AN ABSTRACT OF THE THESIS OF
Samradhni Jha for the degree of Master of Science in Veterinary Science presented on
June 8, 2006.
Title: Macrophage Infection with Wild-Type Mycobacierium ai'ium or Isogenic
Attenuated PPE mutant revealed Differences in Gene Expression and Mycobacterial
Vacuole Proteins.
Abstract approved:
Redacted for Privacy
Luiz E. Bermudez
Mycobacterium avium is a ubiquitous environmental organism found in water and
soil. It can cause disease in patients with pre-existing pulmonary conditions,
immunocompromised patients with the most prevalent being AIDS patients, as well as
apparently healthy people. Studies have indicated that, upon macrophage uptake, Al.
avium prevents phagosome-lysosome fusion, thus avoiding its killing. PPE-PE families
of genes in mycobacteria have been suggested to have a role in mycobacterial virulence
in vivo.
Using an
M avium-attenuated PPE transposon mutant, this study seeks to
examine the differences in the phagosomal proteins expressed upon macrophage infection
with the wild-type and its isogenic PPE mutant. Furthermore, this study aims to
investigate the differences in the transcriptional profile of macrophages infected with
M
avium and the attenuated PPE mutant.
First, the M avium and attenuated PPE mutant phagosome protein expression
were determined by mass spectrometry analysis. It was found that wild-type phagosomes
express proteins different from isogenic PPE mutant phagosomes at 30 mm, 4 h and 24 h.
The proteins identified in the phagosomes of wild-type and the PPE mutant were found to
be involved in bacterial uptake, antigen presentation and recognition, Rab and Rabinteracting proteins, cytoskeleton and motor proteins, proteins involved in biosynthetic
pathways, transcriptional regulators, signal transduction proteins, ion channels and
hypothetical proteins. Fluorescent microscopy studies were carried out to confirm the
protein expression in M avium- PPE mutant-infected macrophages.
After performing a DNA microarray on the macrophages infected with M avium
and the PPE mutant at 4 h, we observed differences in the gene expression patterns of the
wild-type and the PPE mutant infected macrophages.
Finally, real-time PCR was used to confirm the differential expression of three
chosen genes. The genes were found to be upregulated similar to that found in the DNA
array data.
Through these studies, we have determined several proteins, either previously
described or not reported, to be present on the phagosome. Nonetheless, the use of the
PPE mutant established the possibility that one protein may have a key function in
modulating the formation of the phagosome. Another interesting aspect of our findings is
that there is a possible connection between macrophage genes regulated upon uptake of a
bacterium and the downstream effect on vacuole formation.
Macrophage Infection with Wild-Type Mycobacterium
avium
or Tsogenic Attenuated
PPE Mutant revealed Differences in Gene Expression and Mycobacterial Vacuole
Proteins
by
Samradhni Jha
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Master of Science
Presented June 8, 2006
Commencement June 2007
Master of Science thesis of Samradhni Jha presented on June 8. 2006.
APPROVED:
Redacted for Privacy
Major Professor, representing Veterinary Science
Redacted for Privacy
Head of the Department of Biomedical Sciences
Redacted for Privacy
Dean of the Graduate School
I understand that my thesis will become a part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
Jha, Author
ACKNOWLEDGEMENTS
I would like to offer deep respect to my late father Shri. Dhruva. N. Dharaskar for
whatever I am today. He is the building block of my career and was a constant source of
motivation, inspiration, guidance and support, both emotionally and financially. No
words can express my feelings and sincere gratitude towards him because of whom I
stand here today. A sense of satisfaction runs through my heart knowing that I fulfilled
his dream.
I am grateful to my major professor, Dr. Luiz E. Bermudez, for providing me with
the opportunity to work under his guidance. He has been a true mentor, showing me the
right path toward sound knowledge and skills. His timely guidance and suggestions have
helped me achieve my goal here at OSU. I am thankful to my thesis committee, Dr.
Manoj Pastey and Dr. Claudia Hase, for their help and support.
I am indebted to Dr. Lia Danelishvili, Dr. Yoshitaka Yamazaki, Dr. Dilip Patel,
and Dr. Martha Alonso Hearn for their moral support, technical help and useful scientific
discussions. I am thankful to all my lab members, Julie Early, Melanie Harriff, Sanjai
Tripathi, and Dr. Heather Duchow, for keeping up a friendly, pleasant, and cheerful lab
atmosphere full of energy. I enjoyed working with these people.
The care and encouragement I have received from my mother Mrs. Asmita. D.
Dharaskar and my in-laws Shri. Dinesh Jha & Mrs. Kiran Jha was invaluable. I
appreciate all my family members and friends for their unending help. Last but not the
least, I am blessed to have Dr. Shantibhushan Tha as my husband who always stood
beside me. I would like to take this opportunity to thank him for all the love and faith he
bestowed on me.
TABLE OF CONTENTS
Introduction
.1
Macrophage infection with wild-type Mycobacterium avium
or isogenic attenuated PPE mutant revealed
differences in gene expression and
mycobacterial vacuole proteins .............................................................. 8
Discussion .......................................................................... 50
Bibliography ........................................................................ 58
LIST OF FIGURES
Fg
Figure
1.
2.
3.
4.
5.
6.
7.
Fluorescent microscopy images of isolated phagosomes showing
fluorescein-labeled M. avium and 2D6 mutant ..............................................
22
Fluorescent microscopy images of U-937 macrophages infected with
MAC 109 or 2D6 mutant for SP-D protein expression ..................................
27
Fluorescent microscopy images of U-937 macrophages infected with
MAC 109 or 2D6 mutant for I-type Ca2+ channel protein expression .........
28
Fluorescent microscopy images of U-937 macrophages infected with
avium 2D6 mutant for SP-D protein expression ............
complemented
30
Fluorescent microscopy images of U-937 macrophages infected with
complemented M avium 2D6 mutant for T-type Ca2+ channel protein
expression .......................................................................................................
31
M
Upregulation of U-937 macrophage genes upon infection with M avium or
2D6 mutant at 4 h by real-time PCR .............................................................. 36
Real-time PCR graph showing upregulation of genes in macrophage upon
M avium or 2D6 mutant infection .................................................................
56
LIST OF TABLES
Table
1.
2.
3.
4.
List of phagosoma] proteins identified by MS/MS
post-infection with MAC 109 or 2D6 at different time points .....................
23
M
avium 109 but
Macrophage genes upregulated in
downregulated in 2D6 mutant 4 h post-infection .......................................
33
M
avium 109
Macrophage genes downregulated in
but upregulated in 2D6 mutant 4 h post-infection .....................................
34
Sense and antisense primers for real-time PCR ..........................................
55
Dedicated to my parents, in-laws, husband and family members
Macrophage Infection with Wild-Type Mycobacterium avium or
Isogenic Attenuated PPE Mutant revealed Differences in Gene
Expression and Mycobacterial Vacuole Proteins
Introduction
Mycobacteria are a group of gram-positive, acid-fast bacteria capable of
inhabiting a wide range of environments. Of over 60 mycobacterial species are currently
known. Some of these organisms are pathogens, having the ability to infect humans and
animals. There is an increasing evidence of environmental mycobacteria being
pathogenic and causing grave diseases in humans especially immunocompromised
individuals.
Mycobacteria are widespread throughout the environment. Many different
mycobacterial species cause diseases ranging from gastrointestinal infections to skin and
soft tissue infections to disseminated infection. Mycobacterium marinum and
Mycobacterium chelonae, responsible for causing skin granulomas are waterborne
mycobacterial species. Another species Mycobacterium kansasii is associated with
pulmonary diseases. In animals, Mycobacterium avium ssp. paratuberculosis is
responsible for substantial economic losses to cattle industry because of the chronic
inflammatory condition of the intestinal tract. The same bacterium has been proposed to
be the cause of Crohn's disease in humans, but the evidence is still unclear. Probably, the
most important environmental species of significance is the Mycobacterium avium
complex or MAC.
Mycobacterium avium complex is composed of M. avium ssp. avium and
M. intracellulare. M. avium bacilli are extremely widespread in nature. They are
common in surface waters and soils and are frequently isolated from water taps (11). M.
2
avium is an opportunistic pathogen capable of causing diseases in humans and animals.
This complex has been shown to be accountable for most of the cases of non-tuberculosis
mycobacterial infections in the developed countries (20). M. avium affects young, adults,
patients with immunocompromised body system, pre-existing chronic pulmonary
diseases, silicosis, pneumoconiosis, emphysema, bronchiectasis and cystic fibrosis as
well as in people who are apparently healthy (1, 10, 23). M. avium is the most common
isolate from AIDS patients (14) and can cause disseminated disease as well as pulmonary
infections in these individuals (8). M. avium infection is a well-known secondary
complication in people suffering from Mycobacterium tuberculosis and Pneumocytis
carinii infections (35). M. avium organisms are intrinsically drug resistant, making
infections difficult to treat (7).
Mycobacterium avium and the host response
M. avium has predilection for tissue macrophages and blood monocytes. The
bacteria are phagocytosed by host macrophages through complement receptors and CR3
mediated phagocytosis (4). In addition, the integrin fibronectin receptor is also involved
in the internalization of the serum fibronectin bound bacteria (23). Normally,
phagocytosis of the bacteria results in a series of intracellular events specifically
designed to induce killing of engulfed bacteria and include (i) induction of reactive
oxygen and nitrogen intermediates, (ii) gradual acidification of the phagosome due to
the activity of proton-ATPase pumps located on the phagosome membrane, (iii)
phagosome-lysosome fusion which loads the resulting phagolysosome with acidic
proteolytic enzymes and (iv) antigen processing. The resulting lethal environment
effectively kills the majority of ingested bacteria.
