proteins (~30 kDa) that are thought to function as homotrimeric ring structures.
Assembly of the PCNA ring on chromosomes appears to be initiated by the binding of
replication factor-C, which then recruits PCNA and polymerase 5 to form an active
replication complex [13-15],
Dramatic increases in PCNA levels have been shown to occur as animal cells
enter S phase following a period of growth arrest [16]. Although originally thought to be
S-phase specific, it is now accepted that PCNA levels marginally increase (~2-fold) in
cycling animal cells and is more accurately considered an indicator of cell cycle entry
from a Go state [8,16]. In Plasmodium falciparum, PCNA expression is tightly regulated
during intraerythrocytic development [17]. Early, uninucleate ring stages (G1 phase)
contain little, if any, PCNA mRNA or protein. PCNA induction is first detected in the
late ring stage and is maximally expressed in trophozoites. This progression correlates
with the onset of parasite DNA replication in the intraerythrocytic cycle, and thus, PCNA
expression is also observed in schizonts undergoing mitosis and cytokinesis [17-18],
22
These studies illustrate the value of PCNA as a marker to delineate the periods of the cell
cycle during the developmental transitions of other apicomplexan parasites.
In this
chapter, we describe the cloning and expression of two unique PCNA genes in T. gondii.
Materials and Methods
Cell culture and parasite growth
Human foreskin fibroblasts (HFF) were grown in Dulbecco’s Modified Eagle
Medium (Gibco BRE, Grand Island NY) supplemented with either 10% (v/v) newborn
calf serum or fetal bovine serum (Hyclone Laboratories Inc., Logan UT).
T gondii
tachyzoites of three strains: RH (Type I), ME49-PLK (Type II) and VEG (Type III) were
maintained by serial passage in HFF cells according to standard methods [19-21],
Tachyzoites were purified from host cell monolayers by filtration through 3.0 pM
Nucleopore filters (Costar, Cambridge, MA).
PCNA cDNA Cloning
Initial clones were obtained for TgPCNAl by using reverse-transcription/PCR
and degenerate oligonucleotide primers to conserved N-terminal (-Y/FRCDR-) and Cterminal (Y/FLAPK) PCNA sequences. Preliminary TgPCNA2 sequence information
was discovered by examination of the T. gondii EST database (TgESTzy03b07.rl) [4,22].
Complete sequences for each of the TgPCNAs were then isolated by screening a X ZAPII
23
RH strain cDNA library (NIH, AIDS reagent #1896) using [32Pj-Iabeled TgPCNA
specific probes. Full-length cDNA clones for T. gondii PCNAl (TgPCNAl Accession
#AF242301) and PCNA2 (TgPCNA2 Accession #AF242302) were sequenced and
deposited into GenBanlc™.
Based on sequence comparisons, a single divergent region in the 3 ’ end of each
TgPCNA (-11% nt homology and -15% identity) was identified and gene-specific
probes to this region were generated by PCR. The TgPCNAl specific probe corresponds
to amino acids 174 through 284 (-332 bp) and was cloned into pBSK+ after PCR using
primers (S’-CGCGGATCCCTCAAGGAGACCTCGGAGTCGG-S”) and (5’-CGCGGATCCTTTCACGTGGCCGATGCACGAGCG-3’). The TgPCNA2 probe encompasses
amino acids 171 through 274 (-308 bp) and was PCR amplified for cloning using primers
(5’-CCGGAATTCAGACCCACATGCAACTCTCTGCGC-3’) and (5'-CCGGAATTCCAGCGTGCTCCGCGATTGAGG-3 ’).
Phylogenetic Analysis
The putative TgPCNAl and 2 coding regions were aligned with protein sequences
representative of the various eukaryotic PCNAs using the Clustal W program and default
parameter values [23] with some manual refinements. Phylogenetic trees were generated
using PHYLIP (Phytogeny Inference Package v3.5c) [24] with bootstrap analyses (100
replicates) processed through a distance matrix (PAM-350), analyzed by neighbor-joining
methods, and the results compiled into a consensus tree. Accession numbers: Homo
sapiens, NP 002583; Mns musculus,, P17918; Sarcophaga crassipalis, 016852;
24
Drosophila melanogaster, P 17917; Zea mays, AAD10528; Nicotiana tabacum,
AAD19905; Schizosaccharomyces pombe, CAB38513; Saccharomyces cerevisiae,
P15873; P. falciparum [PfPCNAl] CAA48673; P. falciparum [(PfPCNA2) Contig id
13124];
Cryptosporidium parvum
[(CpPCNAl)
Contig id
1079];
C. parvum
[(CpPCNA2) Contig id 1123].
Southern and Northern Blot Analysis
T gondii RH genomic DNA was isolated as previously described [25], digested
with restriction enzymes BgZII and EcdRl, electrophoresed through a 1% agarose gel and
transferred to nitrocellulose. Hybridization of duplicate blots was performed at 42°C for
16 h in a solution containing 50% formamide and 6 X SSC using [32P]-labeled inserts
specific for each TgPCNA gene (see above). Following hybridization, nitrocellulose
blots were washed three times in 2 X SSC containing 0.1% SDS at room temperature (10
min each) and then twice in 0.1 X SSC containing 0.1% SDS at 42°C (30 min each).
Blots were exposed to x-ray film at -80°C.
Total RNA was prepared from RH, ME49-PLK, and VEG strain tachyzoites by
TRIzol (Gibco-BRL, Grand Island NY) extraction and analyzed by Northern blot.
Briefly, equal amounts of total RNA (4.5 pg) for each strain were loaded into duplicate
lanes, separated on a 1.2% (w/v) formaldehyde-agarose gel and transferred to
nitrocellulose. In the Northern blot hybridization experiment, the two probes had equal
specific activities (TgPCNAl-1.67 x IO8 cpm/pg DNA and TgPCNA2-1.61 x IO8 cpm/pg
DNA) as determined following purification of the labeled probes using ELUTIP
25
minicolumns (Schleicher and Schuell5 Keene, NE).
Hybridization and washes were
completed as described above. To normalize the level of TgPCNA mRNA species within
the blots, a T. gondii specific IBS rRNA oligonucleotide probe (5'-AGACCGAAGTCAACGCGACC-3 ’) [26] was 5’-end [32P]-labeled (8 x IO7 cpm/pg DNA) and used to
reprobe the blots after stripping the previous probe in boiling water. Blots were exposed
to x-ray film at -BO0C and mRNA expression levels were quantified using a Bio-Rad
Molecular FX Imager and the Quantity One software package (Ver 4, Bio-RAD,
Hercules CA).
Production of Recombinant Protein and Antibody Reagents
The coding regions for TgPCNAl and 2 were cloned into the Sphl and Hindlll
sites of the pQE-30 (Type I) plasmid vector (Qiagen, Chatsworth CA) and transformed
into the expression bacteria M15[pREP4],
amplified
from
the
cDNA
insert
The TgPCNAl coding sequence was
using
the
ACAT GCAT GCAT GTT GGAAGCCAAGCT CCAG-3 ’)
following
and
primers
(5’-
(5 ’-CCCAAGCTT-
CTCATCCATCATAGAGTCGTC-3 ’). TgPCNA 2 coding region was amplified from
cDNA inserts using (5’-ACATGCATGCATGTTTGAGTGCACGATCGAGGGG - 3 ’)
and (5’-CCCAAGCTTCTCTTCCTCGTCCTCTATTCTC-3 ’).
The pQE-30 vector
added six adjacent histidine residues to the N terminus (6xHis-tag) and allowed the
recombinant proteins to be purified (using native conditions) to virtual homogeneity in
one step using nickel-chelate affinity chromatography, as described by the manufacturer.
Immune serum for each purified TgPCNA protein was prepared by immunizing mice
26
(BALB/c) 3 times intraperitoneally at 2-week intervals with 25 jag of purified
recombinant protein emulsified in Hunter’s Titermax® adjuvant (Sigma, St. Louis MO).
SDS-PAGE and Western blot analysis
Tachyzoites from three genotypic strains (RH, ME49-PLK and VEG) were lysed
using M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) containing
10
PgZml
each
of ■ antipain,
leupeptin,
chymostatin,
pepstatin
phenylmethylsulfonylfluoride and phenanthroline (Sigma, St. Louis MO).
A,
Aliquots
equivalent to 15 X IO6 tachyzoites were subjected to 12% SDS-PAGE under reducing
conditions and transferred to nitrocellulose. Following transfer, the blots were blocked
with 10% horse serum and then incubated overnight with anti-TgPCNAl or 2 mouse
polyclonal sera (1:2500). Antigen detection was accomplished by incubating blots with
alkaline phosphatase-conjugated anti-mouse IgG (Promega, Madison WIS) diluted
1:7,500, followed by development in nitroblue tetrazolium and 5-bromo-4-chloro-3indolylphosphate [27].
Results
GIoninP and sequence comparison of the PCNA genes in T sondii
Analysis of EST database entries along with conventional cDNA strategies
identified two unique PCNA cDNAs in T. gondii, designated TgPCNAl and TgPCNA2.
27
The 951 bp open reading frame of TgPCNAl predicts a protein of 316 amino acids with a
predicted molecular weight of 34,476 daltons. The TgPCNA2 clone contains a smaller
open reading frame of 864 bp encoding a protein of 261 amino acids and a predicted
molecular weight of 32,263 daltons. Consistent with all Icnown eukaryotic PCNAs, the
TgPCNA sequences contain basic helix-loop-helix DNA binding motifs (Fig. 2-1) that
demonstrate >50% similarity to the equivalent region in human PCNA [8],
Other
conserved motifs include the polymerase-5 and p21 putative binding sites (D-SHV-, Fig.
2-1) as well as the C-terminal sequences (-F/YLAP, Fig. 2-1) that appear essential for
proper folding [28].
PfPCNAl contains 10 and P1PCNA2 contains 9 additional C-
terminal amino acids while T gondii PCNAl and 2 contain C-terminal insertions of 53
and 28 amino acids, respectively.
Southern analysis of T gondii DNA with TgPCNAl- and 2-specific-probes shows
a different hybridization pattern, confirming the existence of two unique PCNA genes
(Fig. 2-2). A search of the P. falciparum genome sequence data (The Sanger Centre,
Cambridge UK) also revealed the existence of two distinct PCNA genes. PfPCNAl was
found in sequence data from chromosome thirteen and is identical to the PCNA gene
previously described by Kilbey et al. (1993). A second putative PCNA gene (designated
PfPCNA2) was found in contig #13124 (The Sanger Centre) from chromosome twelve.
TgPCNAl and PfPCNAl share the highest amino acid identity at 49%, compared to 38%
identity for TgPCNA2 and PfPCNA2.
Comparison of TgPCNAl and CpPCNAI
demonstrated 42% identity, whereas the identities for TgPCNA2 and CpPCNA2 are 32%.
Interestingly, intraspecies PCNAs share only 27-30% identity.
28
T gP C N A l
P fP C N A l
CpPCN Al
H um an
TgPCNA2
P f PCNA2
CpPCNA2
T gP C N A l
P fP C N A l
C pPCN Al
Hum an
TgPCNA2
P f PCNA2
CpPCNA2
T gP C N A l
P fP C N A l
CpPCN Al
Hum an
TgPCNA2
P f PCNA2
CpPCNA2
26 VNLDCDERCL
26
26
26
25
25
26
3 ASEc
s |N
56
56
56
56
55
55
56
270
251
245
244
262
251
250
55
55
55
55
54
54
55
83
83
83
82
83
83
82
TQNBjV
H T sk
PNgLL
!nfSl
IQQgS
RRQAQQPRSCI
------------- t l k i B f E I
aiAIGGE 299
MgMDNKD 273
TMgDE-QE 262
B
--------- EGPQSRST
------- D— P ----- S
eg s- -
258
JjgEE----- 286
RgLLVCRN 270
R261
Figure 2-1. Amino acid alignments of the putative functional domains of
two unique classes of PCNAs found in Toxoplasma gondii, Plasmodium
falciparum and Cryptosporidium parvum compared to human PCNA.
Conservation of amino acid sequences found in all PCNA sequences is
illustrated by the DNA binding domain (aa 61-64 underlined) consisting of a
basic helix-loop-helix sequence (-RCDR-). Motifs involved in polymerase-5
and p21 putative binding (D-SHV-) as well as the C-terminal sequence (F/YLAP-, double underline) essential for proper folding are also found in the
apicomplexan PCNAs. Note that PCNAs from each class are more similar
than each pair of PCNAs from a single Apicomplexan. Black squares
represent identical amino acids and gray represents conserved residues.
29
TgPC N A l
B E
TgPCNA2
B E
12 Kr.
<:
Kb
I Kb
Figure 2-2. Southern analysis of T. gondii RH strain genomic DNA hybridized with
TgPCNAl- and 2-specific probes. The hybridization pattern indicates the presence of
two unique PCNA genes. DNA was digested with either Bgl II (lanes I and 3) or Eco Rl
(lanes 2 and 4). Lanes I and 2 were hybridized with the TgPCNAl specific probe and
lanes 3 and 4 were hybridized with the TgPCNA2 probe. Molecular mass markers (kb)
are indicated to the left of the figure.
Phylogenetic analysis of Apicomplexan PCNA sequences
Distinct lineages within the eukaryotic PCNA family of proteins emerge on the
basis of taxonomic assignment at the phylum level (Fig. 2-3). The grouping of humans
and mice represents the phylum chordata while Drospohila and Sarcophaga represent the
arthropoda. Zea and Nicotiana represent streptophyta and the two yeast organisms from
the phylum ascomycota reside independent of any other group.
Toxoplasma,
Plasmodium and Cryptosporidium PCNAs group together representing the Apicomplexa.
Although together, the apicomplexan sequences maintain an independent segregation
between PCNA I and 2 (Fig. 2-3).
30
TgPCNAZ
PfPCNAZ
CpPCNAl
PfPCNAl
CpPCNAZ
TgPCNAl
S .c e r e v i s i a e
S .p o m b e
.m a y s
M.musculus
N .t a b a c v m
H. s a p i e n s
S .c r a s s i p a l i s
D .m e l a n o g a s t e r
Figure 2-3. Consensus tree of Apicomplexa and representative eukaryotic PCNAs.
Numbers along the branches represent the frequency of that group occurring in 100
bootstrap replicates. Lineages within the eukaryotic PCNA family of proteins emerge on
the basis of taxonomic assignment. All six apicomplexan PCNAs group together, but the
PCNAl and 2 sequences segregate at the interspecies level.
