chlamydia-infected cells by a project
TRANSCRIPT
The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected cells
by William G. Valiant
A PROJECT
submitted to
Oregon State University
University Honors College
in partial fulfillment of the requirements for the
degree of
Honors Baccalaureate of Science Microbiology (Honors Scholar)
Presented May 13, 2015 Commencement June 2015
AN ABSTRACT OF THE THESIS OF
William Valiant for the degree of Honors Baccalauriate of Science in Microbiology presented on May 13, 2015. Title: The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected cells.
Abstract approved: __________________________________________________________________
Daniel Rockey
Our laboratory is exploring the mechanism of action of a novel broad
spectrum antiviral, ST-‐669. This compound has activity against a variety of
different viruses and the obligate intracellular bacterium, Chlamydia. In this
study, we explored the effects of ST-‐669 when the cell cycle of the host cells
was altered. Chlamydia spp. were grown in Vero cells in the presence of
various compounds that inhibit the eukaryotic cell cycle, and examined for
inclusion structure and production of bacteria. ST-‐669 was used to examine
the inclusion structure of C. caviae: when treated with ST-‐669, C. caviae
appears to fuse vacuoles to form an inclusion with a single lobe. Most of the
cell cycle inhibitors did not alter the anti-‐chlamydial effects of ST-‐669.
However, treatment of infected cells with vincristine led to an increase in
bacteria production and change in inclusion morphology in the presence of
ST-‐669. It is hypothesized that a protein that is a target of ST-‐669 is
differently present or activated when Vero cells were treated with
vincristine. These results open the door for future proteomic studies that
might elucidate ST-‐669’s mechanism of action.
Key Words: Chlamydia, Inclusion, ST-‐669, Anti-‐chlamydial
Corresponding email address: [email protected]
©Copyright by William Valiant May 13, 2015
All Rights Reserved
The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected cells
by William G. Valiant
A PROJECT
submitted to
Oregon State University
University Honors College
in partial fulfillment of the requirements for the
degree of
Honors Baccalaureate of Science Microbiology (Honors Scholar)
Presented May 13, 2015 Commencement June 2015
Honors Baccalaureate of Science in Microbiology project of William Valiant presented on May 13, 2015. APPROVED: Daniel Rockey, Mentor, representing Biomedical Sciences Ling Jin, Committee Member, representing Biomedical Sciences Brian Dolan, Committee Member, representing Biomedical Sciences Toni Doolen, Dean, University Honors College I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request.
William Valiant, Author
Acknowledgements
Most importantly I would like to thank Dr. Rockey for all the assistance
and mentorship he has provided over the last four years. Thanks to him I will
continue to participate in research for many more years. I would also like to thank
the entirety of the Rockey lab for their continued assistance as well as Dr. Hruby
and Dr. Allen at Siga technologies for their technical assistance.
Table of Contents
Page
Introduction ......................................................................................................................... 1 Chlamydia ....................................................................................................................... 1 ST-669 ............................................................................................................................. 3 The Eukaryotic Cell Cycle .............................................................................................. 5
Thesis Statement ................................................................................................................. 8 Methods ............................................................................................................................... 9
Chlamydial Strains .......................................................................................................... 9 Cell Culture and Chlamydial Infection ........................................................................... 9 Genome quantification .................................................................................................. 10 Fluorescence Microscopy ............................................................................................. 11
Results ............................................................................................................................... 13 ST-669 treatment affects C. caviae inclusion structure ................................................ 13 Anti-chlamydial effects of ST-669 in the presence of Cell Cycle Inhibitors ............... 13
Discussion ......................................................................................................................... 27
References ......................................................................................................................... 29
List of Figures and Tables
Page
Figure 1: The eukaryotic cell cycle……………………………………………………….5
Figure 2: Description of chlamydial growth method……………………………………12
Table 1: Contents of Media……………………………………………………………...12
Figure 3: The morphology of ST-669 treated C. caviae through an infection…….……17
Figure 4: Lobed inclusions in the presence of ST-669……………………………….…18
Figure 5: qPCR to determine the effects of colchicine and ST-669………………….…19
Figure 6: Visualization of the effects of colchicine and ST-669…………………..……20
Figure 7: qPCR to determine the effects of thymidine and ST-669…………………….21
Figure 8: Visualization of the effects of thymidine and ST-669………………………..22
Figure 9: qPCR to determine the effects of low serum and ST-669……….……………23
Figure 10: Visualization of the effects of low serum and ST-669………………………24
Figure 11: qPCR to determine the effects of vincristine and ST-669……..….…………25
Figure 12: Visualization of the effects of vincristine and ST-669………………………26
1
Introduction
Chlamydia
The genus Chlamydia contains multiple obligate intracellular pathogens of
importance to both human and veterinary medicine. C. trachomatis is the most
commonly reported sexually transmitted infection (STI) in the United States and
can cause pelvic inflammatory disease in women. The pathogen also causes
trachoma, which is the leading cause of infectious blindness worldwide[1-2].