Pathogenic species of mycobacteria have developed strategies to circumvent the
major killing mechanisms employed by macrophages. Mycobacteria not only have the
ability to adapt to a changing host environment (19, 50) but also actively interfere with
the signaling machinery within the host cell to counteract or inhibit parts of the killing
apparatus employed by the macrophage. The infected macrophages show impaired
antigen processing (32), reduced responsiveness to interferon-y (IFN-y) (26, 42), reduced
production of cytokines, and reactive oxygen and nitrogen intermediates (5). The
observation that attenuated strains of mycobacteria such as H37Ra and Mycobacterium
bovis BCG are significantly more potent inducers of apoptosis than their corresponding
virulent strains, H37Rv and wild-type M.bovis, suggest that apoptosis functions as a host
defense mechanism which is suppressed by the virulent strains (24).
In case of M. avium, once inside the macrophage, the bacterium is capable of
inhibiting the vacuole acidification and thus avoids the phagosome-lysosome fusion
leading to its survival in the macrophages. It does so partiy by residing in the vacuoles
with limited proteolytic activity, maintaining cathepsin D in an immature form, and
remaining accessible to transferrin (45). A study suggested that M. avium requires Rab5
and accessibility to iron in order to arrest the phagosome maturation (25). M. avium
vacuoles acquire lysosomal membrane protein LAMP-i but not the vesicular protonadenosine triphosphatase (ATPase) responsible for phagosomal acidification (46).
Similar studies on M. tuberculosis vacuoles have revealed incomplete lumenal
acidification and absence of mature hydrolases leading to inhibition of phagosome-
lysosome fusion (48). In addition, Malik and colleagues (28-30) suggested that calcium
modulation by M. tuberculosis is one of the factors responsible for phagosome
maturation arrest. The maturation block in M. tuberculosis has been shown to be
occurring between the stages controlled by RabS which is an early endocytic marker and
Rab7, a late endosome marker (49). Phagosomes containing live virulent M. tuberculosis
show the presence of early endosomal markers like the transferrin receptor, MHC class II
molecules and the ganglioside GM1 (6, 45) whereas exclude the proton ATPase and late
endosomal markers (46), mannose-6-phosphate receptor (51) and lysosomal protease
cathepsin D (6). The absence of proton ATPase is thought to account for reduced
acidification of mycobacterial phagosomes (46) with pH of 6.2-6.3 as compared to a pH
of 5.3-5.4 which is normally associated with the lysosomal compartments. Some studies
suggest that macrophages infected with M. tuberculosis show markedly reduced
expression of MHC class II molecules (18, 22, 33).
Other bacteria, like Salmonella spp., invade and replicate inside the professional
phagocytes and epithelial cells in a membrane bound compartment called the Salmonella-
containing vacuole (SCV). Previous studies have established that the SCV transiently
interacts with early endosomes but only acquires a subset of late endosomal/lysosomal
proteins (43). Studies on SCV revealed transient association of EEA- 1, transferrin
receptor (44) and GTPase Rab5 (38) on early SCVs which are rapidly lost followed by
accumulation of late endosome/lysosome markers such as Rab7 (31), lysosomal
membrane proteins LAMP-i, LAMP-2 and CD63 and vacuolar ATPase (44). SCV
selectively acquires lysosomal glycoproteins and lysosomal enzyme without fusing
directly with lysosomes (12). Another study by (41) showed that phosphatidylinositol 3phosphate (PI3P) is transiently accumulated on SCVs and that P13K is involved in the
development of SCV but is not essential for intracellular survival and proliferation. SCV
5
biogenesis and maturation in macrophages revealed that SCV acidifies and eventually
merge with the lysosomes (2, 34). More recent studies show that the maturation of SCV
in HeLa cells and in primary or cultivated macrophages is quite similar (13, 37).
Coxiella burnetii, another obligate intracellular bacteria which causes Q fever in
humans resides within acidified vacuoles inside the Vero cells with secondary lysosomal
characteristics (3). The bacterium inhabits a cholesterol-rich vacuole and requires free
access to host cholesterol stores for optimal replication (21).
Legionella pneumophila, an intracellular pathogen replicates inside the eukaryotic
cells in a unique vacuole similar to endoplasmic reticulum (ER) and avoids endocytic
maturation. Studies have shown that the host vesicles that attach to L. pneumophilacontaining vacuole (LCV) are derived from ER based on the presence of resident ER
proteins like glucose-6-phosphate, protein disulphide isomerase (PDI) and proteins
having the ER-retention signal lysine-aspartic acid-glutamic acid-leucine (KDEL) (39).
Derre and colleagues reported the recruitment of calnexin, Sec22 and Rabi to the vacuole
shortly after bacterial uptake (9). They also suggested a potential role of Rabi in ER
recruitment process. After ingestion by macrophages, the bacteria inhibit acidification
and maturation of its phagosome. As the bacteria replicates exponentially, a significant
number of the vacuoles acquire LAMP-i, cathepsin D and the pH of the vacuoles average
5.6 (47). The authors also showed that bacteria are able to survive and replicate within
the lysosomal compartments.
Mycobacterial virulence factors
PPE and PE gene families, which encode numerous proteins of unknown function
account for 10% of Mycobacterium tuberculosis genome. Mycobacterium avium has
genome similar regarding the PPE and PE gene families. Few reports have suggested
that these two families might be involved in several roles such as antigen variation,
binding to eukaryotic cells, survival within macrophages and persistence in granulomas
(36, 40). Camacho and colleagues identified a PPE gene (Rv3018c) associated with M.
tuberculosis virulence in vivo. Another study by Ramakrishnan and colleagues reported
that inactivation of PE-PGRS gene in M. marinum resulted in attenuation of virulence of
the bacteria in macrophages. Sampson and colleagues identified a PPE protein (Rvl917)
that is expressed on the bacterial surface. Li and colleagues (27) suggested a M. avium
PPE gene (similar to Rv1787 in M. tuberculosis) to play a role in the inhibition of
acidification and phagosome-lysosome fusion of infected macrophages. The mutation
did not alter the ability of the bacterium to enter macrophages but decreased virulence
and survival in vitro. In addition, the 2D6 mutant was severely attenuated in mice. The
PPE gene was expressed early only in macrophage and not in broth suggesting its role in
the phagosome formation or vacuole maturation as well as in the mechanisms of bacterial
adaptation to the intracellular environment. The vacuole containing the attenuated PPE
mutant underwent acidification to pH 4.8 16 h after phagocytosis.
The inactivated PPE gene is part of the M. avium genome region with five PPE
and PE genes. The organization suggests the existence of three promoters, one upstream
of the inactivated PPE gene in the 2D6 mutant, one between the second and third genes
and another between the fourth and fifth genes in downstream region. Therefore the
phenotype observed could be because of a polar effect of the inactivated PPE gene and
that three or all the five genes are responsible for the phenotype. But since the
complementation of the inactivated PPE gene reversed the phenotype, it is more likely
7
that only the PPE gene inactivated and probably the downstream gene are responsible for
the phenotype.
The vacuoles containing the 2D6 strain were significantly more acidic than the
vacuoles with the wild-type bacterium. This suggests that the PPE gene or the complete
genome region is able to influence vacuole acidification. Since acidification is secondary
to docking of acid pumps to the phagosome, it is plausible to hypothesize that the PPE
gene would either influence change in the phagosome membrane thereby preventing the
acquisition of acid pump or transporting other molecules to the bacterial surface where
they would prevent the binding of proton pump.
An important aspect in the study of M. avium and the PPE gene inactivated
mutant phagosomes would be to identify the proteins expressed on the wild-type
phagosomes and compare them with those expressed on PPE gene inactivated mutant
phagosomes. This study would reveal the differences if any, on both the bacterial
phagosomes and would also shed light on the variety of proteins involved in the
phagosome biogenesis. Additionally, it would correlate the underlying mechanisms
employed by the wild-type bacteria to prevent the phagosome maturation and ultimate
fusion with lysosomes. Furthermore, identification of the genes upregulated in the
macrophages upon M. avium or 2D6 mutant infection would explore the different
pathways triggered upon wild-type or 2D6 mutant invasion in macrophages. Any
differences in the macrophage gene expression would help understand the altered
trafficking stimulated by the wild-type in comparison with the 2D6 mutant.
Differences in Macrophage Transcriptional Profile and Mycobacterial
Vacuole Proteins following infection with Mycobacterium avium and
Attenuated PPE Mutant
Samradhni Jha
Lia Danelishvili ',Yong-jun Li 2 Yoshitaka Yamazaki and Luiz E.
Bermudez
To be submitted to Cellular Microbiology
1*
Abstract
Mycobacterium avium is an environmental opportunistic pathogen which infects and
replicates in mononuclear phagocytes. The phagosome-lysosome fusion is an important
step of the host defense pathway, which culminates in bacterial killing and degradation.
Previous study reported an attenuated mutant 2D6 in which a PPE gene (52%
homologous to Rv1787 in M. tuberculosis) was interrupted by a transposon. This
isogenic mutant, in contrast to wild type, was shown to have an impaired ability to
replicate intracellularly and to prevent the fusion of phagosome and lysosome. To study
the gene expression profile of macrophages infected with the wild-type bacterium and
2D6 mutant, we performed a DNA microarray analysis at 4 h post infection. Differences
were observed indicating that the two bacteria interact with mononuclear phagocytes in
unique fashions. We then hypothesized that the wild-type bacterium environment in
macrophages might differ from the mutant 2D6 environment, and compared the
phagosomal proteins expressed in M. avium infected cells and its isogenic mutant at
different time points. M. avium phagosomes expressed a number of proteins different
from those of it's isogenic mutant. Proteins identified belonged to several functional
classes and many of these proteins have not been previously described to be present on
the bacterial phagosomes. The expression of some selected proteins on the phagosomes
was confirmed by immunofluorescence microscopy.