31
TgPCNAl and 2 mRNA expression
Previous molecular characterization of two nucleoside triphosphate hydrolase
genes in T. gondii revealed the presence of two isoforms whose expression was strain
specific [29].
Because TgPCNA cDNAs were identified from a single isolate (RH
strain), we examined the expression of PCNA mRNA in tachyzoite isolates representative
of the three genotypic classes (RH, ME49-PLK, and VEG). In Figure 2-4, duplicate
Northern blots were hybridized with probes specific for each TgPCNA gene. TgPCNA I
and 2 mRNAs were expressed in all three tachyzoite strains (Fig. 2-4 inset A). The probe
specific for TgPCNAl hybridized to multiple mRNA species. In all three strains, the
largest species was ~3.6 kb, based on a comparison with 28S rRNA (Fig. 2-4 inset, upper
arrow), while a second species (-2.2 kb) migrated a little slower than the I SS-Iike rRNA
(Fig. 2-4 inset, lower arrow) [30-31], A third mRNA species appeared in the Type-Ill
(VEG) strain and was -1.9 kb.
The second TgPCNAl mRNA species was greatly
diminished in the Type-II strain (ME49-PLK) examined here. The basis for multiple
PCNAl mRNA species was not investigated. A single mRNA species of -2.8 kb was
detected by the TgPCNA2 probe (Fig. 2-4 inset A). Following normalization for equal
RNA loading with a T. gcWii-speciflc 18S rRNA probe, the combined expression of all
forms of TgPCNAl was found to be four to seven fold higher in all three tachyzoite
strains, when compared to the levels of TgPCNA2 mRNA (Fig. 2-4 graph).
32
90
I
II
TgPCNAl
III
I
Ii
in
TgPCNAZ
Figure 2-4. Relative levels of TgPCNAl and 2 mRNAs from three different T. gondii
tachyzoite strains. RH-Type I (I), ME49-PLK-Type II (II) and VEG-Type III (III) were
quantified following normalization of RNA loading with a T. gondii 18S rRNA
oligonucleotide probe. Inset A displays the autoradiographic results of hybridization with
the TgPCNA probes. Two mRNA species are present for TgPCNAl in Type I and II
strains and three species are present in the Type III strain (inset blot A). TgPCNA2 (inset
blot A) probe detected one mRNA species at a four to seven fold lower level of
expression. The upper arrow indicates the location of the large subunit rRNA species
(~28S) and the 18S rRNA is indicated by the lower arrow. Inset B displays the
hybridization signal from reprobing each blot with a [32P]-end labeled T. goWh-specific
18S rRNA oligonucleotide probe.
TgPCNAl and 2 protein expression
Recombinant TgPCNAl and 2 were purified to homogeneity from E. coli
extracts on nickel columns and evaluated by SDS-PAGE. Due to the addition of Nterminal histidine tags, TgPCNAl and TgPCNA2 migrated at 45 kDa and 42 kDa,
respectively (Fig. 2-5, A). Purified proteins were used to generate anti-PCNAl- and 2specific mouse polyclonal antibodies. Immune serum prepared from animals immunized
33
with recombinant TgPCNAl reacted with TgPCNAl but failed to recognize recombinant
TgPCNA2 (Fig. 2-5, B- Lanes I and 2). Similarly, serum from TgPCNA2 immunized
mice reacted only with TgPCNA2 recombinant protein (Fig. 2-5, B- Lanes 3 and 4). A
two fold serial dilution of TgPCNAl (Fig. 2-5, C) and TgPCNA2 (Fig. 2-5, D)
recombinant protein demonstrated that each TgPCNA specific antisera has similar
binding/affinity characteristics at the selected titer (1:2500). Preimmune mouse serum
showed no reactivity against either recombinant protein (data not shown).
A
B
1 2
1 2
3
—
__
4
97.4 66
—
31 —
21.5 -
1
2
3
4
5
Figure 2-5. Cross reactivity of polyclonal antiserum to TgPCNAl and 2 recombinant
proteins. (A) Coomassie brilliant blue staining of affinity-purified (native conditions)
recombinant TgPCNAl and 2 (1.5 pg of each protein) separated on a 12% reducing SDSPAGE gel. (B) Western blot analysis of polyclonal antisera produced against each
TgPCNA recombinant protein. Lanes I, 4 contain 30 ng of purified recombinant
TgPCNAl; Lanes 2, 3 contain 30 ng of recombinant TgPCNA2. Lanes I and 2 were
reacted with anti-TgPCNAl (1:2,500) polyclonal serum and lanes 3 and 4 with antiTgPCNA2 polyclonal serum (1:2,500). Molecular mass standards are indicated in kDa
on the left. (C & D) Two fold serial dilution of TgPCNAl (C) and TgPCNA2 (D)
recombinant protein from 120 ng (lane I) to 7.5 ng (lane 5) probed with the respective
polyclonal antisera at identical titers (1:2500).
34
Anti-TgPCNAl and 2 polyclonal antisera reacted with distinct antigens on
Western blots of T. gondii tachyzoites from three genotypic strains. ■In contrast to the
difference in mRNA levels, polyclonal antisera reactivity appeared similar in all extracts
suggesting the levels of each TgPCNA protein are equivalent.
Due to potential
differences in antisera binding to native antigen, the absolute levels of TgPCNA proteins
remain to be confirmed. In all three strains, anti-TgPCNAl serum recognized an antigen
of ~40 IcDa (Fig. 2-6, lanes 1-3) corresponding to native TgPCNAl without the 6xHistag. Likewise, anti-TgPCNA2 recognized a native PCNA2 that was 2-3 kDa smaller
(-37 kDa) but expressed equally in the strains tested (Fig. 2-6, lanes 5-7). RH extracts
were probed with a combination of anti-PCNA I and 2 sera (Fig. 2-6, lane 4) to verify the
TgPCNAl and 2 size differences. The slower migration of native TgPCNAs than what is
predicted by cDNA coding size is likely due to anomalous electrophoresis that has been
noted for other eukaryotic PCNAs [32].
The minor bands that are seen below the
TgPCNA antigens (Fig. 2-6 non-specific) in all the lanes were detected in secondary
antibody controls (data not shown).
35
1 2 3 4 5 6 7
97.4 66 —
45
non-specific
31
21.5
Figure 2-6. Western blot analysis of T. gondii tachyzoite lysates. Three genotypic
lineages (RH, lanes I, 4, 5; ME49-PLK, lanes 2, 6; VEG, lanes 3, 7) were
electrophoresed under reducing conditions (12% SDS-PAGE) and incubated with
TgPCNA specific antisera (1:2500). Lanes 1-3 were reacted with anti-TgPCNAl serum
and lanes 5-7 were reacted with anti-TgPCNA2. Lane 4 was incubated with an equal
mixture of anti-TgPCN A I and anti-TgPCNA2 antisera. Molecular mass standards are
indicated in kDa.
Discussion
To facilitate studies of DNA replication in Toxoplasma, we have identified and
characterized the expression of two PCNA genes in tachyzoites. Comparison of the
putative TgPCNAl and 2 coding sequences with PCNA from humans (Fig. 2-1) and
other eukaryotes reveals the presence of conserved regions that include DNA binding
motifs and replication-associated protein binding domains [8-9,28]. The C-termini of the
TgPCNAs and PfPCNAs contain unique amino acids that extend the domain length
36
beyond that observed for human PCNA. This is significant because it has been suggested
that the C-terminal region plays a key role in determining the specificity of PCNA
interactions with other proteins associated with DNA replication, such as replication
factor C and DNA polymerase 8 [10].
Eukaryotic PCNAs are thought to form active clamp structures as homotrimers
with a six-fold axis of symmetry as the result of having two globular domains per
monomer [8],
Exceptions to this model may occur in the case of rare C-terminal
extended domains as found in carrot and Xenopus [33-34] where these larger PCNAs
may form a dimeric ring structure similar to the prokaryotic p-subunit clamp protein [12].
The presence of these alternate forms of PCNA is not well understood, however, they
appear to be expressed in circumstances where cell division is unusually rapid [8], The
coding sequence of TgPCNAl is predicted to encode a protein that contains a longer Cterminal domain than TgPCNAl as well as human PCNA, and the relative size difference
was confirmed by Western blot analysis (Fig. 2-6). Thus, TgPCNAl may fit within the
group of larger eukaryotic PCNAs, and therefore capable of forming different tertiary
structures.
The situation in Apicomplexa where two distinct forms of PCNA are
simultaneously expressed in a single species provides a unique opportunity to explore the
functional consequence of alternate PCNA structures.
Identification of two distinct PCNA genes within one species is unusual and to
date has only rarely been observed in eukaryotes (with over 40 different species
examined so far). In the eukaryotic family of PCNAs, two cDNAs have been identified
only in carrots [33]. Examination of PCNA-Iike sequences in prokaryotic and viral
genomes have revealed several duplications. Paramecium bursaria Chlorella virus I
37
contains two putative PCNA-Iike sequences [35-36] and recent reports have identified
two distinct (3-subunit proteins (PCNA-Iike) in the Archea, Sulfolobus solfataricus [37].
Within T. gondii, the amino acid identity of TgPCNAl and 2 is only -27%,
representing a higher degree of divergence than would be predicted by a recent
duplication event. TgPCNAl is in fact more similar to PfPCNAl and CpPCNA I (49 and
42% identity respectively) and likewise TgPCNA2 appears more related to PfPCNA2 and
CpPCNA2. Thus, if these forms of PCNA arose from a single gene, the duplication
likely occurred in the most common ancestor of Toxoplasma, Plasmodium and
Cryptosporidium [38-39],
Mechanisms for duplicate gene acquisition are highly
speculative, but one model involves horizontal gene transfer, and a recently published
report suggests this process has been extensive among prokaryotes [40]. This is an
intriguing hypothesis given the identification of two PCNA-Iike sequences in the genome
of a virus that infects P. bursaria [35-36], a prokaryotic organism that may have
similarities to the apicomplexan ancestor [38-39].
Given that the acquisition of two PCNAs in apicomplexan parasites is quite old,
the question must be raised as to what has preserved their existence in the genome over
time.
Current models for the maintenance of a duplicated gene suggest a process
whereby the duplicate gene mutates to acquire a new function (neofunctionalization) or
both genes undergo rapid sequence divergence to a point where only partial function is
retained (subfunctionalization) [41]. In the case of subfunctionalization, both genes are
necessary to fulfill the function of the ancestral gene.
Functional dissection of the
TgPCNAs awaits further studies; however, preliminary cellular localization of ectopically
expressed TgPCNAl and 2 indicates each protein targets to the parasite nucleus (data not
38
shown) allowing for potential interaction. Analysis of TgPCNAl and 2 mRNA levels
demonstrate that in the tachyzoite stage both PCNA genes are actively transcribed (Fig.
2-4). Moreover, the expression pattern in all three Toxoplasma lineages demonstrate that
PCNA expression is not genotypically restricted as has been found for the NTPase
isoforms [29]. The difference in the number of mRNA species and comparative mRNA
levels suggests each TgPCNA gene is independently controlled and alternative
polyadenylation signals might be present in TgPCNAl. The experimental data presented
here suggests that post-transcriptional control might be at work given the nearly equal
levels of protein in spite of a 5-7 fold difference in mRNA levels.
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42
CHAPTER 3
CHANGE IN THE PREMITOTIC PERIOD OF THE CELL CYCLE IS ASSOCIATED
WITH BRADYZOITE DIFFERENTIATION IN TOXOPLASMA GONDII.
Introduction
In the United States, it is estimated that 1.5 million individuals are exposed
annually to Toxoplasma gondii such that by age 50, there is a >35% chance of having
contact with this pathogen [I]. While most of these infections are tolerated. Toxoplasma
is dangerous to the unborn, to patients undergoing chemotherapy or organ transplant, and
to people with AIDS [2-5]. As a result, toxoplasmosis is the third leading cause of
foodborne deaths in the U.S. [I]. The unique ability of bradyzoites and tachyzoites to
interconvert is considered to be the underlying cause of Toxoptoma-encephalitis in
AIDS patients [4,6]. The bradyzoite form is slow growing, immunologically inert and
not readily eliminated by the host immune system, and for this reason, in immunecompromised hosts, recurrent toxoplasmosis can occur each time latent bradyzoites
recrudesce and proliferate as tachyzoites. Understanding the mechanism(s) that regulates
development in this pathogen so that tachyzoite-to-bradyzoite interconversion may be
prevented is of critical importance.
Several lines of evidence suggest that a developmental timer may operate in
tachyzoite-to-bradyzoite switching, although the molecular details are not understood [7],
The differentiation of tachyzoites into bradyzoites occurs in conjunction with a change in
43
the cell cycle that happens spontaneously during emergence from a sporozoite infection
[7] or is induced when tachyzoites are stressed [8-10].
Tachyzoites emergent from
sporozoite-infections differentiate within a relatively tight window of proliferation,
suggesting that the bradyzoite switch is linlced to a division counting mechanism.
Differentiation does not occur if tachyzoite growth is blocked [11], and thus, cell cycle
progression is a prerequisite for development.
However, it is unknown whether the
switch to differentiate is restricted to a specific cell cycle phase.
Tachyzoite replication is characterized by a three-phase cell cycle comprised of
Gl and S phases with mitosis following immediately upon the conclusion of DNA
replication [11-12]. Parasite budding is evident with the formation of daughter apical/
inner membrane complexes which are well developed prior to nuclear division, and thus,
budding may in fact initiate in S phase [11]. The S phase distribution of asynchronously
growing tachyzoites is bimodal suggesting that chromosome replication may be
nonuniform [11]. There is no evidence to support an extended G2 period in dividing
tachyzoites, which is consistent with observations of other apicomplexans undergoing
schizogony [13-17].
In this chapter, we demonstrate that bradyzoite recrudesence
follows a developmental pathway, which is similar to development initiated by
sporozoites. Furthermore, we describe a G2 population that arises during tachyzoite-tobradyzoite differentiation, which is not detected in bradyzoite populations from mature
tissue cysts.
44
Materials and Methods
Cell culture and parasite growth.
Human foreskin fibroblasts (HFF) were grown in Dulbecco’s Modified Eagle
Medium (Gibco BRE, Grand Island, NY) supplemented with 10% (v/v) fetal bovine
serum (Atlanta Biologicals, Atlanta, GA). Sporulated oocysts from a Type III strain
(VEG) were produced as previously described in Jerome et al. [7] and served as the
source of inocula for sporozoite-initiated cultures. Parasites subjected to alkaline media
(pH 8.3) were grown as described in Bohne et al. [18].