Human chlamydial infections can often be asymptomatic, but persistent or
recurring infections can still lead to serious consequences such as infertility and
ectopic pregnancy. Therefore, routine screening for Chlamydia in at risk
populations is very important [3].
A similar bacteria, C. caviae, infects all mucosal membranes in guinea
pigs, and has been used as a model for human infection [4]. Initially, C. caviae
was considered to be a subtype of C. psittaci although it is now considered a
separate species [5]. Other chlamydial species include C. abortus, which causes
abortions in small ruminants, C. psittaci, which is a zoonotic respiratory pathogen
in wild and farmed birds, C. suis, which causes chronic diseases in pigs, and C.
muridarum, which causes mouse pneumonitis [6-9]. C. caviae is not pathogenic to
humans and grows faster than C. trachomatis, so C. caviae is used in study of
chlamydial biology.
The Chlamydia are gram negative bacteria that exhibit an alternating
developmental cycle between infectious elementary bodies (EBs) and dividing
2
reticulate bodies (RBs)[10]. The outer membrane of both EBs and RBs are
decorated with lipopolysaccharide that is conserved across the genus [7, 11].
Within a host cell, Chlamydia grows inside a non-acidified vacuole termed
an inclusion [14]. The inclusion contains chlamydial proteins involved in
virulence, called inclusion membrane proteins (Inc)[14]. The function of most
Incs is unknown, however the lack of IncA in C. trachomatis is associated with
nonfusogenic inclusions in cell culture [15]. Within a species, Incs share little
sequence identity. However this family of proteins are characterized by a
hydrophobicity domain [14, 16]. Although IncA from C. trachomatis and C.
caviae only have about 20% sequence similarity, both are present in the inclusion
membrane and these proteins share an overall structure of coiled-coil structures
[17]. When multiple C. trachomatis EBs enter a host cell, only one inclusion will
be formed if the bacteria have a functional copy of IncA [15]. Unlike C.
trachomatis, C. caviae grows in an inclusion with multiple lobes containing
bacteria regardless of how many bacteria the cell is infected with [14]. The lobes
of the C. caviae inclusion are produced early in the chlamydial developmental
cycle when the C. caviae inclusion membrane is tightly associated with the
surface of the bacteria and division of the bacteria causes division of the inclusion
membrane [18]. Late in the developmental cycle of C. caviae, the inclusion
membrane separates from the surface of the bacteria and bacterial division no
longer causes the inclusion membrane to split [18].
The only instance of observed naturally acquired antibiotic resistance in
Chlamydia was in a strain of C. suis that gained tetracycline resistance through
3
horizontal gene transfer[13]. Because of this, most chlamydial infections are
easily treated in a clinical setting with antibiotics, unlike other STIs, such as
Neisseria gonorrhea, that are becoming increasingly resistant to antibiotics [14].
ST-669
ST-669 is an acyl thiourea-based molecule that was developed by Siga
technologies as a broad spectrum antiviral compound [19]. The antiviral
properties of ST-669 were discovered by screening a compound library for
activity against Bunyaviridae [19]. A structure-activity relationship analysis was
then performed on a derivative, which was then optimized as an antiviral
compound to produce ST-669. In previous studies, ST-669 has shown antiviral
activity against Orthomyxoviridae, Arenaviridae and Flaviviridae, among others
[19].
In a previous study, ST-669 was shown to be effective against intracellular
bacteria in the genera Chlamydia and Coxiella [20]. Treatment of infected cells
with ST-669 resulted in a reduction of genome copies as well as a change in
chlamydial inclusion morphology [20]. The reduction of genomic copies was only
observed when the intracellular bacteria were grown in cells of primate origin—
Chlamydia and Coxiella grown in murine cells were not susceptible to ST-669.
Additionally, when Chlamydia were grown in the presence of cycloheximide, a
eukaryotic protein synthesis inhibitor, the anti-chlamydial effects of ST-669 were
lessened and the chlamydial inclusions partially returned to their normal
phenotype[20].
4
When C. trachomatis was grown in ST-669, the observed inclusions were
markedly smaller than when grown without drug, although the overall structure
was preserved. When C. caviae was grown in the presence of ST-669, the typical
lobed structure was no longer observed. Instead a single vacuole containing the
Chlamydia was present and was markedly smaller than the lobed inclusions
observed in the absence of drug . When treated with cycloheximide as well as ST-
669, C. caviae inclusions were lobed although smaller than when they were
treated with cycloheximide alone. The phenotypic changes of the C. caviae
inclusions allow for the effects of ST-669 to be measured using fluorescence
microscopy [20].