10
Introduction
Mycobacterium avium is an environmental pathogen commonly found in soil and
water (11, 21). M. avium causes disseminated disease in immunocompromised people
such as in AIDS patients, and is a known secondary complication in patients suffering
from chronic pulmonary conditions (1). M. avium preferentially infects tissue
macrophages and blood monocytes. Once inside a macrophage, the bacterium has shown
to inhibit the acidification of the phagosome subsequently preventing the fusion between
the phagosome and lysosome (38). Acidification of the vacuole followed by its fusion
with the lysosomal compartment is part of the host cell mechanisms for intracellular
killing of microorganisms (30). Similar to M. tuberculosis (41), Salmonella (4) and
Leishmania (18), M. avium interferes with endosome maturation process which precedes
phagosome-lysosome fusion. The mechanisms that M. avium uses to survive within
macrophages have been an active area of research. Previous reports have shown that M.
avium has the ability to modulate the intracellular environment by remaining accessible
to internalized transferrin and limiting the proteolytic activity, maintaining cathepsin D in
an immature form (37). Malik and colleagues have suggested inhibition of calcium
signaling by M. tuberculosis is responsible for the prevention of phagosome-lysosome
fusion (26). A study carried out by Li and colleagues (2005) involving screening of M.
avium transposon mutants for clones with decreased ability to replicate intracellularly in
human macrophages identified an attenuated 2D6 mutant in which the transposon
interrupted a PPE gene (52% homologous to Rv1787 in M. tuberculosis). The 2D6
mutant was significantly attenuated in macrophages in contrast to the wild type bacteria
although both bacteria had comparable ability to enter the phagocytic cells. In addition,
11
vacuoles containing the 2D6 mutant could not prevent the acidification of the
phagosomes and fusion with the acidic lysosomes leading to reduced survival in
macrophages. The PE and PPE families of genes in mycobacteria are found disperse
throughout the genomes of M. tuberculosis, M. bovis, M. avium and M. paratuberculosis.
It is assumed that M. avium and M. paratuberculosis lack PE-PGRS proteins (10)
although we have recently found a PE-PGRS protein in M. avium (Li, Y and colleagues,
submitted for publication). These families of proteins have been associated with
virulence of mycobacteria (5, 9, 23).
Early events in the infection are likely to have an influence on the vacuole
characteristics and formation. The PPE gene in M. avium homologue to Rv1787 is
expressed upon entry in macrophages, and therefore may participate in the development
of the M. avium environment. Because the mycobacterial vacuole is probably a
consequence of infection of M. avium and the macrophage from the early moments of
binding and uptake, we want to investigate the differences related to the lack of the
Rv1787 homologue gene. DNA microarray and study of the phagosome proteins were
performed using macrophages infected with the wild-type bacterium and the 2D6 mutant.
12
Materials and Methods
Bacterial strains and growth conditions
Mycobacterium avium strain 109 (MAC 109), a virulent strain in mice, was cultured from
20% glycerol stock onto Middlebrook 7H1 1 agar supplemented with oleic acid, albumin,
dextrose and catalase (OADC; Hardy Diagnostics, Santa Maria, CA) at 37°C for 21 days.
Bacteria were suspended in Hank's buffered salt solution (HBSS) and passed through a
26-gauge needle for 10 times to disperse the clumps. The suspension was then allowed to
rest for 5 minutes and the upper half was used for the assays. The bacterial concentration
was adjusted to
1x109
bacteria mY' using a Mc Farland standard. Microscopic
observations of the suspensions were carried out to verify whether the inoculums were
constituted of dispersed bacteria. Only well dispersed inocula were used in the described
experiments. The 2D6 mutant was cultured from 20% glycerol stock on Middlebrook
7H1 1 agar containing 400pg/ml kanamycin. The 2D6 mutant suspension was made as
described above. The complemented 2D6 strain (23) was also cultured from 20%
glycerol stock and grown on Middlebrook 7H1 1 agar plates containing 200.tg/ml
apramycin (23).
Cells and culture conditions
Human monocytic cell line U937 (ATCC CRL-1593.2) was cultured in RPMI- 1640
(Gibco Laboratories) supplemented with 10% heat-inactivated fetal bovine serum (FBS;
Sigma Chemical), 2 mM L-glutamine. U937 cells were used between passages 15 to 20
and concentrations of 7x106 were seeded in 75cm2 flasks. The cells were grown to 90100% confluency, and allowed to differentiate overnight by incubation with 500ng mY1
phorbol 12-myristate 13-acetate (PMA; Sigma). U-937 and human monocyte-derived
13
macrophages were shown to behave similarly when infected with M. avium and 2D6
mutant (data not shown). The MAC 109 or 2D6 mutant were added to the monolayers at
a multiplicity of infection (MOl) of 10, and the infection was allowed to take place for 2
h at 37°C in 5% CO2. The supernatant was then removed and the cell monolayer was
washed three times with HBSS. The tissue culture medium was then replenished.
Phagosome Isolation and Microscopy
Phagosomes containing M. avium 109 and 2D6 mutant were isolated according to a
protocol described previously (38) with minor modifications. Briefly, infected
macrophages were added homogenization buffer and scraped from tissue culture flasks.
The cells were lysed by approximately 30 passages through a tuberculin syringe (at least
90% of the cells were lysed) and the lysate was carefully deposited over a 12% to 15%
sucrose gradient. The preparation was then centrifuged at 2000 rpm for 40 minutes at
4°C. After centrifugation, the interface was collected and centrifuged in 10% Ficoll
solution at 2500 rpm for 40 minutes at 4°C. A small pellet containing phagosomes was
visible at the bottom of the tube. Phagosomes were analyzed for purity visually on glass
slides by staining MAC 109 or 2D6 with lOjig/ml N-hydroxysuccinimidyl ester 5-(and-
6)- carboxyfluorescein (NHS-CF; Molecular Probes, Eugene, OR) for 1 h at 37°C.
Phagosomes containing live M. avium or 2D6 showed green fluorescence/green
fluorescein stain when observed under lOOx oil immersion (Leica DMLB Scope, Spot
Party Interface; Diagnostics Instruments Inc.). More than 95% of the phagosomes
observed showed bacteria in them.
3rd
14
Mass Spectrometry
The phagosome samples were run by ic/ms-ms using a Waters (Miliford, MA)
NanoAcquity HPLC connected to a Waters Q-Tof Ultima Global. 5 ul of a sample was
loaded onto a Waters Symmetry C18 trap at 4uLlmin, then the peptides were eluted from
the trap onto the 10cm x 75um Waters Atlantis analytical column at 3SOnlJmin. The
HPLC gradient went from 2% to 25% B in 30 minutes, then to 50% B in 35 mins, then
80% B in 40 mins and held there for 5 minutes. Solvent A was 0.1% formic acid in
water, and B was 0.1% formic acid in acetonitrile. Peptide "parent ions" were monitored
as they eluted from the analytical column with 0.5 seconds survey scans from mlz 400-
2000. Up to 3 parent ions per scan that had sufficient intensity, and had 2, 3, or 4
positive charges were chosen for ms/ms. The ms/ms scans were 2.4 seconds from mlz
50-2000.
The mass spectrometer was calibrated using the ms/ms spectrum from glu-fibrinopeptide.
Masses were corrected over the time the calibration was used (one day or less), using the
Waters MassLynx DXC system.
The raw data was processed with MassLynx 4.0 to produce pkl files, a set of smoothed
and centroided parent ion masses with the associated fragment ion masses. The pkl files
were searched with Mascot 2.0 (Matrix Science Ltd., London, UK) database searching
software, using mass tolerances of 0.2 for the parent and fragment masses. The Swiss
Prot database was used, limiting the searches to human proteins. Peaks Studio
(Bioinformatics Solutions Inc., Ontario, Canada) was also used to search the data, using
mass tolerances of 0.1, and the IPI human database.
15
Immunofluorescence Analysis
Because some of the proteins identified in the phagosomes have not been previously
described as part of the vacuole membrane, we examined their presence by using
immunofluorescence. Primary antibodies against pulmonary surfactant protein D (SP-D)
and T-type Ca alphall protein were purchased from Santa Cruz Biotechnology, Santa
Cruz, CA. Primary antibodies used were rabbit anti-SP-D and goat anti-T-type Ca
alphall. Secondary antibodies were Texas-Red conjugates (TR) and included donkey
anti-rabbit IgG-TR (Amersham Biosciences, Piscataway, NJ) and mouse anti-goat IgG-
TR (Santa Cruz Biotechnology, Santa Cruz, CA). The two-well chamber slides from
Nalge Nunc (Rochester, NY) were employed for macrophage monolayer preparation and
fluorescent microscopy. The numbers of U937 cells were determined in a
hemocytometer before seeding. A total of 5x105 cells were added in each tissue culture
well of the two-chamber slides and were differentiated with 2g/ml of PMA overnight.
The monolayers were then infected with MAC 109 and 2D6 labeled with NHS-CF as
described above using a MOI of 10. The cells were incubated for 4 h at 37°C for SP-D
protein expression and 24 h for T-type Ca++alphall protein expression. The timepoints
were chosen because of the expression results. The chambers were washed three times
with sterile phosphate buffer saline (PBS) and treated with 2OOtg/m1 amikacin to kill
extracellular bacteria. The cells were subsequently washed and allowed to air dry. Cells
were then fixed with 100% methanol at room temperature for 10 minutes, permeabilized
in cold 0.1% Triton X-100 (J.T Baker) and 0.1% sodium citrate for 20 minutes on ice.
Next, the monolayers were washed with PBS and blocked with 2% BSA (BSA, Sigma) in
PBS for 20 minutes at room temperature. The 2% BSA was replaced with 1 ml of
16
specific primary antibody and allowed to incubate for 1 h. All the antibodies were
prepared in 2% BSA in PBS to prevent non-specific binding. The cells were then washed
three times with sterile PBS and reincubated with the appropriate Texas-Red conjugated
secondary antibody for an additional 1 h. Macrophages were washed three times with
sterile PBS and allowed to air dry before adding Aqua-mount mounting media (Lerner
laboratories, Pittsburgh, PA) and cover slips (Corning, Corning, NY). Cell preparations
were visualized with a Leica DMLB Microscope. The microscope was operated by Spot
3rd
Party Interface Software with a Photoshop C.S version 8.0 on a Macintosh OS
(version 4.0.9) based system.