To produce tissue-cyst
bradyzoites, CD-I mice (Charles River Laboratories, Wilmington, MA) were inoculated
(s.q.) with 3000 oocysts (VEG strain).
Forty days post-inoculation the brains were
removed from the cranial cavity and placed in phosphate buffered saline (PBS) at room
temperature for two hours. Brain tissue was homogenized in 7.5 mis of PBS using a
hand-held tissue grinder. Following homogenization, Percoll (Sigma, St. Louis, MO)
was added to a final concentration of 24% (v/v) and the solution was centrifuged at
1875xg for 30 minutes. The cysts sedimented in the lower 1/3 of the gradient and were
recovered by centrifugation at I OOOxg for 10 minutes. The cysts were resuspended in 6
mis of PBS and filtered through a large pore screen followed by filtration through Nitex
membrane. The cysts were washed from the top of the Nitex using 2-3 mis of PBS then
pelleted at I OOOxg for 10 minutes and resuspended in 0.5 ml of PBS. The bradyzoites
were excysted for 20 seconds in excystation fluid (0.01 gm Pepsin, 0.2 gm NaCl and
0.0015% concentrated HC1). The excystation was stopped with the addition of DMEM
45
containing 10% FBS, and the bradyzoites were recovered by centrifugation at ISOOxg for
15 min then resuspended in PB S.
Immunofluorescence analysis:
HFFs grown in 8-well chamber slides were inoculated with IO5 parasites. At
various intervals (2h, I Bh, 24h, Day3 and Day 17) following parasite inoculation,
parasites were washed in IX phosphate buffered saline (PBS), fixed with 3.7% formyl
saline and permeabilized with 0.25% Triton X-100. Rabbit polyclonal antiserum [19]
specific for either TgPCNAl (1:5,000) or TgPCNA2 (1:500) was added to the wells and
incubated for 30 minutes. The wells were washed, fluorescein (FITC)-conjugated anti­
rabbit immunoglobulin G diluted 1:100 (Sigma, St. Louis, Mo.) was added. The
bradyzoite-specific monoclonal antibody BAG-1 (1:500) [20] recognizes a cytoplasmic
antigen and was used to quantify the fraction of bradyzoites in each population. The
mouse anti-BAGl antibody was detected with phycoerethrin (PE)-conjugated anti-mouse
IgG (Sigma, St. Louis, Mo.) diluted 1:64.
Surface antigen I (SAG-1) [21] expression in recrudescing parasites was analyzed
by immunofluorescence analysis as previously described in Speer et al. [22], Briefly,
parasites were washed twice in IX phosphate buffered saline (PBS), fixed with 3%
paraformaldehyde for 30 min, treated in acetone for 10 min at 4°C, and air dried. The
wells were treated with DG52 (a-SAGl, provided by Dr. J. Boothroyd) at 1:1,000 for I
hour then washed with PBS and treated with fluorescein. (FITC)-conjugated anti-rabbit
immunoglobulin G diluted 1:100 (Jackson Immunoresearch, West Grove, PA). In all
46
experiments, the slides were evaluated with an epifluorescence microscope (Eclipse
TE300, Nikon Inc., Melville NY), and images were collected with a digital camera
(SPOT™, Dynamic Instruments Inc., Sterling Heights MI).
Fluorescent-Activated-Cell-Sorting (FACS).
Parasites were harvested for DNA content analysis by scraping monolayers,
needle passing, filtering and pelleting (1300 x g). After harvest, parasites were fixed in
ethanol and stored at -20°C for 24h prior to analysis [12].
Fixed parasites were
resuspended in PBS and treated with 10 pg/ml propidium iodide (PI) (Sigma, St. Louis,
MO) and 7.Spl of RNase cocktail (Ambion, Austin, TX) for 30 minutes in the dark. In
order to examine the surface expression of SAGl during bradyzoite development, ethanol
fixed (>24 hr), parasites were centrifuged (1300 x g), re-suspended in 200 pi of antiSAGl antibody at 1:1000, and incubated on ice for 30 minutes. The parasites were
washed (PBS), centrifuged as above, and treated with 200 pi of goat anti-mouse
secondary IgG (FITC) at 1:250 (Jackson). Parasites were washed and resuspended in
PBS for analysis. Two-color analysis of SAGl expression versus DNA content was
performed by staining the DNA following antibody treatment with 10 pg/ml PI and 7.Spl
of RNase cocktail. Parasites were analyzed on a FACSCalibur flow cytometer (BectonDickinson Inc., San Jose CA). A minimum of 8,500 parasites was collected for analysis
(collection rate of ~500-800cells/sec). Antibody fluorescence was collected on a log scale
(FE-1 channel), while propidium iodide fluorescence was collected on a linear scale (FL2).
47
Results
Expression of TgPCNA I and 2 during sporozoite-initiated development.
Parasite development in the intermediate host proceeds via the non-replicative
sporozoite that differentiates into the rapidly growing tachyzoite ultimately giving rise to
bradyzoites in the tissue cyst. At critical transitions, changes in growth rate signal the
appearance of stage specific antigens, indicating that cell cycle mechanisms are somehow
linlced to the developmental program [7,20]. We have recently defined the major features
of the tachyzoite cell cycle [11-12] in part through the use of immunfluorescent markers
that are diagnostic of cell cycle phases. TgPCNA staining patterns are one set of markers
used to detect parasites engaged in DNA synthesis—TgPCNAl stipling and TgPCNA2
shuttling into the nucleus occurs in S phase parasites [11,19]. We have explored the
changes in TgPCNAs during sporozoite-to-tachyzoite-to-bradyzoite development.
TgPCNAl was detected in the nucleus of all three developmental stages (Fig. 3-1 A-E)
with the stipling characteristic of S phase occurring only in dividing tachyzoites (data not
shown). Expression of TgPCNA2 was not evident in the newly excysted sporozoite (data
not shown), but was detectable in parasites 18 h post-sporozoite inoculation (pre­
replication) (Fig. 3-1 B).
In emergent dividing tachyzoites, TgPCNA2 expression
displayed the characteristic asynchronous pattern (-60% nuclear concentrated; 40%
cytoplasmic) as reported (nuclear concentrated. Fig. 3-1 C) [19]. TgPCNA2 was detected
in parasites at day 17 (Fig. 3-1 E) but similar to sporozoites (Fig. 3-1 B), parasites that
co-expressed BAGl at this stage of development no longer had TgPCNA2 concentrated
48
in the nucleus suggesting they were growth-arrested. This was corroborated by the low
frequency of mitotic forms (double nuclei or internal daughters; [11,23]) in the BAGl+
population (data not shown). In contrast, TgPCNAl was concentrated in the nucleus
regardless of the growth state.
Analysis of DNA content changes during sporozoite-initiated development.
Previously, we determined by FACS that VEG sporozoites possess a haploid
genome, which is consistent with this cell cycle assignment; however, microfluorimetric
measurements of T. gondii cystozoites were reported to have heterogenous genome
contents indicating a more active cell cycle distribution [13]. We determined the DNA
content by FACS of relatively pure populations of VEG strain sporozoites (from oocysts)
(Fig. 3-2 A), emergent tachyzoites (day 3-post-sporozoite inoculation) (Fig. 3-2 B), and
in bradyzoites from murine brain cysts (40 day post-oocyst inoculation) (Fig. 3-2 C). At
least 10,000 events were collected for each population and the PI fluorescence of
sporozoites was used as a standard for IN DNA content upon which the other populations
were compared. It is clear from these results that tissue cyst bradyzoites (Fig. 3-2 C)
were more uniformly IN than sporozoites, whereas, populations of emergent VEG
tachyzoites displayed the characteristic fluorescence distribution of asynchronous
replicating parasites [11-12].
49
I T O i 1" " " I i r 1",iiijr'r: R
, iI'!1!, ; ; l n i '|
1 6 HOUR
S p o r o z o ite
2 HCUR
S p o r o z o ite
M
DAY 3
T a e H y s o ite
ilI; ,
i
........-
n
n
2 4 HOUR
T a c h y z o ite
-
—
DAY 17
B ra d y s o ifc e
Figure 3-1. Developmental expression of TgPCNAs. (A) Sporozoites 2 h post­
infection; (top panel) anti-TgPCNAl antiserum, (bottom panel) overlay of antiTgPCNAl and DAPI counterstain. (B) Sporozoites 18 h post-infection; (top) antiTgPCNAl, (bottom) anti-TgPCNA2. Homogenous, whole parasite staining of
TgPCNAZ 18 h post-infection is representative of the staining (2-24 h) prior to
tachyzoite proliferation. (C) Newly emergent tachyzoites 24 h post-sporozoite
infection, (top) anti-TgPCNAl, (bottom) anti-TgPCNA2. (D) Day 3 post-sporozoite
infection, (top) anti-TgPCNAl, (bottom) anti-TgPCNA2. Nuclear recruitment of
TgPCNAZ is evident in all proliferating populations Day-1-6 post-infection. (E) Day
17 parasites; (top panels) anti-TgPCNA 1/anti-BAG I (Ej), (bottom) antiTgPCNAZ/anti-BAG I (Ei). Following the shift to slower growth there is no nuclear
recruitment of TgPCNAZ.
50
Figure 3-2. Flow cytometric analysis of sporozoite, tachyzoite and bradyzoite DNA
content. Parasites were fixed in ethanol and stained with propidium iodide (lOpg/ml) for
30 minutes. The histogram in each panel is the result of -8500 events collected in the
FL2-FI channel. Arrows indicate IN (peak mode FL2=400) and 2N (FL2=800) DNA
contents. Sporozoites (A) and bradyzoites (C) are >95% IN. Asynchronously growing
tachyzoites (B) show the characteristic bimodal distribution [11].
In order to follow cell cycle changes of dividing tachyzoites as they differentiated
into bradyzoites, we measured the DNA content by FACS and monitored a variety of cell
cycle markers and the bradyzoite marker, BAG I, by immunofluorescence [11,20] (Table
3-1). In this series of experiments, VEG tachyzoites emergent from sporozoite-infections
were allowed to differentiate in HFFs spontaneously or were subjected to alkaline stress,
which triggers a more synchronous and robust differentiation [7],
The cell cycle
distributions of Day-3 tachyzoites (Table 3-1) served as a starting reference for these
experiements [I I]. Parasites at Day-9 post-sporozoite inoculation are growth-shifted [7],
which was confirmed here by a higher fraction of parasites with IN DNA complement
(-71%) and a decrease in the proportion of S-phase parasites (Table 3-1). A shift in the
fluorescence of the near-diploid subpopulation from an FL2-H mean of 735 (Day-3,
parasite average value) to 759 was also noted.
Evaluation of parallel cultures by
51
immunofluorescence assay (IFA), correlated well with the FACS results. Based on antiTgPCNAl staining, we determined that only 22% of the parasites contained the
characteristic stipling pattern of S-phase [11], and the fraction of mitotic forms had also
decreased (Table 3-1).
At Day-9 post-sporozoite inoculation, we observed the first
evidence that the proportion of 1.8-2N parasites was not correlated with the fraction of
parasites in mitosis and cytokinesis.
This was in contrast to synchronized RHTK+
parasites (RHAZzxg^ri parasites expressing an active thymidine kinase) [12] where
changes in the fraction of 1.8-2N parasites occured in parallel with the proportion of
parasites that were engaged in mitosis and cytokinesis [11-12],
As differentiation
progressed in the VEG cultures, the fraction of 1.8-2N parasites increased such that by
Day-15 post-sporozoite inoculation the population was comprised of 84% IN and 15%
1.8-2N parasites with relatively small fractions containing characteristic S-phase (>1%),
TgPCNAl stippling or mitotic/cytokinetic forms (Table 3-1).
The two predominate
populations at Day-15 had average FL2-H values of 402 and 772, indicating they were
much closer to the expected values for IN and 2N contents, respectively. Similar results
were obtained with sporozoite-infected cultures exposed to alkaline stress (96 h timepoint shown here in Table 3-1). Nearly all of the parasites treated in pH 8.3 medium for
96 h expressed BAGl (-99%), and the population could be divided into two major
fractions based on DNA content; -76% of the parasites were IN (Table 3-1) and 21%
were near-diploid (760 FL2-H) with relatively few parasites in S-phase or
mito sis/cytokinesis.
52
Dav
0
3
7
9
15
Gl
95
61
64
71
84
S phase
5
30
29
22
>1
G2
0
0
0
5
15
M/C
0
9
8
2
>1
pH shift
76
3
21
>1
%BAG1
0
0
2
12
51
99
Table 3-1. Cell cycle and developmental distributions during sporozoite-to-bradyzoite
differentiation. Parallel cultures were analyzed by flow cytometry (propidium iodide
stain) and immunofluorescent microscopy to provide estimates of the proportion of
parasites in G l, S, G2 and mitosis/cytokinesis (M/C) and to detect parasites that had
begun to differentiate (BAGl+). The proportion of S phase parasites was estimated by
flow cytometric analysis and confirmed by counting nuclei that showed stipled antiTgPCNAl staining [I I]. G2 parasites are defined here as the fraction of 1.8-2N parasites
that are not accounted for by S-phase nuclei or parasites in mitosis/cytokinesis (double or
u-shaped nuclei, daughter parasites). Gl was estimated from the proportion of IN
parasites. Sporozoite infected cultures were shifted pH 8.3 media at 72 hr post­
inoculation and cell cycle distributions were determined 96 hr later.
Parasites expressing both SAGl and BAGl markers comprise most of the G2 fraction.
During tachyzoite-to-bradyzoite development a number of stage markers change
expression in opposite directions; e.g. SAGl decreases while BAGl increases. Taking
advantage of these markers, we employed a two color FACS analysis (Fig. 3-3) to
evaluate 96 h alkaline-treated parasites for DNA content versus SAGl expression.
Identical cultures, prepared with the same sporozoite inoculum and alkaline medium were
also analyzed for BAGl expression. We found that virtually all the parasites in these
cultures had begun to differentiate by 96 h (99% were BAGl+ by IFA, data not shown).
By FACS, 96 h alkaline-treated populations were heterogenous for the SAGl marker,
with 22.7% of the population at or below the background fluorescence cut-off (FL1 <15)
53
(Fig. 3-3 A, top). In contrast, undifferentiated tachyzoite populations were relatively
homogenous for SAGl expression as expected (Fig. 3-3 A, bottom). The DNA content
of subpopulations, based on their SAGl expression (Fig. 3-3 A, top) (bright, FL1>139 vs
negative, F L K l5), demonstrated that the SAG!" parasites were primarily haploid (IN)
(Fig. 3-3 B top) whereas, SAGl+ parasites were comprised of nearly equal fractions of
IN (55%) and 2N (45%) (Fig. 3-3 B, bottom).