Because ST-669 is effective at inhibiting the growth of both viruses and
bacteria in only cells of primate origin, it is hypothesized that ST-669 acts on a
host cell process that is uniquely present in primate cells. This hypothesis is
supported by the fact that when host protein synthesis is arrested, the effects of
ST-669 are lessened.
5
The Eukaryotic Cell Cycle
Figure 1: The eukaryotic cell cycle During S-phase of the cell cycle, the cell replicates its DNA to have two copies of each chromosome. The cell fully divides during M-phase as microtubules pull the chromosomes to opposite ends of the cell. Two gap phases, G1 and G2, separate M-phase and S-phase. During the gap phases, checkpoints must be reached before the cell cycle can move forward. When a cell is not moving though the cell cycle, it rests in G0. When eukaryotic cells undergo mitosis, they proceed through a tightly
regulated set of stages collectively termed the cell cycle. The cell cycle has 4
main stages: G1, S, G2 and M [21-22]. During S phase, the cell duplicates its
chromosomes to produce sister chromatids [21]. During M phase, the sister
chromatids are separated into two new cells completing cell division [21]. The
purpose of the G1 and G2 phases is to act as temporal gaps between S phase and
M phase to ensure the cell has enough of the components needed to divide and to
allow cell growth [22]. When a cell is not dividing or preparing to divide, it is said
to be in the G0 [23]. (Figure 1)
M
G1
S
G2
G0
6
There are many avenues of control involved in the cell cycle, including
cyclin-dependent protein kinases, which are involved in a cascade of
phosphorylation that leads to continuation through the cell cycle [22-24]. The
tight regulation of the cell cycle is necessary; many cancers stem from mutations
in genes that regulate this process [25-26]. As a cell progresses in the cycle,
different proteins are required to accomplish each step of the cell cycle so the
cell’s transcriptome and proteome, or the collective mRNA or proteins, change
[25, 27]. The division of cells in vitro can be experimentally disrupted using
different strategies.
Low Serum
Cell culture media commonly contain 5-10% fetal bovine serum (FBS) to
supply the eukaryotic cells with the necessary nutrients to grow including
macromolecules, trace elements and growth factors [28]. Within the serum are
mitogens, or factors that encourage the cells to begin to divide [29]. When the
mitogens are not present in the media for the cells, they will enter G0 and not
divide. Therefore, reducing the amount of serum present in the medium will lead
to cells arrested in G0[29].
Vincristine and Colchicine
Vincristine and colchicine bind to microtubules and prevent proper
formation of the mitotic spindle, causing an M phase arrestment of the cell
cycle[30]. During mitosis, sister chromatids are pulled away from the plane of
division by mitotic spindles, which are made of microtubules. Before sister
chromatids can be pulled apart, the cell must pass the spindle checkpoint, which
7
ensures the spindles are properly connected to the chromatids[31]. If the spindle
checkpoint is not met, the cell will arrest in M phase until they eventually move
into G1 as abnormal tetraploidal cells[30] [31].
Thymidine
As the cells divide, the synthesis of nucleotides is tightly regulated. Cells
that are treated with extracellular thymidine show elevated levels of
deoxythymidine triphosphate (dTTP), which leads to inhibition of ribonucleotide
reductase [32]. The elevated levels of ribonucleotide reducatse lead to lowered
levels of deoxycytosine triphosphate, which leads the cell to be unable to fully
synthesize DNA and replicate its chromosomes.
8
Thesis Statement The goal of this thesis is to help determine the molecular target of ST-669 by
measuring its anti-chlamydial abilities when cells are exposed to differing mitotic
inhibitors. We also hypothesize that ST-669 can be used as a tool to investigate
the inclusion formation of C. caviae.
9
Methods Chlamydial Strains
All assays were conducted with Chlamydia caviae strain GPIC. A
concentrated solution of EBs was produced by scraping an infected monolayer of
McCoy cells then sonication for 15 seconds to disrupt the membrane in PBS. The
cell debris was pelleted by 15-minute centrifugation at 1000 RPM and frozen.
Cell Culture and Chlamydial Infection
Vero cells were grown at 37°C in 5% CO2 in Minimal Essential Medium,
10%FBS and 5mM L-glutamine (MEM-10). At 0 hours, Vero cells were set into
24 well trays at 30% confluence. If the cells were to be used for microscopy, the
cells were laid into 12mm glass coverslips. At 12 hours, the media was removed
from the wells and half the wells received media containing cell cycle inhibitors,
as outlined in Table 1, while the other half received control media. At 36 hours,
the media were removed from the wells and the cells were washed with PBS. C.
caviae EBs were diluted in PBS and inoculated onto cells at a multiplicity of
infection equal to 1. The trays were centrifuged at 2000rpm at 37°C for 1 hour.