RNA extraction
For the DNA microarray, the U937 infection assay for MAC 109 and 2D6 mutant
followed by RNA isolation was carried out as described previously (28). Briefly, U937
monolayers of approximately 108 cells were infected with MAC1O9 or 2D6 (lx 108
concentration) for 4 h. The cells were washed to remove extracellular bacteria and total
RNA was isolated using Atlas pure Total RNA labeling System (Clonetech Laboratories,
Palo Alto, CA) according to the manufacturer's instructions. The resultant RNA was
treated with DNase for 30 mm at 37°C followed by phenol-chloroform extraction and
precipitation with ethanol. The RNA was run on 1% denaturing agarose gel and
quantified by UV spectrometer at 260/280 nm.
To confirm the expression and to determine the relative transcriptional levels of Gprotein coupled receptor kinase 4 (GRK-4), diacylglycerol kinase delta (DGKD) and
lymphocyte cytosolic protein 2 (LCP2) by real-time PCR, similar U937 infection assay
was performed as described above and modifications in the RNA extraction method were
17
made. After 4 h, the monolayers were washed with HBSS and scraped and collected in a
50 ml falcom tube and placed on ice. The cells were centrifuged at 500 rpm for 5
minutes to remove any residual extracellular bacteria. Then, 2 ml of Trizol (Invitrogen,
Carlsbad, CA) was added to the falcom tube. The suspension was then passaged 20 times
through 21-gauge needle to lyse the mononuclear cells. The lysate was then centrifuged
at max (14,000) rpm at 4°C. The supernatant was then transferred to heavy Lock Gel I
(Eppendorf, NY) and to it chloroform:isoamyl alcohol (24:1) (Sigma) was added and
mixed. After centrifugation, the aqueous phase was precipitated in isopropanol followed
by 75% ethanol wash to remove isopropanol. DNase treatment of total RNA was carried
out before probe synthesis using the protocol described by Atlas Pure Total RNA labeling
system (Clonetech, Mountain View, CA). The quality of RNA was verified on a 1%
denaturing agarose gel and the concentration was calculated based on the absorbance at
260nm.
cDNA was synthesized as per the protocol described by Invitrogen (Carlsbad, CA). Total
RNA (Sjig) with oligo(dT)20 and dNTP mix was incubated at 65°C for 5 mm and cooled
on ice for 1 mm. For each total RNA sample, 10 i1 cDNA synthesis mix was made: lOx
RT buffer, 25mM MgC12, 0.1 M DTT, 40U/il RNaseOUT
and 200U/pJ
Superscript III
RT. The samples were mixed gently and collected by brief centrifugation. Then, the
samples were incubated in a thermal cycler at 42°C for 50 mm and the reaction was
terminated at 70°C for 15 mm and cooled on ice. Finally, the reactions were collected by
brief centrifugation and 1 .tl of RNase H was added to each sample and incubated for 20
mm at 37°C. The cDNA prepared was used for real time PCR.
DNA Micro-Array
Yongjun Li carried out DNA microarray. The protocol is summarized below.
Preparation of 32P-labelled cDNA probes
32P-labelled cDNA probes were prepared using the Atlas pure Total RNA labeling
System (Clonetech Laboratories) as previously described (28). In brief, 5tg of total
RNA was reverse transcribed using the primer mix supplied with each array. The
mixture was heated to 65°C for 2 mm in a PCR thermal cycler followed by 50°C for 2
mm. At this time, a master mix containing Sx Reaction buffer, dNTP, dATP. DTT and
MMLV reverse transcriptase was added, mixed and incubated for 25 mm at 50°C. lox
termination mix was then added to end the reaction. Unincorporated nucleotides were
removed using a Nucleospin Extraction Spin Column (Clonetech Laboratories, Palo Alto,
CA) as per the manufacturer's instructions. Scintillation counting was done to measure
the incorporation of radionucleotide into the probe.
Hybridization and Data Analysis
In brief, the Clonetech Human Nylon Filter Arrays (Clonetech Laboratories) were
prehybridized in 5 ml of Express-Hyb solution supplemented with 0.5mg salmon testes
DNA at 68°C for 30 mm. The radiolabelled cDNA probe was heated in boiling waterbath
for 2 mm followed by keeping 2 mm on ice. Then it was added to the hybridization
solution and was allowed to hybridize to the filter array overnight. The membranes were
washed in SSC plus 0.1% sodium dodecyl sulphate (SDS) at 68°C for 30 mm and further
rinsed in SSC for 5 mm at room temperature. Next, the filters were wrapped in plastic
wrap and exposed to phosphor imaging screen for 24 h. Analysis of the phosphor
imaging screens was done by using a phosphor imager (Perkin Elmer, Boston, MA) and
19
Atlaslmage 2.0 software. Global normalization method was used to normalize the arrays
to the uninfected control array. The following genes were used as housekeeping genes
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), tubulin alpha 1 (TUBA 1),
hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), major histocompatibility
class 1 C (HLAC), beta-actin (ACTB) and 23-kDa highly basic protein (PRL13A). Only
genes that showed differential expression atleast by two-fold were incorporated in the
results. The experiment was repeated two times.
Real Time PCR
SYBR technology was used to perform real time PCR. Run protocol for the LightCycler
was as follows: denaturation 95°C for 5mm; amplification and quantification repeated
for 35 times: 95°C for 30 sec, 59°C for 30 sec and 72°C for 1 mm with one fluorescence
measurement followed by 72°C for 5 mm and 4°C.
The threshold cycle (Ct) is defined as the fractional cycle number at which the
fluorescence reaches lOx the standard deviation of the baseline and was quantified as
described in User Bulletin #2 for ABI PRIIMS 7700 sequence detection system (ABI).
The fold change in gene expression was determined using an amplification-based
strategy. For each gene amplification, before calculating the fold change, the Ct values
were normalized to the Ct of f3-actin using the following formula:
Fold Change
2-A(ACt)
where ACt = Ct,
target
Ct,
3-Actin ,
A(ACt) = ACt,
expressed
- ACt, control
Quantitative analysis was performed using iCycler I software (BIORAD, Hercules, CA).
A relative quantification was used in which the expression levels of macrophage target
genes were compared to data from a standard curve generated by amplifying several
dilutions of a known quantity of amplicons. Real time PCR efficiency was determined
using a dilution series of cDNA template with a fixed concentration of the primers.
Slopes calculated by the LightCycler software were used to calculate efficiency using the
following formula: E
10(1/slope)
These calculations indicated high real time efficiency
with a high linearity. Because expression of 13-actin is constant independent of
conditions, target genes from both, control and experimental groups were normalized to
the expression level of the 13-actin gene.
21
Results
Mass spectrometry analysis of isolated wild-type M. avium and 2D6 phagosomes
To identify the phagosomal membrane proteins expressed at various time points in
MAC 109 and 2D6 infected U-937 cells, we infected mononuclear cells with the
fluorescein-labeled bacteria and at 30 mm, 4 h and 24 h after infection, isolated the
phagosomes. As shown in Figures 1. A & B, both MAC 109 and 2D6 showed
comparable ability to enter the macrophages and be within phagosomes. MS/MS results
of the phagosomes revealed several proteins with function in bacterial uptake, antigen
presentation and recognition, Rab-interacting proteins, cytoskeleton and motor proteins,
proteins involved in biosynthetic pathways, transcriptional regulation, and signal
transduction proteins. Several of the proteins also have function as ion channels. A
number of hypothetical proteins were also identified (Table 1). Some proteins identified
were unique to MAC 109 or the 2D6 vacuoles at different time points. In addition, some
proteins identified were expressed in MAC1O9 as well as 2D6 vacuoles at single time
point or multiple time points. Together, these results suggest that the wild-type
phagosomes express a number of proteins different from the vacuole of the isogenic 2D6
mutant.
Immunofluorescence analysis
Many proteins identified in the mass spectrometry data have not previously been
described in M. avium phagosomes. To confirm the initial observation, we carried out
assays using fluorescent microscopy to establish the presence of some selected proteins.
One of the proteins chosen, pulmonary surfactant protein
D (SP-D)
was observed to be
selectively expressed in the MAC1O9 phagosomes at 4 h time point but not in the 2D6
22
A.
Figure 1. Fluorescent microscopy images of isolated phagosomes showing fluoresceinlabeled M. avium (A) and 2D6 mutant (B).
23
Table 1. List of phagosomal proteins identified by MS/MS post-infection with MAC 109
or 2D6 at different time points.