Thus, the SAGl+ subpopulation
comprised most (85%) of the parasites that possesed a diploid genome in the
differentiating population.
Bradyzoite de-differentiation is kineticallv similar to development initiated bv
sporozoites.
Because mature sporozoites and bradyzoites appear to share an identical cell cycle
pattern, we examined whether the bradyzoite-infection of HFF cells would follow a
developmental course that was different or similar to that initiated by the sporozoite.
Bradyzoites were purified from brain cysts of animals that had been infected with VEG
oocysts 40 days prior to cyst harvest and inoculated into HFF monolayers. SAGl and
BAGl expression and population growth-rate were followed in these cultures for a period
of two weeks (Fig. 3-4). Bradyzoites purified from tissue cysts were SAG!" but re­
expressed this marker by 18 h post-inoculation and prior to the onset of parasite
replication in these cultures (Fig. 3-4 A).
By 24 h, SAGl+-parasites had begun to
replicate and by 80 h a relatively fast growing tachyzoite population had emerged (Fig. 34). BAGl expression steadily declined in these cultures and was undetectable by day 5.
54
Between Day 7 and 8 post-bradyzoite inoculation, the population spontaneously shifted
to a slower growth rate (Fig. 3-4 B), and evidence of BAGl expression was observed
(Fig. 3-4 C). BAGl expression increased in these populations such that by Day-15 the
populations were again 75% positive for this antigen (Fig. 3-4 C).
A________
i Bradvzoitc
SAGl -ncg ~25%
Tachyzoite
SAGl -ncg <5%
Fluorescence
Figure 3-3. Two color flow cytometric analysis of differentiating parasite populations.
Day-3 post-sporozoite VEG tachyzoites were shifted into pH 8.3 media for 96 hr and
stained for the tachyzoite-specific marker SAGl (mAB-DG52 [21]) (Panel A top) and
DNA content with propidium iodide (panel B, top and bottom). Parallel cultures
analyzed by immunofluorescence for the bradyzoite-specific marker BAGl [20] showed
that 99% of the parasites in these populations were BAGl+. Undifferentiated tachyzoites
stained for SAGl and analyzed by FACS are included here as a reference (panel A,
bottom). Gated subpopulations in the differentiated cultures (panel A, top) were analyzed
for DNA content (panel B). SAG I-negative parasites (FEI <15) were 90% IN (panel B,
top), whereas SAGl-positive (FL1 >139) were divided nearly equally into IN and 2N
distributions (panel B, bottom). Note that the SAGl+, 2N parasites represent 85% on the
total parasites that are diploid in this differentiated population. The DNA content of dull
versus bright SAGl parasites in the population of undifferentiated (A, bottom)
tachyzoites were also analyzed and found to be identical (data not shown).
55
Figure 3-4. Analysis of the growth rate and BAGl expression during recrudescence. (A)
VEG strain bradyzoites purified from brain cysts were seeded into HFF monolayers and
analyzed for BAGl [20] (black bars) or SAGl [21] (hatched bars) expression during the
first 5 days post-inoculation. Note that SAGl expression was first detected I Bh post­
inoculation and prior to the onset of parasite replication in these cultures. (B) Average
vacuole size decreases as emergent tachyzoites shift to a slower growth phenotype day-6
post-bradyzoite infection. (C) The number of BAGl+ parasites increases following the
growth shift. D = Day, h = hours.
56
Discussion
The precise mechanisms that control apicomplexan development have not been
uncovered, but like other protozoa, the results shown here are consistent with a linlc
between chromosome replication and the parasite developmental pathway (Fig. 3-5
presents a cell cycle model for the T. gondii intermediate-host developmental pathway).
VEG sporozoites (Day-O) are >90% haploid and do not begin to proliferate until ~20
hours post-inoculation, at which time their conversion into the tachyzoite is nearly
complete [11-12], At Day-3 (post-sporozoite), VEG tachyzoites replicate rapidly and
display a DNA profile of ~60% IN, -30% 1N-2N, and -10% 2N. The fraction of 2N
parasites in Day 3-parasites corresponds closely to the number of parasites in mitosis and
cytokinesis. A G2 population is undetectable at this time; however, as parasites reach
growth restriction at Day-9, a new population (G2) emerges which contains a diploid
DNA content and is not engaged in mitosis/cytokinesis. By Day-15 post-sporozoite
infection, G2 parasites are -15% of the population, while the other 85% contains a
haploid DNA content.
Alkaline shifted VEG parasites follow a similar, if abridged
developmental course (Table 3-1). Here, 99% of the population express BAGl+ four
days post-sporozoite inoculation, indicating that the entire population has begun to
differentiate, with some parasites intermediate (BAGl+ZSAGl+) and others further along
the pathway (BAG I+ZSAGV). Importantly, analysis of these parasites based on their
SAGl expression and DNA content (Fig. 3-3) demonstrates that the G2 population is
predominately double positive (BAGI+ZSAGl+). Parasites from tissue cysts are SAG!"
57
and uniformly haploid, and thus, the G2 fraction in differentiating populations is likely an
intermediate stage in the developmental pathway.
Bradyzoite
Sporozoite
G 0(
Definitive cycle
Figure 3-5. Model of Toxoplasma gondii development in the intermediate host.
The observation of VEG tachyzoite-to-bradyzoite develoment is consistent with a
process whereby a change in the chromosome cycle is manifest by a late-S/G2 slowing or
pause.
Parasites emerging from this pause are committed to differentiate into
bradyzoites. This process is likely stochastic, as suggested for differentiation of Theileria
schizonts [24]. Thus, the probability of differentation is dependent on a change in the
steady-state level of a factor(s) above (or below) a threshold margin.
Consequently,
differentiation is not synchronous as one or more cycles may be required to achieve the
level needed to commit to differentiation. Laboratory-adapted strains, such as RH, have a
fast cell cycle with a very short or non-existent G2, and therefore, a lowered potential to
differentiate. Differentation does not occur if growth is blocked [11] because cell cycle
58
progression is required. Mechanisms that control protozoan chromosome replication are
commonly intertwined with those that regulate cellular development. For example, it is
well established that the differentiation of Dictyostelium and Acanthamoeba brought on
by starvation conditions is triggered specifically when these protozoa occupy Iate-S
phase/G2, suggesting DNA replication is likely a prerequisite for the change in
developmental program [25-26].
Differentiation of Trypanosoma brucei in the
bloodstream into pro cyclic forms [27], Plasmodium merozoite into gametocytes [28],
Theileria annulate schizonts into merozoites [24], Leishmania amastigotes into
promastigotes [29], and importantly, T gondii tachyzoites into bradyzoites [8-10] are all
induced to varying degrees by DNA synthesis inhibitors.
In each of these models,
changes in the chromosome cycle [30] (typically a slowing of DNA synthesis) appear
crucial for development. It is thought that remodeling of nucleoprotein complexes may
result when replication forks pass, and via this mechanism, changes in transcriptional
patterns are connected to DNA replication [31]. Such a model has been proposed to
account for the complex changes in the developmental program that occur during
metazoan development [32] and could explain the linlc between DNA replication and
protozoan development.
A prediction of the above model is that any condition that slows DNA synthesis
appropriately has the potential to alter late-S/G2 and induce bradyzoite differentiation.
This would explain why a variety of drugs with different modes of action induce
differentiation in T gondii.
The model does not explain why sporozoite-derived
tachyzoites spontaneously growth shift after ~20 divisions.
Reconciliation of this
question might be found when comparing early VEG tachyzoite division with higher
59
eukaryotic embryonic development. The events that follow fertilization are rapid nuclear
division events controlled by a maternal cyclin [33-35], The degradation of maternal
cyclin regulates the timing and number of divisions in embryonic systems and dominates
over the internal cycle of each cell. By analogy, factors carried in by the sporozoite or
synthesized during sporozoite differentiation into the tachyzoite could similarly control
the limited expansion of early tachyzoites revealing the underlying bradyzoite pathway
only when their levels drop below a threshold. We have now demonstrated that dedifferentation of bradyzoites back into tachyzoites (recrudescence) is kinetically
indistinguishable from the sporozoite-induced developmental pathway (Fig. 3-4).
Bradyzoites from in vivo tissue cysts differentiate into tachyzoites (as quantified by
SAGl+ expression) in the same time-frame as sporozoites (12-18 h post-invasion) and
only parasites that express tachyzoite markers proliferate. Like emergent tachyzoites
from sporozoite-initiated infections, those emergent from bradyzoite inoculations grow
relatively fast for a limited number of divisions and then they first spontaneously slow
their growth and subsequently differentiate back into bradyzoites (Day-8 post-invasion).
By Day-15 post-bradyzoite inoculation, 75% of the population has re-expressed the
bradyzoite-specific antigen BAGl. Thus, a single cell cycle mechanism may control both
branches of the intermediate life cycle of T. gondii.
Collectively, these data and data presented in Jerome et al. [7] and Radke et al. [11]
represent investigations into the relationship between the cell and developmental cycle of
the Toxoplasma intermediate life stage. The model put forth (Fig. 3-5) describes the
complete developmental life cycle with respect to the cell cycle. Sporozoites are in a GO
state and differentiate into the tachyzoite which proliferates by means of a three stage cell
60
cycle [11], then tachyzoites differentiate into bradyzoites by entering a G2 period prior to
the final mitotic event that leads to the growth-arrested bradyzoite (Fig. 3-5).
Bradyzoites can de-differentiate, re-enter the cell cycle and apparently follow the same
developmental pathway used by the sporozoite.
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29. Sacks DL, Perkins PV. Identification of an infective stage of Leishmania
promastigotes. Science 1984;223:1417-1419.
30. Nasmyth K. Control of S phase. In:Concepts in Eukaryotic DNA replication.
(DePamphilis, M.L. ed) Cold Spring Harbor Laboratory Press 1999:331-386.
31. Wolffe AP. Chromatin structure and DNA replication: Implications for
transcriptional activity. In:Concepts in Eukaryotic DNA replication. (DePamphilis,
M.L. ed) Cold Spring Harbor Laboratory Press 1999:271-293.
32. Edgar LG, McGhee ID. DNA synthesis and the control of embryonic gene
expression in C elegans. Cell 1988;53:589-599.
33. Ducommun B. From growth to cell cycle control. Semin Cell Biol 1991;24(4):23341.
63
34. Hunt T. Cyclins and their partners:from a simple idea to complicated reality. Semin
Cell Biol 1991;2(4):213-222.
35. Pines J. Cyclinsrwheels within wheels. Cell Growth Differ 1991;2(6):305-310.
64
CHAPTER 4
PROLIFERATING-CELL-NUCLEAR-ANTIGENS OF TOXOPLASMA GONDII APE
DISSIMILAR IN EXPRESSION AND POTENTIAL FUNCTION.
Introduction
DNA replication in eukaryotic and prokaryotic cells requires a scaffold onto
which the replisome assembles and is stabilized [1-3]. In prokaryotes, the P-subunit
serves this role whereas eukaryotes utilize proliferating-cell-nuclear-antigen (PCNA) [4].
Although PCNA was initially mis-assigned as a cyclin, its core function is to increase the
processivity of DNA polymerase, and in some cells, the cycling of PCNA is coincident
with the turnover of cyclin complexes [5], Within the eukaryotic replisome, PCNA
functions as a sliding clamp through which chromosomal DNA passes and upon which
delta polymerase can process lengthy templates [6-7]. Given its central position in DNA
metabolism, it is not surprising that PCNA has been demonstrated to play a role in DNA
repair, cell cycle control, transcriptional regulation, and chromatin assembly [6-7].
Consequently, attempts to delete the PCNA gene have been unsuccessful because it is
essential to cell division [8-9].
Our laboratory previously described two unique PCNAs [Chapter 2 and ref. 10] in
the protozoans, Toxoplasma gondii and Plasmodium falciparum. The PCNAs from T.
gondii are distinct, and more similar to each of their counterparts in P. falciparum than
they are to each other, suggesting they did not arise from a simple gene duplication [10].
65
Expression of TgPCNAl and 2 was detected in tachyzoite strains representing the three
genotypic lineages, and each PCNA appears to be encoded by a single gene.
The
occurrence of multiple PCNAs has been found in the genome analysis of
Cryptosporidium parvum [M. Abrahamsen, personal communication, Chapter 2], a
related apicomplexan, and Drosophila melanogaster [11]. Genomic sequencing of
thermophilic Archea have uncovered several instances of multiple genes encoding
diverse (3-subunits [12-13], No significance for having multiple PCNAs, such as a gain
or division of function, has emerged from studies of cells that possess two copies of
PCNA. Multiple (3-subunits from two bacteria [12-13] have been cloned; however, only
subtle differences in their respective activities have been noted [12-13].
In the studies presented here, we demonstrate that Toxoplasma TgPCNAl and 2
are differentially expressed with respect to the tachyzoite cell cycle and that each PCNA
forms homotypic but not heterotypic interactions, indicating they likely act
independently. We also provide evidence that TgPCNAl likely serves as the major
replisomal PCNA in this microorganism as it is capable of complementing the PCNA
function in yeast and appears to be essential in Toxoplasma.
66
Materials and Methods
Cell culture and parasite growth
Human foreskin fibroblasts (HFF) were grown in Dulbecco’s Modified Eagle
Medium (Gibco BRL, Grand Island NY) supplemented with 10% (v/v) fetal bovine
serum (Atlanta Biologicals, Atlanta GA). The T. gondii strain 'RHiShxgprt [14] was used
as the parental strain for gene knockout experiments.
Production of Antibody Reagents
TgPCNAl and 2 coding regions were cloned into pQE-30 (Qiagen, Chatsworth
CA) expression vectors in order to produce recombinant proteins as described [10].
Immune serum for each TgPCNA was prepared by immunizing New Zealand White
rabbits with 100 pg of purified recombinant TgPCNA (rTgPCNA) in Hunter’s
Titermax® adjuvant (Sigma, St. Louis MO). The antisera was titered and verified for
TgPCNA specificity as described in'Guerini efal. (2000).
Cell cycle specific expression of TgPCNA I and 2
TgPCNA protein localization was examined in synchronized RHTK+ tachyzoites
(RHAhxgprt parasites expressing an active thymidine kinase) [15]. HFF’s were infected
with -80,000 RHtk+ tachyzoites in 8-well chamber slides.