Protein
Accession
number
MAC 109
(h)
24
0.5
4
2D6 mutant
0.5
(h)
4
24
Bacterial Uptake
Complement clq tumor necrosis factor related protein 5
Complement receptor 2
Macrophage receptor MARCO
Pulmonary surfactant protein D
Scavenger receptor with C type Lectin type 1
TNFRSF1A-associated via death domain! TRADD
Tumor necrosis factor receptor member 1A ITNFRSF1A
Q9BXJO
P20023
Q9UEW3
P35247
Q9BYH7
Q15628
1NCFA
x
x
x
x
x
x
x
x
x
x
x
Antigen Presentation & Recognition
Integrin alpha 1
Integrin alpha JIb
MHC class I
MHC class II
P56199
x
x
x
x
P085 14
Q951D4
154427
Rab Interacting
EEA1
Peroxin 5-related protein
Rabaptin 5
Q15075
Q81YB4
Q15276
x
x
x
x
Cytoskeletal Proteins & Motors
Alpha actin
Ankyrin 1
Beta actin
E-MAP-115
Keratin, type II cytoskeletal I
KinesinlKIF3
Kinesin family member 26A
L-plastin
Myosin heavy chain non-muscle type A
Myosin X with ATPase activity
RHOC
TCP- 1 zeta
Tubulin alpha
Tubulin alpha 2
Tubulin alpha 3
Tubulin beta 2
P62736
Q6S8J3
Q96B34
Q14244
P04264
014782
Q8TAZ7
P13796
P35579
Q9HD67
P08134
P40227
Q71U36
Q13748
Q71U36
P07437
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Proteins in Biosynthetic Pathways
ABC transporter 2
ADP-ATP translocase
Aldoketo- reductase 3
ALG12
ATP synthase
Fatty acid synthase
Fatty acid transporter member 1
GAPDH
Phosphatidyl inositol glycan class T
Q9BZC7
P05141
x
x
x
Q9UFE 1
x
x
x
Q9BVJO
P06576
P493 27
Q6PCB7
P04406
Q92643
x
x
x
x
x
Table 1. continued
Protein
IMP dehydrogenase
K-Cl cotransporter
Mitochondrial dicarboxylate carrier
Neutral amino acid transporter
NUAK family, SNF1-like kinase 1
PI4KII
Pyruvate kinase
Ribophorin II
Trehalose precursor
Accession
number
MAC 109
(h)
4 24
0.5
2D6 mutant
0.5
P 12268
x
T17231
Q9UBX3
Q15758
x
24
x
x
060285
x
x
Q9BTU6
PSOO1 10
(h)
4
x
x
x
Q5JYR7
x
043280
x
Transcriptional Regulators
Lysine-specific histone demethylase 1
CRSP complex subunit 2
CREB binding protein
DEAH box polypeptide 9
WI 16
Msx2-interacting protein
p-lOO co-activator/SND 1
p-300 /CBP associated factor
Zinc finger & BTB domain containing protein 4
Zinc finger protein 588
Zinc finger protein 43
60S acidic ribosomal protein P2
60S ribosomal protein L6
60S ribosomal protein L9
60S ribosomal protein L14
060341
060244
Q75MY6
x
x
x
Q08211
Q16666
Q96T58
Q7KZF4
Q92831
Q9P1ZO
Q9U115
P 17038
P05387
Q02878
P32969
P50914
x
x
x
x
x
x
x
x
x
x
x
x
x
Proteins Interacting with Signal Proteins
cAMP-specific 3',S'- cyclic phospho-diesterase
Calcium & lipid binding proteinlNLF2
Chondroitin sulfate synthase 3
CXC3C membrane-associated chemokine
Doublecortin & CaM kinase like- 3
Dystrobrevin alpha
Golgin subfamily A member 5
Microtubule associated-Ser/The kinase 3
Protein kinase A anchoring protein 9
Protein kinase N
Serine/theonine kinase 16
TER ATPase
Q08493
388125
Q86Y52
P78423
Q9C098
Q9Y4J8
Q8TBA6
x
x
x
x
x
x
x
x
060307
Q99996
Q8NF44
x
x
Q5UOF8
x
P55072
x
Ion Channels
Amiloride-sensitive cation channel
Voltage dependent-N-type calcium channel alpha lB subunit
Voltage dependent-T-type calcium channel alpha 11 subunit
Q96FT7
Q00975
x
x
x
Q9PDX4
Other Proteins
AFG3 like protein 2
APBB1
Astrotactin 2
Q9Y4W6
000213
075129
x
x
x
25
Table 1. continued
Protein
Apoptotic chromatin-condensation inducer in the nucleus
Clathrin heavy chain 1
EphrinB3
48kda histamine receptor subunit peptide 4
HSP60
HSP90
Importin alpha 2
Interferon regulatory factor 6
LRCH4/Ligand binding receptor
NADPH oxidase activator 1
Protein disulfide- isomerase A6
p-53-associated parkin-like cytoplasmic protein/PARC
Serine protease inhibitor
Vitrin
14-3-3 protein/Tyrosine 3-monoxygenase
Accession
number
Q9UKV3
Q00610
Q15768
MAC1O9
(h)
24
4
0.5
2D6 mutant
0.5
(h)
4
x
x
x
x
AAB 34251
x
x
x
P10809
P07900
P52292
014896
075427
x
x
x
Q2TAM 1
P07237
Q8IWT3
Q9NQ38
24
x
x
x
x
x
Q6UX 17
P62258
x
Proteins with Unknown Function
Hypothetical protein DKFZp434A 128
Hypothetical protein LOC 136288
Hypothetical protein K1A1783
Hypothetical protein K1AA1783
Hypothetical protein FLJ32795
Hypothetical protein FLJ42875
Hypothetical protein FLJ4549 1
Hypothetical protein DKFZp434G131
Hypothetical protein FLJ46534
Hypothetical protein FLJ00361
T34567
Q8NEG2
Q96JP2
Q96JP2
Q96M63
Q8N6L5
Q6ZSI8
x
x
x
x
x
x
x
x
Q9HOH4
Q6ZR97
Q8NF4O
x
x
26
phagosomes. The second protein, T-type Ca2 channel 11 alpha showed selective
expression in 2D6 phagosomes at 24 h time point and not in the MAC1O9 phagosomes.
SP-D is a carbohydrate binding glycoprotein and has been shown to be involved
in ligand binding and immune cell recognition (37). To confirm the expression of SP-D
on MAC 109 vacuoles at 4 h time point, cells were infected in parallel with fluorescein-
labeled bacteria (MAC 109 and 2D6) for 4 h. The SP-D protein was observed to be
present in nearly all on the MAC 109 vacuoles (Fig 2. A, B, C) whereas in 2D6-infected
as well as in uninfected macrophage control, no SP-D was expressed (Fig 2. D, E, F &
Fig 2. G, H respectively). This finding confirmed our data that SP-D is expressed in
MAC 109 phagosomes but not 2D6 mutant phagosomes at 4 h time point.
T-type
Ca2
channel is an integral membrane protein, which controls the rapid
entry of Ca2 into excitable cells, and is activated by CaM-Kinase II (Swissprot
database). To verify our initial observation by MS/MS, we carried out parallel infection
assays with fluorescein labeled 2D6 and MAC 109 bacteria for 24 h. As shown in Figures
3. A & B, the majority of the cells infected with 2D6 mutant showed T-type Ca2 channel
protein staining whereas those infected with the wild type MAC 109 and uninfected
control U937 cells, all did not show any expression for the protein (Figures 3. C, D & Fig
3. B & F respectively). The finding confirmed that T-type Ca2 channel is expressed in
mononuclear phagocytes infected with 2D6 attenuated mutant, but not when infected
with MAC1O9.
To further investigate whether the complemented M. avium 2D6 mutant
phagosomes showed similar protein expression as that of wild type, we infected the cells
27
A.
M.avium 109
Green
D.
2D6 Mutant
Green
G. Uninfected Control
B.
SP-D Protein
Red
C.
No SP-D
F.
Merge
Merge
H. Uninfected Control
Figure 2. Fluorescent microscopy images of U-937 macrophages infected with MAC 109
or 2D6 mutant for SP-D protein expression.
2D6 Mutant
Green
A.
C.
M.avium 109
Green
E. Uninfected Control
T-Type Ca2+
Channel protein
B.
Red
n
NoT-TypeCa2+
F. Uninfected Control
Figure 3. Fluorescent microscopy images of U-937 macrophages infected with MAC 109
or 2D6 mutant for T-type Ca2+ channel protein expression.
with 2D6 complemented bacteria (23) for 4 h with MAC 109 as a positive control. The
vacuoles containing the complemented M. avium 2D6 mutant showed expression of SP-D
protein (Fig 4. A, B, C) similarly to vacuoles containing the wild type bacterium (Fig 4.
D & E), though the percentage of infected cells showing the expression was less than in
macrophages infected with the wild type bacterium. The results showed that the
complementation of the strain of M. avium 2D6 restores the phenotype of the wild-type
MAC 109 to some extent.
To determine whether the complemented M. avium 2D6 mutant phagosomes were
deficient in T-type Ca2 channel expression similar to the wild-type bacterium,
mononuclear cells were infected with complemented M. avium 2D6 bacteria. The 2D6-
attenuated mutant was employed as a positive control. As shown in Fig 5. A & B, the
complemented bacteria did not show the expression of T-type Ca2 channel protein
expression similar to wild type whereas the 2D6 mutant showed the protein to be
expressed on its phagosomes (Fig 5. C & D). On visual screening of the monolayers, it
was observed that as compared to the wild-type bacterium, only a smaller percentage of
cells infected with M. avium complemented 2D6 mutant did not express the T-type Ca2
channel protein. Again, this showed that the complemented M. avium 2D6 mutant strain
only partially restores the phenotype of wild type bacterium.
Analysis of differential gene expression in U937 cells after infection with MAC1O9
and 2D6 attenuated mutant by DNA microarray
The expression of genes in mononuclear phagocytes infected with MAC 109 and
2D6 was examined at 4 h post-inoculation. This time point was chosen based on
previous results (28). The majority of the genes examined did not show any significant
30
Complemented
M.avium
A.
2D6 Mutant
Green
B.
SP-D Protein
Red
C.
Merge
M.avium 109
Green
D. Positive Control
E.
SP-D Protein
Red
Figure 4. Fluorescent microscopy images of U-937 macrophages infected with
complemented M. avium 2D6 mutant for SP-D protein expression.
31
Complemented
M. avium
No T-Type Ca2+
Channel Staining
2D6 Mutant
Green
I
2D6 Mutant
Green
C. Positive Control
T-Type Ca2+
Channel protein
D.
Red
Figure 5. Fluorescent microscopy images of U-937 macrophages infected with
complemented M. avium 2D6 mutant for T-type Ca2+ channel protein expression
32
difference in expression (2 fold or above) after infection with MAC 109 and 2D6 mutant.
M. avium-infected cells showed low numbers (19) of differentially expressed genes
(Table 2), as compared to 2D6-infected cells which showed greater number (35) of
differentially regulated genes (Table 3). The mononuclear cells infected with wild-type
or 2D6 mutant showed no similarities in gene expression pattern. The genes upregulated
in cells infected with wild-type bacteria consisted mainly of those involved in
intracellular signaling such as LCK, PKIA, DGKA, DGKD, INPP1, APBA2 and PDE1C.
Few of the other genes were involved in the metabolic pathways like GPD2 (involved in
glycerol-3-phosphate metabolism) and CYP4F2 (involved in leukotriene metabolism).
Other genes that showed upregulation were PPM1G (cell cycle arrest), HIPK3 and
RORC (inhibition of apoptosis), ITK (T-cell proliferation and differentiation), GRK4
(regulation of G-protein coupled receptor protein signaling), NFKB 1 (transcriptional
regulator) and others. The genes downregulated in wild-type but upregulated in 2D6
mutant included genes involved in signal transduction (BMX, CCR3, GPR17, GABBR1,
GABBR2, YWHAZ, RAB7, RAB 13, IFNA1, DGKZ and DGKG), apoptosis (BLK,
GZMA), bacterial uptake (ITGB 1, CR1), immune response (IL1ORA, TNFRSF17,
MS4A1, LCP2), metabolic pathways (DDOST, PLTP), and others like bacterial killing
(cathepsin G), negative regulators of G-protein signaling (RGS 12 & RGS 13), potassium
channel regulator (CHP), microtubule movement (TUBB, DCTN1, CETN2 & S100A1 1).
Macrophage gene expression analysis by quantitative real time PCR
To confirm the changes in macrophage gene expression level upon infection with M.
avium or its isogenic 2D6 mutant from the DNA microarray data findings, three genes
were selected for real time PCR analysis, GRK4 (G-protein coupled receptor kinase 4),
33
Table 2. Macrophage genes upregulated in M avium 109 but downregulated in 2D6
mutant 4h post infection
Gene
Gene Bank ID
Name
Amyloid beta (A4) precursor protein
binding
Cytochrome P450
APBA2
ABO 14719
CYP4F2
U02388
DGKA
DGKD
GPD2
AF064767
D63479
AB002323
NM_000408
GRK4
NM_005307
HIPK3
AF004849
ThPPl
NM_002 194
ITK
D13720
LCK
M36881
NFKBI
M58603
PDE1C
U40371
PKIA
S76965
PPM1G
Yl3936
PTPN1 1
Dl 3540
Calcium/calmodulin-dependant 3',
5'- cyclic nucleotide
phosphodiesterase I C
Protein kinase (cAmp-dependent)
inhibitor alpha
Serine/threonine protein phosphatase
PP1-gamma I catalytic subunit
Protein tyrosine phosphatase
RGS3
AF006610
Regulator of G-protein signaling- 3
RORC
U16997
RAR-related orphan receptor C
ROR1
M97675
Receptor tyrosine kinase-like orphan
receptor 1
DYNCIHI
Function
Signal transduction
Inactivation & degradation of
leukotriene B4
Intracellular signaling
Diacylglycerol kinase alpha
Phosphatidylinositol signaling
Diacyiglycerol kinase delta
Microtubule reorganization
Cytosolic dyenin heavy chain
Glycerol-3-phosphate dehydrogenase Glycerol-3-phosphate
metabolism
2
Regulation of G-protein coupled
G-protein coupled receptor kinase 4
receptor protein signaling
Inhibition of apoptosis
Homeodomain interacting protein
kinase 3
Phosphatidylinositol signaling
Inositol polyphosphate-1phosphatase
T-cell proliferation &
1L2-inducible T-cell kinase
differentiation
Intracellular signaling
Lymphocyte-specific protein
tyrosine kinase
Transcriptional regulator
Nuclear factor of kappa light
polypeptide gene enhancer in B-cells
Fold
induction
10.7
2.6
2.3
6.7
17.4
3.5
3.5
2.05
2.0
2.4
3.5
2.3
Signal transduction
17.4
Negative regulation of protein
kinase A
Negative regulator of cell stress
response/cell cycle arrest
Intracellular signaling, cell
migration
Inhibition of G-protein mediated
signal transduction
Inhibition of Fas ligand and 1L2
expression
Unknown
2.0
3.2
2.4
3.4
3.1
4.0
iI
Table 3. Macrophage genes downregulated in M avium 109 but upregulated in 2D6
mutant 4 h post infection
Gene
Gene Bank
Name
AMBP
ID
X04494
Alpha-1-microglobulin
BLK
BMX
CCR3
CD53
CETN2
CHP
BC004473
AF045459
AF247361
BC040693
X72964
NP_009 167
B-lymphoid tyrosine kinase
BMX non-receptor tyrosine kinase
Chemokine receptor 3
CD53 molecule
Centrin, EF-hand protein 2
Calcium binding protein P22
CR1
CTSG
DCTN1
Y00816
NM_001911
NM_004082
Complement receptor I
Cathepsin G
Dynactin I
DDOST
D29643
DGKG
DGKZ
EMILIN2
FOXC2
GABBR1
GABBR2
GPR17
GZMA
IFNA1
AF020945
Dolichyl-diphosphooligosaccharideprotein glycosyltransferase
Diacylglycerol kinase gamma
Diacylglycerol kinase zeta
Elastin microfibril interfacer 2
Forkhead box C2
GABA receptor I
GABA receptor 2
G-protein coupled receptor 17
Granzyme A
Interferon alpha I
Interleukin 10 receptor alpha
US 1477
ILI0RA
AF270513
Y08223
Yl 1044
AF069755
NM_005291
NM_006 144
NM_024013
U00672
ITGB1
LCP2
MCAM
MS4AI
PBX2
BCO20057
NM_005 565
X68264
M27394
NM_0025 86
PLTP
RAB7
RABI3
NM 006227
X93499
X75593
Fibronectin receptor
Lymphocyte cytosolic protein 2
Melanoma cell adhesion molecule
Membrane-spanning 4-dmains
Pre-R-cell leukemia transcription
factor 2
Phospholipid transfer protein
RAS related GTP binding protein
RAS related GTP binding protein
RGS 12
AFO3 5152
Regulator of G-protein signaling 12
RGS13
AF030107
Regulator of G-protein signaling 13
SIOOA11
NM_005620
Calgizzarin
TNFRSF17
TUBB
YWHAZ
Z29574
AB062393
NM_003406
TNF receptor
Tubulin beta
Tyrosine 3 -monooxygenase
.
Function
Negative regulation of immune
response/Protease inhibitor
Apoptosis
Intracellular signaling
Signal transduction
Growth regulation
Microtubule organization center
Potassium channel
regulator/Signal transduction
Bacterial uptake
Bacterial killing
Lysosome and endosome
movement
N-linked glycosylation
Intracellular signaling
Intracellular signaling
Cell adhesion
Transcription factor
Signal transduction
Signal transduction
Signal transduction
Apoptosis
Intracellular signaling
Inhibition of proinflammatory
cytokine synthesis
Bacterial uptake
Immune response
Cell adhesion
Immune response
Transcriptional activator
Lipid metabolism
Vesicular transport regulation
Small GTPase mediated signal
transduction
Negative regulation of G-protein
signaling
Negative regulation of G-protein
signaling
Motility, invasion & tubulin
polymerization
Immune response
Microtubule based movement
Signal transduction
Fold
induction
4.2
3.3
18.6
4.1
4.1
6.3
20.8
4.3
2.9
35.8
3.3
5.3
48.1
14.7
5.9
6.1
2.8
83.2
2.1
2.6
2.3
4.6
37.5
4.7
9.6
3.0
3.6
3.4
7.5
3.0
2.6
9.6
2.6
4.0
4.3
35
DGKD (Diacyiglycerol kinase delta), and LCP2 (Lymphocyte cytosolic protein 2). The
gene 13-actin was used as a positive control, while the uninfected cells were used as a
negative control. The two genes GRK4 and DGKD were found to be upregulated in the
wild-type but downregulated in the 2D6 mutant infected macrophages whereas LCP2 was
found to be downregulated in wild-type but upregulated in the 2D6 mutant in DNA array
data analysis. The two genes GRK4 and DGKD showed significant upregulation upon
M. avium infection of macrophages in contrast to infection by 2D6 mutant (Fig 6). In
addition, LCP2 gene showed significant upregulation in macrophages upon infection with
2D6 mutant in contrast to wild-type infected macrophages. None of the three genes
showed upregulation in the uninfected negative control cells. These results confirmed
our DNA array data.
36
14
12
10
Macrophage Genes
Figure 6. Upregulation of U937 macrophage genes upon infection with M. avium or 2D6
mutant at 4 h as determined by real-time PCR. The data represents the average of 3
independent experiments ± SD.
37
Discussion
M. avium like M. tuberculosis, primarily infect the host mononuclear phagocytes.
Targeting mononuclear phagocytes and being able to survive within them indicate
efficient mechanisms evolved by the virulent mycobacteria capable of neutralizing the
bactericidal activity of macrophages.
In M. tuberculosis, PE-PGRS and PPE are two glycine-rich protein families
which constitute approximately 10% of the M. tuberculosis genome. Recent reports have
suggested that these two gene families might be involved in antigen variation, eukaryotic
cell binding, survival within macrophages and persistence in granulomas (31, 35).
Richardson and colleagues (2001) showed that a PPE protein (Rv1917) is expressed on
the bacterial surface. Using signature-tagged mutagenesis, Camacho and colleagues
identified a PPE gene (Rv3018c) associated with M. tuberculosis virulence in vivo (6).
In addition, Ramakrishnan and colleagues observed that inactivation of PE-PGRS gene in
M.marinum resulted in attenuation of bacterial virulence in macrophages.
A study by Yongjun Li and colleagues (2005) has suggested a role of M. avium
PPE protein (Rv1787) in inhibition of acidification and phagosome-lysosome fusion in
infected macrophages. They demonstrated the M. avium PPE transposon mutant had
comparable ability to enter the mononuclear phagocytes as wild-type but was defective in
survival and virulence in vitro as well as in mice. This gene was upregulated following
uptake of the wild-type bacteria by macrophages, but not in culture media, indicating a
possible role in the early events of the phagosome formation. This observation suggests
that this particular PPE gene might play a role in the modulation of the phagosome
membrane thereby having an important participation on the dynamics and creation of the
vacuole membrane and the vacuole content. This would enable the bacteria to create an
environment inside the phagosome suitable for its growth and survival. The fact that the
2D6 mutant vacuole undergoes acidification implies that this PPE gene might play a role
in inhibition of acquisition of proton pumps on the wild-type phagosome.