Following 16 hours of
67
growth, exogenous thymidine [10 jjM] was added to the media and monolayers were
incubated an additional 4h at 37°C allowing the RHTK+ parasites to arrest at the Gl/S
boundary. Replacing the thymidine containing media with normal I % DMEM releases
the parasites from the block thereby allowing the synchronized population to proceed
through at least one complete cell cycle. The intracellular localization of PCNAl and 2
was determined by immunofluorescence imaging.
Parasites were washed in IX
phosphate buffered saline (PBS), fixed with 3.7% formyl saline and permeabilized with
0.25% Triton X-100.
Wells in each slide were treated with either oc-TgPCNAl
(1:10,000) or a-TgPCNA2 (1:500) rabbit polyclonal antisera for 30 minutes, washed and
then treated with fluorescein (FITC)-conjugated anti-rabbit immunoglobulin G diluted
1:100 (Sigma, St. Louis, Mo.). Parasites were simultaneously stained with anti-mouse
C4F3 antibody at 1:500 [16] and 4,6-diamidino-2-phenylindole (DAPI, 0.16 pg/pl) to
detect daughter apical complex formation and nuclear division. The mouse anti-C4F3
antibody was detected with secondary antibody phycoerethrin (PE)-conjugated anti­
mouse IgG (Sigma, St. Louis, Mo.) diluted 1:64. The slides were evaluated with an
epifluorescence microscope (Eclipse TE300, Nikon Inc., Melville NY), and images were
collected with a digital camera (SPOT™, Dynamic Instruments Inc., Sterling Heights
MI).
68
TgPCNAl and 2 protein interaction
Recombinant TgPCNAl and 2 were allowed to interact at physiological
temperature (37°C) and pH (7.5). Prior to electrophoresis the recombinant proteins were
incubated 15 min. in 0.5 M Tris (pH 7.5) at 37°C [17] and loaded in native sample buffer
(62.5 mM Tris, pH 6.8, 40% glycerol, 0.01% Bromophenol Blue).
Homotypic and
heterotypic interactions were detected by Coomassie Blue staining following
electrophoresis on a 4-15% gradient gel (Bio-Rad, CA) under native conditions at IO0C
[17] .
TgPCNAl and 2 interaction was also assessed by yeast two-hybrid analysis.
TgPCNA coding regions were amplified from cDNA [10] using the polymerase chain
reaction (PCR) and cloned (TgPCNAl, EcoRHSall\ TgPCNA2, EcoRI/XhoT) into pBDGAL4 Cam phagemid vector (Stratagene, La Jolla CA).
The pBD-GAL4 has the
tryptophan (W) selectable marker. TgPCNAl and 2 were both cloned into the pADGAL4-2.1 phagemid vector (Stratagene) using EcoRI/Xhol. This vector can be selected
for in yeast by using the Leucine selectable marker. The four plasmid combinations (two
homotypic and two heterotypic) were transformed into yeast strain YRG-2 using LiAc
[18] and plated on media lacking leucine and tryptophan (SC -W-L). Protein interaction
was assessed on yeast complete medium lacking histidine (SC -W-L-H).
69
Transcomplementation of a cold-sensitive S. cerevisiae POL3Q (TCNA) mutant
The Saccharomyces cerevisiae strain PY 44-52, contains a cold sensitive mutation
in the POL30 gene (yeast PCNA) (kindly provided by Dr. Burgers, [19]). In order to test
the ability of TgPCNAl or 2 to complement this defect in yeast, TgPCNA coding regions
were cloned into a plasmid (pMET426) [20] that contains a selectable marker (URA) and
an inducible promoter (MET). Strain PY44-52 was transformed individually with each
TgPCNA and, following selection on media lacking uracil (-Ura), single colonies were
inoculated onto two selective medias (-Ura -Met or -Ura + 1.3 pM methionine) and
incubated at 150C.
Generation of TgPCNA gene “knockout” constructs
T. gondii genomic clones for TgPCNA I and 2 were isolated from a XDASH-II
genomic library (AIDS Research and Reference Reagent program, Cat. No 2863) and the
flanicing, exon and intron structures were sequenced. To prepare the TgPCNAl lcnockout
construct [21-22], a 6.5 kb genomic fragment was cloned into pBluescript KS+ and the
hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXPGRT) [14] minigene
was subcloned into a unique Munl site in exon 4. Similarly, a 10.2 kb genomic fragment
spanning the TgPCNA2 locus was cloned into pBluescript KS+ and an ~3.3 kb region
(BamHl/Munl) encompassing exons 4 and 5 was replaced with the HXGPRT minigene.
The linearized targeting constructs (50 pg) were transfected into IO7 parasites by
70
electroporation, as previously described [23], and stable transformants were selected
using 50 pg ml'1 mycophenolic acid and 50 pg ml'1 xanthine. After two rounds of
selection, parasites were cloned in 96-well plates by limiting dilution in the presence of
drug selection. TgPCNAl knockouts were screened by PCR of genomic DNA using
primers 5’-GGCTGGAGATGAGTCGCAGG-3’ (TgPCNAl-gPIFya; sense) and 5’CCCTCTG-CGTCATCCACACC-3 ’ (TgPCNAl-TgPCNAl revKO; antisense), while
TgPCNA2 lcnockouts were identified with primers 5'-CCGAGCAACTGGAAGTCCC-3'
TgPCNA2-P2;
sense)
and 5'-GGGGGCAAGACAGCGAGTCAC-3'
(TgPCNA2-
P2r21c; antisense).
Analysis of TsPCNA2 “knockout” parasites
To confirm TgPCNA genetic deletions, genomic DNAs were isolated and
Southern analysis was DNA performed. Additionally, total KNA was prepared from
RHAhxgpA and TgPCNA2 knockout parasites by TRIzol (Gibco-BRL, Grand Island NY)
extraction and analyzed by Northern blot for the presence of TgPCNA2 mRNA.
Hybridization was performed at 42°C for 16h in a solution containing 50% formamide
and 6 X SSC using [32P]-labeled inserts. Following hybridization, nitrocellulose blots
were washed three times in 2 X SSC containing 0.1% SDS at room temperature (10 min
each) and then twice in 0.1 X SSC containing 0.1% SDS at 42°C (30 min each). Blots
were exposed to x-ray film at -80°C. Southern blots were hybridized with either the 6.5
kb TgPCNAl or 10.2 kb TgPCNA2 genomic fragments labeled with [32P] by nick-
71
translation. The probes used for Northern analysis represent a single divergent region of
each TgPCNA that is capable of distinguishing each mRNA species [10].
A variety of methods were used to characterize TgPCNA2 knockout parasites.
Growth rate and virulence were tested as described in Jerome et al. [24]. The DNA
content of parasites was measured using flow cytometry [25]. After harvest, parasites
were fixed in ethanol and stored at -20°C for 24h prior to analysis then resuspended in
PBS and treated with 10 pg/ml propidium iodide (PI) (Sigma, St. Louis, MO) and 7.5pl
of RNase cocktail (Ambion, Austin, TX) for 30 minutes in the dark. A variety of cell
cycle markers were used to visualize parasite growth, including markers for daughtrer
parasite formation (C4F3) [16] and nuclear division [25]. The ability of parasites to
differentiate into bradyzoites was examined under alkaline conditions (pH 8.3) [26].
Two biochemical strategies were employed to assess in vitro DNA synthesis [17]
and incision repair [27]. DNA polymerase activity from protein extracts of “knockout”
and “wt” parasites was analyzed by measuring product length resulting from the
extension of an oligo dT primer (18 bases) annealed to a polydA template (size range 300
to 100 nucleotides) annealed at a ratio of 20:1 (poly dA:primer/ pg:pg) [17].
The
reaction was incuabated at 37°C for 90 minutes in the presence of gamma 32P-Iabeled
TTP and then stopped by the addition of SDS, extracted with phenol:chloroform and
denatured for 10 minutes in buffer containing 90% formamide.
Products were
electrophoresed on a 6% denaturing (8M Urea) polyacrylamide gel in IX TBE then the
gel was exposed to x-ray film at -80°C. Products were analyzed for both length and
radiolabel incorporation. The incision repair assay used the same protein extracts and the
72
template consisted of two oligos (one containing a mis-match) annealed together and a
radiolabeled nucleotide. Products were electrophoresed on a 15% polyacrylamide gel
and exposed to x-ray film at -SO0C for analysis.
The frequency of non-homologous integration was measured in “knockout” and
parental parasites using the plasmid pmmCAT [28] which contains the chloramphenicol
selectable marker.
Following electroporation,
selection was
carried out in
chloramphenicol (20 pM) and parasites were plaqued to determine viability and
integration. Stable integration was reported as the number of chloramphenicol resistant
parasites/number of viable parasites.
Results
TgPCNA2 is transported in and out of the nucleus during parasite replication
As mammalian and yeast cells progress through a single division cycle, PCNA
was shown to localize to the nucleus concurrent with replication foci building and firing
[29]. To study the localization of TgPCNAl and 2 during the cell cycle [15, 25], we
analyzed synchronized RHTK+ tachyzoites [15] using rabbit antisera specific for
TgPCNAl or 2. At all cell cycle stages TgPCNAl fluorescence remained concentrated
in the nucleus (Fig. 4-1 -❖ -), and as demonstrated previously, TgPCNAl displayed a
stippling pattern in those parasites that were in S-phase [25].
The localization of
TgPCNA2 changed with respect to the cell cycle phase (Fig. 4-1). In asynchronous
tachyzoite populations, TgPCNA2 was concentrated in the nucleus of -61% of the
73
parasites and this number increased (>80%) in parasites blocked in late GI/early S phase
(Fig. 4-1,
nuclear, late Gl/earlyS, inset A). The remaining 20% of the parasites from
the blocked population showed no evidence of nuclear localization (Fig. 4-1, -Acytoplasmic, inset B). As synchronized parasites transited S phase and entered mitosis,
the fraction of parasites with TgPCNA2 concentrated in the nucleus steadily decreased to
less than 20% of the population. Thus, TgPCNA2 is distinct from TgPCNAl in that it
concentrates in the nucleus only during late Gl and S phases and in other stages of the
cell cycle it is distributed throughout the parasite cytoplasm.
HO
100
90
CZ2 80
M
H 70
W
60
I
50
Cu 40
30
20
10
0
G 1/S
s
M/C
Gl
e a r ly
S
la t e
Figure 4-1. The cellular distribution of TgPCNA changes during the tachyzoite cell cycle.
R H T K + tachyzoite growth was synchronized with thymidine (4h block) at the Gl-S
boundary of the cell cycle (B). At hourly intervals from 1-6 h post thymidine release
(points 1-6), parasites were stained with TgPCNA I or 2 specific antisera in order to
determine the cellular location of each protein in 100 vacuoles picked at random. Inset A
and B show representative changes in the TgPCNA2 staining patterns during the course
of tachyzoite division.
74
TgPCNAl and 2 form homotypic but not heterotypic complexes
PCNA in human and yeast cells is thought to function as a homotypic tim er that
encircles DNA during replication [6-7, 17, 30]. The expression of distinct PCNAs in
Toxoplasma raises the question of whether TgPCNAl or TgPCNA2 also interact, either
separately or with one another, to form a multimeric structure. To explore this question,
we determined whether purified recombinant proteins might interact under native
conditions in vitro [17] or in a yeast two-hybrid assay. Recombinant TgPCNAs were
incubated separately or in equal mixtures and then resolved on native gels. By itself,
rTgPCNAl remains primarily monomeric (Fig. 4-2A, lane 2) with approximately 5-10%
of the total protein migrating at a molecular mass consistent with dimerization (Fig. 42A, band a). Conversely, rTgPCNA2 alone readily forms a homodimer (Fig. 4-2A, band
b) with a small amount of monomer and tim er (Fig. 4-2A, band c) also evident in these
gels. When rTgPCNAl and rTgPCNA2 were combined (Fig. 4-2A lane 4) the protein
complexes were additive of the patterns seen individually (compare Fig. 4-2A lane 4 with
lanes 2, 6). No new complexes were detected nor were there any apparent shifts in the
complexes, suggesting rTgPCNAl and 2 do not interact.
To independently confirm this pattern of interaction, we analyzed TgPCNAl and
2 binding in the yeast two-hybrid system.
For analysis of homotypic interactions,
plasmids (binding domain (BD) and activation domain (AD)) containing either
TgPCNAl (I-BDT-AD) or TgPCNA2 (2-BD:2-AD) were co-transformed into yeast
strain YRG-2.
Additionally, TgPCNAl and 2 in BD and AD plasmids were co­
transformed into yeast giving the following two heterotypic combinations (I-BD:2-AD
75
and 2-BD:l-AD). The interaction between TgPCNAl fused with the activation and bait
domains reconstituted the GAL4 transcription factor and activated expression of the HIS3
reporter gene allowing yeast to grow on synthetic complete media lacking histidine (Fig.
4-2B, right plate). Likewise, TgPCNA2 fused to both domains grew on selective media
(Fig. 4-2B, right plate). Thus, both sets of homotypic interactions found in the
recombinant protein system are confirmed with the two-hybrid approach. In contrast,
yeast co-transformed with the heterotypic plasmid combinations did not grow in either
configuration (Fig. 4-2B, right plate).
TgPCNAl but not TgPCNA2 complements a POL3Q yeast mutant
Unsuccessful attempts to generate POL30 (PCNA) deletion mutants in yeast
demonstrate that PCNA is an essential gene [9]. S. cerevisiae strain PY44-52 [19] is a
PCNA (POZdO) cold sensitive mutant that is capable of growth at 3O0C but not at 15°C
(Fig. 4-3, left panel, top and bottom). To determine whether TgPCNAl or 2 could
complement PY44-52, their coding regions were cloned downstream of the methionine
repressible promoter in vector pMET426 [20].
PY44-52 cells transformed with
pMET426-TgPCNAl were able to grow at 15°C in low methionine demonstrating that
TgPCNAl is capable of rescuing the POL30 mutation in yeast (Fig. 4-3, right panel,
bottom). As little as 1.3 pM methionine blocked TgPCNAl complementation at the
restrictive temperature (data not shown).
In contrast, PY44-52 was not rescued by
pMET426-TgPCNA2 (Fig. 4-3, right panel, bottom), although these transformants grew
normally at 3O0C in methionine-deficient medium indicating heterologous TgPCNA2
expression in yeast is not toxic.