The gene homologue to Rv1787 is part of a M. avium chromosomal region with
five PPE and PE genes. The organization of this region suggests the existence of three
promoters, one upstream of the PPE gene inactivated in the 2D6 mutant, one between the
second and the third genes and another between the fourth and fifth genes in the
downstream region (23). Therefore, it is possible that the inactivation of the PPE gene in
2D6 mutant would have a polar effect in three or all the five genes responsible for the
observed phenotype. The fact that complementation of the 2D6 mutant significantly
reversed (not completely) the phenotype, strongly suggest that only the PPE gene
inactivated and the two additional downstream genes are responsible for the phenotype
observed.
Normally upon uptake of a bacterium, macrophage undergoes a series of events
specifically designed to eliminate the engulfed microorganism, which include (i)
induction of reactive oxygen and nitrogen intermediates, (ii) gradual acidification of the
phagosome due to the activity of proton-ATPase pumps located on the phagosome
membrane, (iii) phagosome-lysosome fusion which loads the resulting phagolysosome
with acidic proteolytic enzymes, (iv) antigen processing and (v) antigen presentation.
The resulting lethal environment effectively kills the majority of the ingested bacteria.
Pathogenic mycobacterial phagosomes in contrast, show incomplete lumenal
acidification and absence of mature lysosomal hydrolases (41). Recent findings by Malik
et a! (25-27) suggested that the bacterial manipulation of calcium is in part responsible
for the phagosome maturation arrest. The pathogenic mycobacterial phagosomes have
been shown to alter the trafficking of the plasma membrane markers including MHC
molecules (7), EEA-1 and LAMP-i (42). Via and colleagues also suggested that the
block in phagosome maturation occur between the stages controlled by early endocytic
marker RabS and late endocytic marker Rab7, and that Rab5 is accumulated on the
mycobacterial phagosome membrane whereas Rab7 is not (42). Altogether, the
published data indicate that virulent mycobacterial phagosomes are selective in their
fusion with various cytoplasmic organelles and do not mature into a phagosomelysosome.
Although a good deal of information has been generated about the M. tuberculosis
phagosome, relatively little is known about M. avium vacuole. Once inside a
macrophage, M. avium resides in compartments secluded from the terminal stages of the
endocytic pathway (8, 39). The bacterial vacuoles have less proteolytic activity and
maintain some markers like LAMP-i and cathepsin D in an immature form (38, 39). The
vacuoles also remain accessible to internalized transferrin (38). The wild-type bacterium
requires RabS and not Rab7 as well as acquisition of iron for arresting the phagosome
maturation (22). Tn addition, Kelley and colleagues proposed that absence of Rab5 limits
the concentration of iron within the M. avium phagosome resulting in its inability to
inhibit phagosome maturation as evident by the decreased LAMP-i staining on M. avium
phagosomes after addition of ferric citrate in the dominant negative RabS expressing
macrophages (22).
EI
We have identified several proteins expressed on the phagosomes of macrophages
infected with wild-type M. avium and its isogenic 2D6 mutant in which a PPE gene
(homologue to Rv1787) was interrupted by the transposon. An earlier study has shown
the impaired ability of 2D6 mutant to replicate inside the mononuclear phagocytes (23).
Mass spectrometry analysis of the phagosomal proteins of 2D6 mutant and the wild-type
bacterium yielded several differences in the protein expression in the vacuole membrane.
For example, expression of EEA- 1 and Rab5 effector was seen on 2D6 phagosomes but
not on the wild-type phagosomes which is in agreement with the observation reported by
Fratti el al and Via et al (14, 42).
A relatively large amount of published data suggests the role of complement
receptors CR1, CR3 and CR4 (36) and mannose receptor (36) in the uptake of M.
tuberculosis by macrophages. CR3 has been shown to be one of the main receptors
involved in phagocytosis of M. avium by macrophages and monocytes (3, 34). In
addition, integrin fibronectin receptor is also involved in the internalization of the
bacteria (21). CR2 was identified among various receptors on M. avium phagosomes.
Studies have suggested an important role of CR1/2, CR3 and CR4 in host defense against
Streptococcus pneumoniae infections (32). Functional studies have demonstrated that
CR2 mediates tyrosine phosphorylation of 95 kDa nucleolin and its interaction
phosphatidylinositol 3 kinase (2). Though the presence of CR2 on M. avium phagosomes
at 24 h time point does not confirm its role in bacterial uptake, it could be possible that it
might play a role in the uptake and may not have been recycled. Also, the possibility of
its presence on the phagosomal membrane due to just being a membrane protein present
on the surface of plasma membrane cannot be ruled out. It is known that M. tuberculosis
41
and M. avium are similar in their invasion strategy of mononuclear phagocytes. But there
could be little differences in the receptors involved during the uptake of both bacteria.
Thus, CR2 could be another receptor involved in M. avium uptake by macrophages
besides CR3 and CR4.
Surfactant protein A (SP-A) expressed on M.
tuberculosis
vacuoles has been
shown to be involved in enhancing the uptake of bacteria by macrophages (17, 24, 44).
We identified SP-D protein expression on M. avium phagosomes. Surfactant-associated
proteins A and D (SP-D) are pulmonary collectins that bind to bacterial, fungal and viral
pathogens and have multiple classes of receptors on pneumocyte and macrophage
membrane (12). In addition, they act as chemoattractant to phagocytes.
Among the strategies mycobacteria has evolved to compromise the activation of
T-cells, is the down regulation of the expression of MHC class II, CD1 and co-
stimulatory molecules (19, 40, 45). The lack of MHC class II molecule expression in M.
avium phagosomes and its presence in the attenuated 2D6 mutant phagosomes in our data
is in agreement with the above findings. MHC class I molecules are involved in
presentation of the antigens located in the cytoplasm. The fact that MHC class I
molecules were found on 2D6 mutant phagosomes at 24 h time point may reflect altered
trafficking by the bacteria as its expression at early time points on the phagosome would
have suggested that the protein being present on the cell surface, during phagocytosis
would have been taken up during vacuole formation. The presence of MHC class I
molecules on the 2D6 phagosomes could also be due to the fact that mycobacterium
antigens are processed by MHC class 1(13). MHC class I has also been reported to be
present on latex bead phagosomes (16).
42
Several proteins not previously shown to be associated with the mycobacterial
phagosomes were identified in the phagosomal preparations. Because we cannot rule out
the possibility of contamination of the phagosome preparations with other organelles,
which indeed is a limiting factor of most subcellular fractionation techniques, we selected
two proteins which has not been previously described on vacuole membranes, voltage
dependent T-type calcium channel 11 alpha protein (T-type calcium channel), an integral
membrane protein unique to neuron cells and, pulmonary surfactant protein D (SP-D)
which is a carbohydrate binding protein, to confirm the expression of some of these
proteins on the mycobacterial phagosomes. Fluorescent microscopy studies using
antibodies specific to these proteins revealed their presence on the bacterial phagosomes.
Recent studies on Legionella and Brucella state that these organisms reside in
compartments displaying features of endoplasmic reticulum (ER) (29). In addition, there
is an evidence of recruitment of endoplasmic reticulum (ER) to nascent phagosomes
containing inert particles or Leishmania and that it makes a major contribution to the
phagosomal membrane (15). This explains how antigens of vacuolar pathogens are
presented to T lymphocytes via MHC class I machinery located on ER. Considering this
information, it would be plausible to find ER particles on mycobacterial phagosomes.
Some of the mitochondrial proteins like ATP synthase and HSP6O found in our
preparations have also been shown to be present in latex bead containing phagosomes
(16). This data is in agreement that at least some of the proteins present in our
preparations might be genuine constituents of phagosomes.
In the DNA array analysis, upregulation of genes like GZMA (granzyme A),
cathepsin G was observed in macrophages infected with 2D6 mutant. GZMA is a serine
43
protease involved in lysis of target cells. Cathepsin G is a protease which participates in
the killing and digestion of engulfed pathogens. Upregulation of GZMA and cathepsin
G in the macrophages infected with 2D6 mutant might reflect the impaired ability of 2D6
to survive and thus undergoing killing in macrophages and other mononuclear
phagocytes.
Recent report on the elemental analysis of M. avium phagosomes in Balb/c mouse
macrophages revealed high concentrations of potassium and chlorine at 24 h timepoint
and correlated it to the microbiocidal killing similar to that observed in neutrophils (43).
The upregulation of CHP (potassium channel regulator) in the 2D6 infected macrophage
microarray data and the finding of K-Cl transporter on the 2D6 mutant phagosomes at 24
h time point in our mass spectrometry data could represent an evidence at least in part to
the above published report as we know that the 2D6 mutant is unable to survive in the
macrophages (23). Therefore, there is a possibility that K-Cl transporter and CHP could
be involved in the augmentation of the potassium and chlorine concentrations in the
phagosome leading to mutant killing.
Studies in mice suggest that RORC (RAR-related orphan receptor C) may inhibit
the expression of Fas ligand and 1L2 (NCBI database). The upregulation of RORC in
macrophages infected with wild-type bacterium may indicate that this gene plays a role in
inhibition of apoptosis induced by the bacterium. HIPK3 (homeodomain interacting
protein kinase 3) is a Fas/FADD-interacting serine/threonine kinase that induces FADD
phosphorylation and inhibits one of the Fas-mediated apoptosis (33). The upregulation of
HIPK3 in wild-type infected macrophages shows the bacterial ability to inhibit apoptosis
by triggering induction of macrophage genes involved in inhibition of apoptosis.