76
5
6
TgPCNAl BD/AD
pLaminin Cl
pSV40
TgPCNAI BD/
TgPCNAZAD
TgPCNAZ BD/AD
TgPCNAZ BD/
TgPCNAl AD
L egend
SC -W -L
SC -W -L -H
Figure 4-2. TgPCNAl and 2 form homotypic but not heterotypic complexes. (A)
Recombinant TgPCNAl and 2 or a mixture of both proteins were separated on a 4-15%
native gradient gel at 15°C; 5 pg (lane 2) or 10 pg (lane 3) of TgPCNAl alone, 5 pg (lane
6) or 10 pg (lane 7) TgPCNA2 alone, or a mixture (5 pg each) of both proteins (lane 4).
Each protein (5 pg) was run under reducing conditions in order to provide a reference for
monomeric TgPCNAl (lane I) and TgPCNA2 (lane 5). (B) Yeast two-hybrid analysis of
TgPCNA protein interactions.
Bait (TgPCNAl-BD, TgPCNA2-BD) and prey
(TgPCNAl-AD, TgPCNA2-AD) plasmids were constructed, co-transformed into yeast
strain YRG-2, and selected on selective (SC -L -W) media (middle panel). Colonies
were streaked onto SC-L-W-H (right panel) to assay for fusion protein interaction.
Homotypic TgPCNAl or 2 co-transformants grew on triple selection media whereas in
either heterotypic configuration (1-AD/2-BD or 2-AD/l-BD) no growth was detected.
Positive (pSV40-AD/p53-BD) and negative (pSV40-AD/Laminin C-BD) controls
displayed the correct phenotype.
77
P arental
Strains
C om p lem en tation
30°C
15°C
PY 44
P Y 4 4 -5 2
P Y 4 4 -5 2 / P Y 4 4 -5 2 /
PCNAl
PCN A2
Figure 4-3. TgPCNAl complements a POL30 (PCNA) mutant yeast strain. S. cerevisiae
strain PY44-52 contains a mutation in the POL30 (PCNA) gene such that growth occurs
at 30OC but not at 150C. The parental strain PY44 is capable of growth at both
temperatures. Plasmids containing the protein coding sequence of TgPCNAl or 2 were
placed under the control of the yeast promoter MET25 were transformed into PY44-52
(right panels). TgPCNAl complemented the defect in PY44-52 (growth at 15°C, right
panel) while TgPCNA2 did not.
Generation of TgPCNA gene “knockouts”
Although PCNA is thought to be essential to cell growth in eukaryotes, this has
only been demonstrated in cells that contain a single PCNA species [8-9]. To examine
this question in T. gondii, we have attempted to produce a single gene deletion of each
TgPCNA. The Toxoplasma PCNAl gene is comprised of four similar sized exons and
three introns (Fig. 4-4A). The TgPCNA2 gene has a different composition, which is
consistent with the concept that these genes were acquired independently.
The
78
TgPCNA2 locus consists of five exons and four introns. The fourth exon is very small
(82 nucleotides) while the fourth intron spans 1280 nucleotides (Fig. 4-4B), which
appears to be more than twice the average intron size in this parasite.
RWkhxgprt parasites were electroporated with a ATgPCNA2 construct (Fig. 44B), selected in media containing 50pg ml"1 mycophenolic acid and 50pg ml"1 xanthine
and then cloned by limiting dilution in 96 well plates.
From a total of 192 clones
examined by PCR for a gene replacement, clones #3, 53 and 90 were identified as
putative TgPCNA2 knockouts (~1:66).
Southern analysis of the three TgPCNA2
knockout clones confirmed they contained a single gene replacement (Fig. 4-5A).
Further characterization of clone #3 by Northern analysis verified the absence of
TgPCNA2 mRNA (Fig. 4-5B) in this mutant.
An attempt to delete the TgPCNAl gene was also undertaken using both “hit-andrun” mutagenesis [21-22], and homologous gene replacement as was done for TgPCNA2.
Neither strategy resulted in a TgPCNAl gene replacement.
The “hit-and-run”
mutagenesis provided 29 clones, and seven were confirmed as pseudodiploids. However,
several attempts to generate a gene replacement by negative selection (6-thioxanthine at
320 pg/ml) were unsuccessful. Direct gene replacement was also attempted, and while
TgPCNA2 knockouts were found at a frequency of I in 66 mycophenolic acid resistant
clones, no TgPCNAl replacements were found in >200 clones examined.
79
A
_________________
I
.
-6.5 kb
I
i
TgPCNAl locus
2870
x
x
____________62
HXGPRTj^fl-
1000
2870
Targeting
Construct
- 1 0 .2 kb
i
I
I
I
i
I
I
i
TgPCNA2 locus
-1600
202
145
I
I I I
™
941 —
149
82
7 0 7 * 431*
284
1280
X
■
■
-4400
X
149
145
1600
I
HXGPRT
941
707
-3000
Z
Targeting
Construct
Figure 4-4. Genetic structures of TgPCNAl and 2 and targeting construct designs. (A)
The gene for TgPCNAl is comprised of four exons and three introns. (B) TgPCNA2
gene has five exons and four introns.
4#..... 2.8 kb
— 7.3 kb
< — 6 kb
-2 .7 kb
W
T
3
53 90
W
T
3
Figure 4-5. Southern and Northern analysis of ▲TgPCNA2 parasites. (A) Southern
analysis of Toxoplasma gondii TgPCNA2 knockout #3, 53 and 90 and RlIAAxgpr/ (wt)
genomic DNA hybridized with the 10.2 kb TgPCNA2 genomic fragment. DNA was
digested with Nae\ such that it would produce a hybridization pattern of two bands which
resolve to 7.3 and 2.7 kb fragments for the TgPCNA2 locus. Note that the knockout
parasites have a 6 and 2.7 kb fragment indicating a loss of genomic DNA within the
TgPCNA2 gene. (B) Northern blot of TgPCNA2 mRNA from RHAAxgpr/ and
TgPCNA2 knockout #3. In the wt parasite one mRNA of ~2.8 kb is shown (as predicted
[10]). In TgPCNA2 knockout parasite #3 no mRNA species is detectable.
Phenotypic Analysis of ATgPCNA2 parasites
The growth of ▲TgPCNA2 parasites in culture, and similarly, virulence in mice
was unchanged when compared to RHAAxgpr/ (parental) parasites. Analysis of DNA
content by flow cytometry [25] revealed the characteristic asynchronous pattern of 60%
C l, 30% S and 10% G2+M previously described for RH strain tachyzoites [25]. Thus, it
was not surprising that there was no difference in the fraction of asynchronous parasites
81
engaged in mitosis and cytokinesis, as measured using in situ markers [25]. The ability
of ATgPCNA2 tachyzoites to express the bradyzoite specific antigen I [31] in response
to alkaline conditions (pH 8.3) [26] was also unchanged when compared to parental
parasites.
Assay of DNA polymerase activity [17] in ATgPCNA2 parasites demonstrated
there was no difference in overall activity or length of in vitro product as the result of this
gene replacement. Likewise, DNA incision repair activity on an oligonucleotide template
appeared to be normal. Finally, it is known that in RH strain parasites non-homologous
recombination occurs at a high frequency [21, 32]. Using the pminCAT plasmid [28], we
explored whether stable recombination was affected by the deletion of TgPCNA2.
Chloramphenicol resistance occurred in I of 27 viable ATgPCNA2 parasites, which was
nearly identical to the frequency found using the parental strain (1/34).
Discussion
Cellular levels of PCNA do not appear to play a role in regulating DNA
replication as they marginally fluctuate in most cells [5-7]. The mechanisms that control
PCNA nuclear transport/export and assembly into replication complexes appear more
important for regulating DNA synthesis, although nuclear shuttling of PCNA does not
occur universally [33-34], In T. gondii, the TgPCNAs exhibit strikingly different patterns
of cellular localization. TgPCNAl is concentrated in the nucleus during all tachyzoite
cell cycle phases (and in all intermediate host developmental stages [Chapter 2]) where it
becomes associated with replication foci [29] in parasites undergoing chromosome
82
replication [25].
The cellular distribution of TgPCNA2, on the other hand is quite
distinct from TgPCNAl.
In mitotic parasites or those in early C l, TgPCNA2 is
distributed throughout the parasite cytoplasm (Fig. 4-1 inset B). Only in late Gl/S does
TgPCNA2 become concentrated in the nucleus (Fig. 4-1 inset A).
TgPCNA2 is
cytoplasmic in parasites that are growth arrested as a consequence of parasite
development (e.g. bradyzoites) or via stress or drug treatment (data not shown). Thus,
cytoplasmic distribution of TgPCNA2 correlates with a GI/GO state.
The molecular structures that are responsible for differential regulation of
TgPCNA are not known, but there are a number of potential nuclear localization signal
(NTS) sequences in each protein. A conserved NLS site is present in the N-terminus of
both TgPCNAs which is sufficient to confer nuclear targeting to a GFP fusion protein
(data not shown). Two other putative, but distinct NLS sequences, are located in the Cterminal
region
of
each
TgPCNA.
In
TgPCNAl,
residues
277-292
(RSCIGHVKFFLAPKM) are similar to replication factory targeting sequences found in
DNA ligase and replication factor C (RFC) [35], but in TgPCNA2, this sequence is
highly divergent or nonexistent. Conversely, a bipartite NLS in TgPCNA2 (residues 233252; QISFRLKLQAERERD-AKKK) that is not conserved in TgPCNAl, shares strong
similarity to the nuclear import motif of cell cycle control factor, p53 [36].
Eukaryotic PCNAs are thought to form active clamp structures as homotrimers
with a six-fold axis of symmetry [6-7, 37] , as the result of having two globular domains
per monomer [6-7, 17,30,37], There are suggestions that PCNA may form dimers that
have a distinct function, but this model has not been confirmed [6-7,17,30], Results from
the interactor studies presented here using recombinant proteins (Fig. 4-2 A) and yeast
83
two-hybrid (Fig. 4-2 B), coupled with work reported for P. falciparum PCNAs
[Chakrabarti, personal communication], demonstrate that the distinct apicomplexan
PCNAs form homotypic but not heterotypic interactions. Each TgPCNA is capable of
forming higher order structures; however, they likely act independently of one another
which is consistent with their differential roles in cell cycle regulation.
Multiple PCNAs in a single cell is rare and seemingly unnecessary, given most
eukaryotes function perfectly well with one isoform.
Why two distinct PCNAs are
preserved in divergent Apicomplexans is unknown but might be explained by a process
involving either neofunctionalization or subfunctionalization of PCNA-associated
mechanisms in these parasites [10,38],
The results shown here seem to be more
consistent with neofunctionalization, as TgPCNAl appears to serve as the major DNA
clamp protein, while TgPCNAZ is not essential and may serve a function outside the set
of known PCNA activities.
TgPCNAl is expressed in all intermediate-host
developmental stages where it is concentrated in the parasite nucleus and assembles into
replication foci during S phase. TgPCNAl is capable of replacing the function of PCNA
in yeast, forms higher-order structures as seen with other PCNAs, but only with itself and
not with TgPCNAZ.
Several independent attempts to knockout this gene were
unsuccessful, suggesting it is essential to tachyzoite growth.
TgPCNAZ does not
complement yeast strain PY44-52 (Fig. 4-3) and deletion of TgPCNAZ did not appear to
affect parasite growth rate, in vitro DNA polymerase activity, non-homologous
recombination, or DNA repair.
TgPCNAZ, while not essential, displays a striking
regulation in cellular distribution during the parasite cell cycle, and overexpression of this
protein in tachyzoites was toxic. Several attempts to establish transgenic parasites that
84
expressed epitope tagged TgPCNA2 have failed, and parasites that transiently
overexpress TgPCNA2 are abnormal (data not shown). In contrast, transgenic parasites
expressing >10-fold higher TgPCNAl have easily been established (data not shown).
There is a lack of discernable toxicity of TgPCNA2 expression in Saccharomyces,
demonstrating that this protein is not generally disruptive to the eukaryotic replication
machinery (Fig. 4-3). It remains possible that TgPCNA2 has a redundant or subfunction
that was not explored in these studies. Further work is needed to determine the precise
role of the PCNA2 class of proteins in apicomplexan parasites.
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88
CHAPTER 5
GENETIC COMPLEMENTATION OF A TOXOPLASMA GONDII
CELL CYCLE MUTANT.
Introduction
There are large differences in the virulence of Toxoplasma strains, which for the most
part are not understood [1-3].
Notwithstanding the host contribution to virulence,
parasite-encoded determinants have been mapped to chromosome VIII [4] in Type I
strains, and there are numerous examples where gene deletion, insertion or chemical
mutation, and ectopic expression have attenuated the virulence of laboratory strains [510; L. Knoll personal communication]. Most of these alterations appear to affect parasite
growth and/or development. There is no evidence, thus far, to indicate Toxoplasma
produces cytotoxins or that invasion itself generally causes or triggers host cell death via
a biochemical mechanism. Parasitaemia that can be exacerbated by increased growth rate
and/or is increased as the consequence of a failure to switch into the slow growing
bradyzoite form, in combination with deleterious immune reactions [11-12], appears to be
the primary causes of Toxoplasma pathogenesis.
Despite the importance of growth as the underlying cause of toxoplasmosis, we know
very little about the regulation of the parasite cell cycle or how growth mechanisms are
linked to parasite development.
It has long been established that differentiation of
tachyzoites-to-bradyzoites is accompanied by a change to a slower parasite growth rate
89
[13-15], which is programmed [16] and obligatory to development, as fast growing
mutant strains do not spontaneously differentiate but will do so if their growth is slowed
by drug or stress treatment [13-15], Recently, we have discovered that bradyzoite dedifferentiation (recrudescence) commences a developmental cascade indistinguishable
from the pathway initiated by the sporozoite, which includes the obligatory shift in the
parasite cell cycle that precedes re-development of tissue cysts [Chapter 2], Thus, there
may be a common cell cycle mechanism that regulates both branches of Toxoplasma
development in the intermediate host life cycle. Whether this mechanism is tied to a
specific phase of the cell cycle needs to be determined, although we have recently
identified a G2 population that is transiently present during bradyzoite development,
suggesting that the developmental switch my be dependent on chromosome replication.
These and other findings [16] indicate that molecular switches linlc the growth rate and
number of parasite divisions to the developmental fate of this organism, and therefore,
studies of the parasite cell cycle are critical for both an understanding, of chronic as well
as acute disease.
In mammalian cells [17-18], bacteria [19], viruses [20-21] and yeast [22-25], loss-offunction mutants have been utilized to explore the regulation of events within the
eukaryotic cell cycle.
One strategy for finding genes involved in the cell cycle has
utilized temperature sensitive (ts) mutants [24-25], In T. gondii, tachyzoite temperature
sensitive mutants have been generated using a chemical mutagenesis strategy [7,9].
Seven ts tachyzoite mutants were created in the 1970s, but no cell cycle defects were
identified [7]. In recent work by our laboratory [9], 40 temperature sensitive mutants
90
were isolated and 8 (20%) of those were found to have a cell cycle defect [9], A limiting
factor in the large-scale pursuit of T. gondii tachyzoite ts mutants was the absence of a
complementation strategy. Recently, two strategies were developed for complementing
mutants: I) an episomal vector that contains large genomic fragments [26], and 2) an
approach that combines high frequency insertion of cDNAs [27] with the power of phage
recombination [28-29].
In this work, we present the complementation of Al 1C9 [9] —
a putative mitotic checkpoint mutant in Toxoplasma.
Materials and Methods
Cell culture and parasite growth
The temperature-sensitive mutant A11C9 was produced by chemical mutagenesis of
the RHAhxgprt tachyzoite strain [9] and is maintained by serial passage in human
foreskin fibroblasts (HFF) at 35°C. Complete growth arrest of this mutant occurs within
1-2 divisions at 40°C with reversion to a temperature resistant phenotype occurring at a
frequency of I in IO6 parasites [9]. HFF cells were cultured in Dulbecco’s Modified
Eagle Medium (DMEM, Gibco BRL, Grand Island NY) supplemented with 10% (v/v)
fetal bovine serum (Atlanta Biologicals, Atlanta, GA).
91
Construction of destination vector and complementation of tel 1C9
The vector Tsc3ABP-P30 [28] was modified to construct a new destination vector
[29] by replacing the P30 coding region with an EcoRV restriction site into which the
cc<£B-recombination cassette was inserted (reading frame A; Gateway Vector Conversion
System, Invitrogen, Carlsbad, CA).
Bacterial strain DB3.1 (Invitrogen) was used to
propagate the vector under chloramphenicol (30pg/ml) selection. The resulting vector
designated Tg-pgraDEST/DHFR+ is capable of LR recombination [29] and was used to
re-express recovered cDNA inserts in Toxoplasma under pyrimethamine and temperature
selection.
Tachyzoite mutant Al 1C9 was transformed with a T. gondii RH-strain cDNA library
where each cDNA insert is flanked by lambda recombination sites (att-sites) [28]. A total
of 80 million tachyzoites (20 million parasites/cuvette/50 p.g library DNA) were
transfected using a modification of the restriction-enzyme-mediated-integration protocol
described in Black et al. [30]. Briefly, library plasmid DNA was linearized with Ascl
(50U/50p.g for 16h at 37°C), and following ethanol precipitation, air-drying, and re­
suspension in cytomix [31], the DNA was added to 20 million Al 1C9 parasites in a
cytomix that contained 5OU of Notl [28]. Following electroporation, the parasites were
cultured without selection in 1% DMEM parasite-medium [31] for 3 hours at 35°C to
allow invasion to occur and then shifted to 40°C. Recovery of cDNA inserts from the T.
gondii genome by recombination followed published protocols [28]. Genomic DNA was
purified from polyclonal I “-temperature-resistant parasites and used directly to recover en
92
mass cDNA inserts in the pDONOR201 vector (Invitrogen) by BP in vitro recombination
[28-29].
Plasmids were transformed into bacteria, and the resulting colonies scraped,
collected by centrifugation, and plasmids were purified using standard methods. Inserts
in the I0-BP library were then shuttled into the Tg-pgraDEST/DHFR+ destination vector
via a second recombination (LR), and plasmids were recovered and purified as above. In
order to enrich for genetic complements, the I0-LR plasmid library was transfected into
M lC P as above, and the were parasites double selected at 40°C in the presence of I pM
pyrimethamine (Pyr), which was added 24 hours post-electroporation. The complete
process was then repeated once more in order to generate 3“-transformants prior to cDNA
insert sequencing. At each round of electroporation, parasite viability and the rate of
stable transformation was determined by plaquing parasites with or without selection.
Enrichment of cDNA inserts was evaluated by colony PCR or Sfil restriction enzyme
digestion as described [28]. Vector primers proximal to the attL sites of pDONOR201
(sense 5’-TCGCGTTAACGCTAGCATGGATCTC-3’ and antisense 5’- GTAACATCAGAGATTTTGAGACAC-3 ’) were used for colony PCR.
Analysis of the cDNA for TgXPMC2
Single-pass sequencing and BLASTx of selected cDNA inserts was performed to
ascertain the identity of potential gene complements using the attL sense primer. The full
coding region was determined for one cDNA insert, TgXPMC2, and primers were then
designed to clone the allele from parental M1C9 mRNA (sense 5'-TTATCCGACG-
93
TTAGTACGGGGC-3 ’ and antisense 5’-CCGCTGTGGAAGCAA-CGATCG-3’) by RTPCR using PFU polymerase (Stratagene, La Jolla, CA) to reduce PCR-induced base
changes. The coding region of A11C9 XPMC2 was completely sequenced from three
independent isolates.
Phenotypic analysis of TgXPMC2-complemented Al 1C9
A series of experiments were performed to compare parasite division of A11C9 to a
TgXPCM2-complemented temperature-resistant clone (XPMC2-/rl 1C9). Growth was
measured at 35, 37 and 40°C by calculating the average vacuole size 36 hours post­
inoculation.
DNA content of asynchronous growing parasites was analyzed by flow
cytometry as previously described [32-33]. In order to visualize cell division, parental
Al 1C9 was sequentially transformed with the ACP-GFP fusion (apicoplast-specific) [34]
followed by TgXPMC2, and the transgenic parasites were co-stained with DAPI (nuclear
stain) [9]. Two-color analysis was performed using HFF cells grown in 8-well chamber
slides and inoculated with 2.5 X IO4 parasites per well. Parasites were allowed to invade
I h at 35°C (permissive temperature) then shifted to the non-permissive temperature
(40°C). After 24 hours, slides were washed one time in phosphate buffered saline (PBS),
fixed 10 min. using 3.7% formyl saline (pH 7.4) and permeabilized for 20 min in 0.25%
Triton X-100. Monolayers were incubated 30 min. with rabbit anti-GFP antibody (1:150)
(Chemicon, Temecula CA).
Simultaneously, parasite nuclei were stained using 4,6-
diamidino-2-phenylindole (DAPI, 0.16 pg/j-il).
Following treatment with primary
94
antibody, monolayers were washed 3X with PBS and then treated with goat anti-rabbit
secondary at 1:250 (Jackson Immunoresearch, West Grove, PA) for 30 min. Finally,
slides were washed 4X in PBS, and cover slips were mounted using Gel/Mount (Biomeda
Corp., Foster City, CA).
Results
Complementation of cell cycle mutant Al 1C9
Genetic complementation of AllCO was accomplished using an RH-strain cDNA
library [28], where each insert was flanked by lambda-recombination sites (see scheme
Fig. 5-1).
One in 210,000 viable parasites were temperature-resistant after primary
transformation with the cDNA library. This ratio increased 30-fold following secondary
transformation and a further -6-fold enrichment was observed after a tertiary round.
Combined, the three rounds of selection achieved >200-fold enrichment. Restriction
digest of the 3°-LR library revealed three large inserts; 2,300 nt (2/20 colonies); 1,900 nt
(1/20); and 2,700 nt (1/20) with the remaining plasmids containing inserts of <500 nt
(Fig. 5-2A). Single-pass sequence of selected plasmids demonstrated that the 2,300 nt
inserts were identical.
To determine whether any of these inserts would be further
enriched, a fourth small scale transformation of A11C9 was performed using the 3°-LR
library at 1/10th the normal DNA (5 pg) concentration and without the addition of Notl to
the cytomix during electroporation. Temperature-resistant parasites were again selected,
the cDNA inserts recovered by BP recombination, and the colonies analyzed by PCR.
95
Figure 5-2B revealed that the 2,300 nt cDNA insert was further enriched (8/20 colonies).
Thus, this insert was sequenced completely, and a BLAST analysis identified the cDNA
insert as encoding a protein with similarity to XPMC2 from Xenopus laevis [37-38], The
TgXPMC2 was transformed into Asl IC9 and confirmed to be responsible for the
temperature resistant phenotype (Fig. 5-2C).
T achyzoitc
cDNA library.
1° T r a n sfo r m a n ts
K nrichm cnt
I : 2 10,000
2” T r a n sfo r m a n ts
I : 7,000
3 “ T r a n sfo r m a n ts
3" a ll-in s e r ts
reco v ered
m d .sequence!
C o n firm a tio n o f
co m p lem en ta tio n
Figure 5-1. Schematic representation of Asl 1C9 complementation.
96
Figure 5-2. Recovery of cDNA inserts from ts\ 1C9 complements. (A) Sfil digest of 3°LR sublibrary plasmids revealed three large inserts of 2,700 nt (lane I); 2,300 nt (lane
3,18); l,900nt (lane 8) and several inserts of <500 nt. (B) PCR of bacterial colonies (4°
BP) recovered from Al 1C9 parasites complemented with 5pg of the 3°-LR sublibrary.
(C) Complementation of tsl 1C9 with the 2,300 nt containing plasmid in (B) resulted in
the re-recovery of this insert in 8 of 10 BP plasmids.
Molecular Biology of Toxoplasma XPMC2
Toxoplasma XPMC2 (TgXPMC2) cDNA encodes an open reading frame of 361
amino acids (Fig. 5-3) with a predicted molecular weight of ~40kD and an isoelectric
point of 9.59. The N-terminal half of the protein encodes a putative exonuclease domain
(residues 60-219) conserved in all XPMC2-related genes [39].
TgXPMC2 is -30%
identical to the Xenopus laevis homolog known to complement yeast mitotic catastrophy
mutants [37-38].
97
I
48
94
141
188
235
282
329
MVTESHSHSGLAKRGGSIHRSQGRGDGHSNQQLRQLFNHSRTRRIQG
GPSIVRPPAPAVSLDCEMVGCGPDGNISALAQVSICDEKGEVLLDEF
VMPDMRITDFRHHVTGLSWSILRDRGISFNAARTLVTDIIRGKVLVG
HALQHDLQVLALDHPVHMIRDTSKYKPLRPPGLARNAVPSLRRLTSH
WLNREIQTGIHNSVEDCRAAMDLYRKFQSQWERQFLAVNDAQNSLTS
IEYLEDKLDHESGTLQTANTPGQAPRKRARSDHGVARQRGSEVYDSD
DSGTSEAATVVASESSHPSSNVSAQGRKRQRSVENGARDGVSGYDRT
KDADGFPGIDLSGLTKKERKKLKRKLCLGRRG
Figure 5-3. Amino acid sequence of TgXPMC2. Bold text represents the
putative exonuclease domain (aa 60-219) [39].
In order to determine whether the TgXPMC2 allele in Al 1C9 was mutated, RT-PCR
was used to clone the coding region from this mutant, and three independent cDNA
clones completely sequenced. The nucleotide sequence of TgXPMC2 from Al 1C9 was
identical to that obtained by genetic complementation (RH-strain cDNA library),
indicating that TgXPMC2 is likely a suppressor of the temperature-sensitive pathway in
A l 1C9.
Cell cycle analysis of TgXPMC2 transformants
To address whether TgXPMC2 was capable of fully reversing the cell division defect
in Al 1C9, we employed a series of methods and cell cycle markers to evaluate the growth
of XPMC2 complements (XPMC2-trl 1C9). As described previously [9], the phenotype
of A l 1C9 is characterized by an arrest (within 1-2 divisions) following shift to 40°C into
two distinct populations based on DNA content (~50% IN and 2N). The growth of
XPMC2-trl 1C9 parasites was evaluated at three different temperatures (Fig. 5-4A), as
98
compared to RHMixgprt controls and til 1C9 parasites. Based on average vacuole size,
there was no difference between the growth rate of RH controls and XPMC2-trl 1C9
parasites at any of the temperatures evaluated. The DNA content of asynchronously
growing XPMC2-trl 1C9 parasites was also unaffected by temperature and was
indistinguishable from normal RHMixgprt profiles (data not shown, [9,33]) whereas
til 1C9 DNA profiles (bold trace overlay) displayed the characteristic bimodal IN and 2N
distributions when shifted to 40°C (Fig. 5-4B).
A
B
FL2-H
FL2-H
Figure 5-4. Phenotypic rescue of til 1C9 by TgXPMC2. (A) The growth of XPMC2trl 1C9 (white) parasites at 35, 37 and 40°C compared with the parental RHkhxgprt
(shaded) and til 1C9 (black) parasites as assessed by the average number of parasites per
vacuole at 36 hours post-infection of HFF cells. (B) DNA content of asynchronously
growing XPMC2-trl 1C9 parasites (shaded) compared to til 1C9 (bold trace overlay) at
35 and 40°C.
A number of cell cycle markers are now available in T gondii that allow major steps
in parasite replication to be visualized. Markers for plastid division and daughter budding
are particularly useful for monitoring mitotic and cytokinetic processes in these parasites
[33-36],
We introduced ACP-GFP [34] and TgXPMC2 sequentially into til 1C9
99
parasites in order to compare cell division events in the mutant versus complemented
parasites. Following shift to 40°C, 50% of M1C9 parasites arrest with a 2N content that
appears to be the result of a mitotic defect [9]. The labeling of the plastid with ACP-GFP
in this mutant demonstrates that the while nuclear division is blocked, plastid division
and movement to opposite sides of the nucleus do occur at the non-permissive
temperature (Fig. 5, A-B), although the plastids are larger than normal and somewhat
elongated. In contrast, plastids in TgXPMC2-complemented parasites are completely
normal in size and appear to divide just prior to nuclear division (Fig. 5 Ai-Bi) as
previously described for RH parasites [35]. Thus, TgXPMC2 is capable of alleviating the
specific block in Al 1C9 cellular division that occurs between the conclusion of plastid
division and the onset of nuclear division.
100
Figure 5-5. Temperature-sensitive, mitotic block of t s l 1C9 is rescued by
TgXPMC2. Top panel; photomicrographs of til 1C9 made transgenic for ACPGFP(plastid localized), and stained with GFP antiserum (A) and DAPI (B)
following growth 24 hours at 40°C. Phase image is shown in panel C. Bottom
panels; ACP-GFP transgenic tsllC9 parasites were complemented with
TgXPMC2 and plastid/nuclear division evaluated at 40°C as in the top panels.
Plastid division (A
l), nuclear division (BO, and the corresponding phase image
(CO are shown.