As the 2D6 mutant and the wild-type bacterium have comparable ability to enter
the macrophages (23), it would be reasonable to expect that the 2D6 mutant uses similar
uptake pathway as wild-type. This is evident from the upregulation of various receptors
like ITGB1, GPR17, GABBR1, GABBR2, CR1, and CCR3 in the 2D6-infected
macrophages. The upregulation of Rab7 on the 2D6-infected macrophages is an
evidence that the 2D6 mutant expresses late endosome markers and undergoes
phagolysosome fusion (23).
GRK4 (G-protein coupled receptor kinase 4) is normally expressed in cerebellar
purkinje cells where it mediates desensitization of GABA receptor 1 (20). The DNA
array data showed upregulation of GRK4 in macrophages infected with wild-type
bacterium. There is no published data regarding GRK4 expression in M. avium infected
macrophages. Real-time PCR confirmed the upregulation of this gene in macrophages
infected with M. avium. This suggests that GRK4 is differentially expressed in M. avium
infected macrophages. Real-time PCR also confirmed the upregulation of DGKD
(Diacyiglycerol kinase delta) and LCP2 (Lymphocyte cytosolic protein 2) in the wild-
type and 2D6-infected macrophages respectively. These results show that the abovementioned genes are differentially regulated in macrophages when infected with wild-
type or 2D6 mutant. The variation in the fold intensity of these genes in the real-time
data as compared to the array could be because of the degradation of RNA during
handling and cDNA synthesis.
We studied protein expression of the mycobacterial phagosome and compared it
to a isogenic mutant. We identified several proteins either previously described or not
reported to be present on the phagosomes. Nonetheless, the use of the PPE mutant
45
established possibility that one protein may have key function in modulating the
formation of the phagosome. Alternatively, the PPE-PE operon may be part of a complex
system influencing or impacting the expression of other bacterial genes. Another
interesting aspect of our findings is the possible connection between macrophage genes
regulated upon uptake of a bacterium and the downstream effect on vacuole formation.
Future studies will address some of the differences found in this study and possibly
would provide insights on the mechanisms of pathogenesis and survival of mycobacteria
inside the host.
Acknowledgements
This publication was made possible in part by the Mass Spectrometry Core Facility of the
Environmental Health Sciences Center, Oregon State University, from grant number P30
ESOO21O, National Institute of Environmental Health Sciences, National Institutes of
Health.
We thank Denny Weber for preparing the manuscript.
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Discussion
The macrophage provides the primary defense against microbial infection.
Different intracellular pathogens have evolved to exploit diverse niches within their host
cell. Some escape into the cytoplasm; others are immune to digestion within the
lysosomes; and the third group, which includes pathogenic mycobacterial species,
prevents the normal maturation of their phagosome and fusion with the lysosomes.
Although the lack of fusion of mycobacterial vacuoles with lysosomes was observed
years ago by Hart and colleagues
(15-17),
35
the exact nature of this vacuole and its
relationship to other endocytic vesicles remains elusive. PE-PGRS and PPE gene
families have recently been shown to be involved in mycobacterial virulence in vivo.
Some studies have suggested their role in the phagosome maturation arrest phenomenon.
In this study, we determine the phagosomal membrane proteins expressed on the
macrophage upon M. avium infection and compare it with those expressed on
phagosomes in PPE gene inactivated 2D6 mutant-infected macrophages. The intent in
this study was to detect significant differences in the protein expression of the
phagosomes of wild-type M. avium and its isogenic 2D6 mutant. Analysis of differential
gene expression in macrophages infected with M. avium and its isogenic 2D6 mutant was
also carried out to determine the differences in the nature of genes upregulated by wildtype and 2D6-infected macrophages.
In the first part of the study, an array of proteins was identified in the wild-type,
as well as the 2D6 phagosomes. Some of the proteins such as the macrophage receptor
MARCO, complement Clq, pyruvate kinase, etc. were found to be expressed on both
bacterial phagosomes; while the majority of the proteins expressed on wild-type
52
phagosomes were different from the 2D6 mutant phagosomes. This indicates that the
wild-type bacterium potentially live in an environment quite different from its mutant.
Many proteins identified in the phagosomal preparations have not been previously
described to be present on the phagosomes. This could be a result of contamination of
the preparation with cytoplasmic contents. To confirm the presence of some of these
proteins, fluorescent microscopy experiments were carried out, which revealed that these
proteins are actually expressed on the phagosomes. In summary, numerous proteins were
identified in the M. avium and its isogenic mutant phagosomes, some of which have not
been previously reported. These proteins might unveil the underlying mechanisms used
by the bacterium to prevent the phagosome-lysosome fusion.
The second part of the study involved the identification of differential gene
expression in macrophages after infection with the wild-type or the 2D6 mutant. The
DNA array carried out to find this piece of information showed a whole range of genes
which were differentially regulated upon M. avium or 2D6 mutant infection. The genes
expressed in macrophages infected with wild-type was quite different from those
expressed in 2D6 mutant-infected macrophages. The genes upregulated in the wild-type
infected macrophages were fewer in number, compared to its isogenic mutant, and
included a variety of kinases, anti-apoptotic genes, and intracellular signaling molecules;
while the 2D6-infected macrophages showed upregulation in genes involved in apoptosis,
bacterial uptake, signal transduction and immune response. Some genes which showed
upregulation in macrophages have not been previously described to be expressed in
macrophages, for example, the G-protein coupled receptor kinase 4 (GRK4). To confirm
the expression, real time PCR was employed to validate the expression of GRK4 in
53
macrophages upon M. avium infection. Overall, the results reflect the diverse nature of
genes upregulated in macrophages infected with wild-type or the 2D6 mutant.
The study reveals differences in the vacuolar protein expression and gene
expression in macrophages after infection with either the wild-type or the PPE gene
inactivated mutant. Many proteins from the mass spectrometry data and genes from the
DNA microarray data suggest that the 2D6 mutant undergoes phagosome-lysosome
fusion, and killing inside the macrophage suggests a possible role of PPE gene in
mycobacterial virulence in vivo.
While working on the fluorescent microscopy portion of this project, I dealt with
two problems. First, the background on the slides was very strong and caused problem in
identifying the expression of the protein in infected cells after staining with primary and
secondary antibodies. Following the washing protocol several times did not change the
background issue. After increasing the washing steps, the problem still persisted. Then I
did titration experiment using different dilutions of the primary and secondary antibodies
to figure out the correct concentrations to use. The titration experiment did help
somewhat with the background issue. After determining the correct concentrations of the
antibodies, I tried to improve the washing procedure by washing the slides 2 times,
keeping them on the shaker for ten minutes during the wash process, and then aspirating
the fluid. This helped reduce the background to a great extent.
The main aim of the fluorescent microscopy studies was to clearly identify the
labeled bacteria and the protein expression on the phagosome in the infected
macrophages. When observed under the microscope, nearly all cells were flooded with
too many bacteria and it was difficult to detect the protein on a single phagosome.
Initially, cells were infected in a 2-chamber slide with 1 ml of bacterial suspension, so I
tried to infect the cells with fewer bacteria. This did not produce the desired results. Then
a labmate suggested declumping the bacteria by pipetting the bacterial suspension several
times and letting it stand for at least 10 minutes before using the superficial layer of the
suspension to infect the cells with 100 to 150 t1 of the bacterial suspension. This helped
to infect nearly all cells with as less as 3-4 bacteria instead of hundreds per cell. The
technique allowed visualizing the expression of the proteins on the phagosome of the
infected macrophages very clearly.
RNA extraction of eukaryotic cells is easy if done very carefully. The RNA
extracted from the infected U937 cells looked very good on the gels prior to DNase
treatment. After DNase treatment, I amount of RNA decreased and also the quality of
RNA was very poor. I quickly learnt not to treat all the good RNA with DNase at a time.
Treating only one sample of RNA with DNase enzyme, instead of all of it, I saved time
by not having to extract RNA from the first step again. Finally, good DNase-treated
RNA was produced when I used a new DNase enzyme and changed both the incubation
time and place for the RNA. That is, instead of keeping the RNA in the cell culture
incubator for 1 h after adding DNAse enzyme, I kept the RNA at room temperature on
the bench for 2 h. This yielded a good quality RNA after DNase treatment. The
designing of the correct primers that would amplify the genes for the real-time PCR study
took 2 months (Table 4 & Fig 7). The difference in the fold induction in the microarray
data and the real-time PCR could be due to the degradation of RNA, while handling, and
cDNA synthesis.
55
Table 4. Sense and antisense primers for real-time PCR
Primers
Target
PCR product
(bp)
13-actin
5' TGATGGTGGGCATGGGTCAGA 3'
800
5' CCCATGCCAATCTCATCTTGT 3'
GRK4
5' AATGTATGCCTGCAAAAAGC 3'
235
5' GATTGCCCAGGTTGTAAATG 3'
DGKD
5' CTCGGCTTACGGTTATTCCAG 3'
656
5' CCATCTCCATCTTCAGCCTCC 3'
LCP2
5'
CACTGAGGAATGTGCCCTTTC 3'
5' GTGCCTCTTCCTCCTCATTGG 3'
408
1200
1100
1000
900
u
b 800
v
U
Sd
700
10
.1
500
400
300
200
U
.
100
0
-100
-200
o
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
Cycle
Figure 7. Real-time PCR graph showing upregulation of genes in macrophage upon M.
avium or 2D6 mutant infection.
57
Several useful conclusions and technique improvements were achieved during this study.
Most importantly, we have provided an insight into the array of proteins expressed on the
mycobacterial phagosome at different time points, along with proteins which have not
been previously been described to be present on the phagosomes. Furthermore, we have
studied the gene expression profile of the macrophages upon M. avium infection and
generated a useful amount of data reflecting the pattern of gene regulation in
macrophages. A few genes, not previously been shown to be regulated in macrophages,
were observed in our data. Future research will address some of the differences observed
in this study and will provide a detailed view of the pathogenesis and survival
mechanisms of mycobacteria inside the host.
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