101
Discussion
The results of previous studies [9], taken together with the experiments presented
here, demonstrate that til 1C9 is a mitotic mutant. At the non-permissive temperature,
tillC 9 arrests quickly (1-2 division) into two distinct populations (I and 2N) based on
DNA content [9]. Approximately half of the parasites arrest with a single undivided
nucleus that is associated with partially formed daughters [9] and a divided plastid (Fig.
5-5). This potentially places the defect at either the entry into or at a point in mitosis.
The list of proteins that could be responsible for this phenotype is not exhaustive, and
could be limited to the mitotic cyclin pathway as is suggested by the unusual arrest of
tillC 9 into I and 2N subpopulations [9, Fig. 5-4].
Yeast strains deficient in the
cyclinB/cdk pathway arrest in Gl and mitosis [40-43], which reflects the dual action of
this cyclin class on START and mitotic checkpoints [25,43],
Complementation of tillC 9 with a homolog of eukaryotic XPCM2 is consistent with
a mitotic defect, although the TgXPMC2 gene identified here is likely a suppressor of this
defect and not the actual ti allele in this mutant. X. Iaevis XPMC2 was previously shown
to act as a suppressor of a mitotic catastrophe event by complementing both the weel50/mikl and weel-50/cdc2-3w yeast mutants [37-38]. Yeast cells defective in wee I and
chid have been shown to contain a hyperactive cdc2 kinase, and this higher state of
activity might be the cause of a premature entry into mitosis. Collectively, the wee I,
chid and cyclinB/cdk pathways are intertwined and required to function properly for
successful completion of mitosis [37-38, 43-44], Finding that the T. gondii XPMC2
102
homolog complements M1C9 suggests that the cyclinB/cdk or wee I/chid pathways in
this mutant is likely affected. It is not generally understood how XPMC2 acts to suppress
mitotic catastrophe [37]. It may divert inappropriate cdc2 kinase activity by serving as an
alternative phosphorylation substrate, although proteins with similar activity do not
complement mitotic mutants [37].
A complex series of checkpoints regulate the entry and exit of eukaryotic cells from
one part of the cell cycle to another [25]. There are well established checkpoints that
control S phase entry (known as START), the fidelity of DNA replication, and the timing
of mitosis with respect to completion of DNA synthesis, chromosome segregation and
cytokinesis [25]. Recent work in T. gondii, using a variety of cellular markers (plastid
and nuclear and inner membrane complex proteins) [33-36], has begun to characterize the
timing of events in endodyogeny [33-34]. Tachyzoites have a three-phase cycle with Gl,
S, and mitotic/cytokinetic phases [33]. Parasite budding begins in Iate-S phase with the
appearance of conoid structures (apical complexes) in the cytoplasm [35-36,45] and
continues until each daughter parasite has obtained a nucleus and its share of the mother
cell organelles and cytoplasm [35-36,45]. The control of this process is not understood,
but remains a critical area of research. A checkpoint similar to START likely operates in
T. gondii, as tachyzoites arrested in Iate-GI/S by drug treatment or as a result of a Gl
temperature defect do not initiate DNA replication or form mitotic and cytokinetic
structures [32-33], Mitotic checkpoints must also occur in these parasites, as AllCP
demonstrates a durable arrest of mitosis and cytokinesis when shifted to the nonpermissive temperature.
There is no evidence to suggest that parasite budding can
103
continue in the absence of nuclear division in this mutant as has been observed in
parasites exposed to microtubule agonists [46]. This discrepancy could be explained if
mitotic checkpoints in these parasites depended on a physical association with the
parasite cytoskeleton.
A similar tethering-model has been proposed to explain the
discoordinate regulation of nuclei that share the same cytoplasm in Plasmodium schizonts
[47-48],
Forward genetics is a classical strategy to identifying genes associated with a variety
of biochemical events [17-25], although until recently it has not been possible in T.
gondii. We have combined features of phage recombination [29] with the high frequency
of integration in the Toxoplasma genome [27] in order to develop a robust method to
complement T. gondii tachyzoite mutants [28]. This method was used to isolate drug
targets in heterologous apicomplexan models [28] and recently to isolate developmentalspecific antigens in Toxoplasma (Radke and White, unpublished results). The results
here demonstrate that this new approach can be successfully applied to loss-of-function
chemical mutants, which opens the door to generating and complementing numerous
mutants to study cell cycle regulation in T gondii.
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10. Dzierszinski F, Mortuaire M, Cesbron-Delauw MF, Tomavo S. Targeted disruption
of the glycosylphosphatidylinositol-anchored surface antigen SAG3 gene in
Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in
mice. Mol Microbiol 2000;37(3):574-82.
11. Tenter AM, Heckeroth AR, Weiss TM. Toxoplasma gondii: from animals to
humans. Int J Parasitol 2001;30:1217-1258.
12. Jones JL, Kruszon-Moran D, Wilson M, Mcquillan G, et ah, Toxoplasma gondii
Infection in the United States: Seroprevelance and Risk Factors. Am J Epidem
2001;154(4):357-365.
13. Soete M, Camus D, Dubremetz JF. Experimental induction of bradyzoite-specific
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necessary for induction of bradyzoite-specific antigens: a possible role for nitric
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15. Soete M, Fortier B, Camus D, Dubremetz JF. Toxoplasma goWmkinetics of
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17. Talavera A and Basilico C. 1977. Temperature sensitive mutants of BHK cells
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cells. J Biol Chem 265:26-33.
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characterization of temperature sensitive mutants of reo virus. Virology 37:335-342.
22. Hartwell L, Culotti J and Reid B. 1970. Genetic control of the cell division cycle in
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27. Donald RG, Roos DS. Stable molecular transformation of Toxoplasma gondii: a
selectable dihydrofolate reductase-thymidylate synthase marker based on drug
resistance mutations in malaria. PNAS USA 1993;90(24):11703-11707.
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integration elevates transformation frequency and enables co-transfection of
Toxoplasma gondii. Mol Biochem Parasitol 1995;74(l):55-63.
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for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods in Cell
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32. Radke J and White M. 1998. A cell cycle model for the tachyzoite of Toxoplasma
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2000;151(6):1-13.
36. Hu K, Mann T, Streipen B, Beckers CJ, Roos DS, Murray JM. Daughter cell
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108
•CHAPTER 6
GROWTH AND DEVELOPMENT IN TOXOPLASMA
Summary
The significance of Toxoplasma gondii to human and animal health, along with its
value as a model for other pathogenic protozoa, makes this microorganism an important
model in the field of Apicomplexa research [1-3]. It is clear from numerous studies of
the diseases caused by this family of microorganisms that parasitaemia itself is key to
pathogenesis, and thus, an understanding of the growth processes in these pathogens
could provide better treatments which are desperately needed. Without a framework for
parasite-specific models, it seems appropriate to turn initially to models in other
eukaryotes where the relationship between the cell cycle and development is well
understood [4-5]. This is particularly important for T. gondii where growth mechanisms
are linlced with the developmental pathway, and this linlcage underlies chronic disease
[6]. Two theories have been proposed to explain mechanisms that control the eukaryotic
cell cycle [4], One theory suggests that cell division and development follow an ordered
succession of events (the domino model) [4]. According to this model, once an organism
passes the cell cycle restriction point Icnown as “START” [4] a cascade of events occurs
with each subsequent event dependent upon the successful completion of a previous
event [4], This model applies to the classical animal and yeast cell cycle. The other
theory, based on work from Drosophila [4,7] and Xenopus [4,7] embryogenesis, suggests
109
DNA replication and mitosis/cytokinesis are independent events focused on achieving a
quantitative outcome which is controlled by a “central clock” (the clock model) [4,7].
In Chapter one, we presented a description of the three types of nuclear/cellular
division; schizogony, endopolygeny, and endodyogeny (a binary form of schizogony) [812]—one or more of these strategies may be used by a single species during its life cycle.
Thus, Apicomplexan replication is relatively plastic, suggesting that cell cycle controls
are flexible. Additionally, the scale of proliferation appears to be adapted to the host-cell
environment in many of these parasites. For example, Eimeria bovis sporozoites that
invade relatively long-lived endothelial cells in the intestinal central lacteals produce very
large schizonts (with >100,000 first-generation merozoites) [13], while sporozoites of
Cryptosporidium parvum, which invade short-lived epithelial cells of the intestine, form
small schizonts that produce 6-8 merozoites [14]. In each case a counting (clock??)
mechanism appears to adapt the number of divisions to match the host environment,
which ensures further development.
A similar mechanism apparently operates in T.
gondii, where emergent tachyzoites from either sporozoite or bradyzoite infections follow
a flexible but preprogrammed growth expansion that is linlced directly to development of
the tissue cyst (see Chapter 3). Thus, Toxoplasma ensures that its biotic potential is
sufficient but does not lead to the destruction of its host, which would prevent its survival
and transmission to the definitive host. Altogether, the observations of Apicomplexa
growth and development seem more consistent with a developmental timer system or
“clock mechanism” than the classical animal cell cycle paradigm of linear, stepwise
events.
no
A prediction of the clock model is that there is more flexibility in cell cycle
checkpoints than would occur in the “domino model” [4], Although it is too early to
answer this question in apicomplexa parasites, the study of endodyogeny in T. gondii,
demonstrates that the tachyzoite cell cycle is distinct from the classical animal cell in
having a three-phase cycle where DNA replication is non-uniform, cytokinesis begins in
S phase, and there does not appear to be a G2 period [15].
A more thorough
understanding of endodyogeny resulted from ' the use of cellular markers and
synchronized parasites that documented (in real-time) the events of mitosis and
cytokinesis [15-18]. These works provided the tools and have identified the landmarks
needed to begin defining the regulation and control of the cell cycle and development in
this organism. In the work presented here, we examined not only the relationship but
also the regulation of the cell cycle and developmental linkage in Toxoplasma.
We first demonstrated that bradyzoites initiate a growth and developmental
pathway that is indistinguishable from the pathways initiated by sporozoites (Chapter 3).
This finding is significant because bradyzoite recrudescence is the underlying cause of
chronic disease. Moreover, this data also demonstrated that a single mechanism might
control both branches of T gondii development in the intermediate host. The key feature
of this pathway is that a change to a slow cell cycle is required for emergent tachyzoites
to enter the bradyzoite developmental pathway. What cell cycle mechanism is required
or how the cell cycle is altered during the bradyzoite switch was also addressed. There is
no evidence to support an extended G2 period in dividing tachyzoites [15], which is
consistent with observations of other apicomplexans undergoing schizogony [19-23]. In
Ill
this thesis, we describe a G2 population arising during tachyzoite-to-bradyzoite
differentiation that is not found in mature bradyzoites from in vivo cysts.
This G2
population co-expresses tachyzoite and bradyzoite markers indicating it is an
intermediate stage in the developmental pathway. This data suggests that chromosome
replication is required for T. gondii development, and thus, bradyzoite switching could be
similar to other protozoan developmental mechanisms [see Chapter 3],
Chromosome replication and segregation (mitosis) are not understood in these
parasites, but these mechanisms, through their linlcage to development, are important to
understanding chronic disease caused by Toxoplasma. To gain further insight into the
regulation of the parasite cell cycle, we took both a reverse and forward genetic approach
to study the tachyzoite cell cycle. One approach used bioinformatics and cDNA library
screening to identify cell cycle related proteins. We identified two distinct proliferating
cell nuclear antigen (PCNA) genes in T. gondii ([24], Chapter 2) and have extended this
discovery to other divergent Apicomplexans (Plasmodium and Cryptosporidium). This
finding is interesting because PCNA serves as the platform for DNA synthesis and in
virtually all other eukaryotes only a single PCNA is required for chromosome-associated
activities. Why apicomplexan parasites have two PCNAs is unknown but in Chapters 3
and 4 we demonstrated that Toxoplasma TgPCNAl and 2 are differentially expressed
during development and the tachyzoite cell cycle. We also showed that each PCNA
likely acts independently and provided evidence that TgPCNAl potentially serves as the
major replisomal PCNA because it is capable of replacing PCNA function in yeast and is
essential in Toxoplasma.
The function of PCNA2 in T. gondii remains a mystery.
112
although its conservation across deep branches in the Apicomplexa phylogeny indicates it
may serve a role in parasite growth.
Further work aimed at understanding the
mechanisms controlling developmental transitions might shed new light on the potential
role(s) of TgPCNA2.
There are clearly caveats to the above reverse genetics strategy, as the gene
(without function in many cases) ultimately drives the course of experiments in search of
a phenotype. Forward genetics starts with a phenotype and consequently provides a solid
foundation for understanding parasite-specific cellular mechanisms. Chapter 5 focused
on the tachyzoite ts mutant 11C9 which arrests within I to 2 divisions at the nonpermissive temperature (40°C), and approximately half of the parasites arrest with a
single undivided nucleus (2N) [25]. These observations place the defect in M1C9 at
some point in mitosis. Complementation of ts\\C 9 with a homolog of the eukaryotic
XPMC2 [26] suggests that the cyclinB/cdk or weel/chkl pathway in this mutant is likely
affected. A complex series of checkpoints regulate the entry and exit of eukaryotic cells
from one part of the cell cycle to another [4], From this data we can suggest that there
are at least two checkpoints in the T. gondii tachyzoite, one controlling S phase entry
(!mown as START) [4,15,18] and another that controls the timing of mitosis (Chapter 5).
The data presented within show further evidence to linlc growth mechanisms with
development, and ultimately the coupling of these two pathways may influence chronic
disease.
In T. gondii, a proposed linkage between growth and development seem
consistent with a developmental timer system (“clock mechanism”), yet the presence of
checkpoints suggests that the domino model plays at least a partial role in regulating the
113
tachyzoite cell cycle. This integration of the clock and domino theories is not entirely out
of the realm of possibility; for example, Hartwell [4] suggested that a protein kinase
(CDC2/CDC28 as the kinase oscillator) may be the biochemical basis of the “clock”
mechanism and that this clock drives the basic cell cycle but that additional control
circuits (known as checkpoints) might be present to prevent errors. Finally, through the
use of a variety of techniques, including loss-of-function mutants and reverse genetics, a
more complete picture of the cell cycle and development can be formed now that some
landmarks have been identified in Toxoplasma. Some of the current areas of research in
Toxoplasma cell biology that can be addressed in the future are: I) the identification of
proteins that regulate the relationship between development and the cell cycle; 2) how is
the scale of proliferation regulated in the Apicomplexa; and finally 3) how does the
cellular environment influence the rate, mode and number of parasite divisions.
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MONTANA STATE UNIVERSITY - BOZEMAN