The PBS was removed and media were added to the wells containing either 10uM
ST-669 or an equivalent volume of DMSO, plus or minus the cell cycle inhibitor
used in the particular experiment. At 66 hours, the cells were either prepared for
microscopy or genome quantification.
10
To prepare the cells for microscopy, the medium was removed and
replaced with 100% methanol for 10 minutes. After the removal of the methanol,
PBS was added into the wells.
To prepare treated cells for genome quantification, media were removed
and 300uL PBS was added into each well. Trays were then placed at -80°C
overnight to allow for cell lysis. Trays were then thawed at room temperature and
agitated by manual pipetting. DNA was extracted from the PBS using the Qiagen
DNeasy blood and tissue kit using instructions provided by the manufacturer. The
single exception to the described technique was the addition of 5 mM
dithiothreitol to the initial lysis buffer.
To observed C. caviae in Vero cells at varying times during the infection,
fully confluent Vero cells were infected with a multiplicity of infection of 10 with
C. caviae and allowed to grow in MEM-10 in the presence and absence of ST-
669. At 11, 14, and 30 hours post-infection, cells were fixed with methanol and
labeled with antibodies as described below.
Genome quantification
All TaqMan reagents for quantitative real-time PCR were purchased from
Applied Biosystems. TaqMan universal PCR Master mix was used with an input
of 2 µl of template DNA and primers and probes with 5′-6FAM and 3′-MGBNFQ
labels. Primers and probes were specific to C. caviae GPIC ompA (probe 6FAM-
CATCACACCAAGTAGAGC-MGBNFQ) [19]. Tenfold serial dilutions of
quantitated target DNA were included in the analyses at concentrations ranging
from 103 to 107 genome copies per assay. Genome copy number and standard
11
error were automatically calculated using an ABI StepOne real-time PCR
machine with standard curve settings and extrapolated to reflect total number of
bacterial genome copies per mL.
Fluorescence Microscopy
Methanol-fixed cells were washed three times with PBS. Monoclonal
antibodies against chlamydial LPS and C. caviae IncA were diluted 1:5 and 1:200
respectively in FA Block (2% BSA in PBS). Diluted primary antibody was added
to the cells and incubated for 1 hour. The primary antibody was removed and the
cells were washed three times with PBS. Secondary antibodies conjugated with
either rhodamine or fluorescein were diluted 1:1000 in FA Block. Diluted
secondary antibody was added to the cells and incubated for 1 hour in the dark.
The secondary antibody was removed and the cells were washed three times with
PBS. The coverslips were affixed to were mounted on mounting media containing
4µg/mL 4′,6′-diamidino-2-phenylindole (DAPI) and Vectasheild (Vectasheild,
Vector Laboratories). Pictures were taken at 1000x magnification with a Lecia
fluorescence microscope and a PC-based digital camera imaging system
(Qimaging Co, Surrey BC, Canada).
12
Figure 2: Description of chlamydial growth method A: Vero cells were set 30% confluence in a 24-well tray. B: MEM-10 was removed and replaced with MEM-10 containing the cell cycle inhibitor of interest. C: Vero cells were infected with C. caviae in PBS, MEM-10 with cell cycle inhibitors and ST-669 was added. D: Cells were fixed or DNA was collected. Table 1: Contents of Media
Control Minimum essential medium, 10% FBS, 5mM L-Glutamine and equivalent volume of DMSO or PBS
Low-Serum Minimum essential medium, 1% FBS, 5mM L-Glutamine
Vincristine Minimum essential medium, 10% FBS, 5mM L-Glutamine, 1uM vincristine in PBS
Colchicine Minimum essential medium, 10% FBS, 5mM L-Glutamine, 100uM colchicine in DMSO
Thymidine Minimum essential medium, 10% FBS, 5mM L-Glutamine, 1uM thymidine in DMSO
0 hours 12 hours 36 hours 66 hours
A B C D
13
Results
ST-669 treatment affects C. caviae inclusion structure
Beginning in early points following infection of cells, C. caviae can be
observed growing in a lobed inclusion. However, when treated with ST-669, an
inclusion with a single lobe is observed (Figure 3). This single lobed morphology
is observed late in the infection even when Vero cells are initially infected with a
high multiplicity of infection of C. caviae. When Vero cells infected with a high
multiplicity of infection of C. caviae were fixed and labeled with fluorescent
antibodies at both 11 and 14 hours post infection, inclusions with multiple lobes
were observed (Figure 3).
Anti-chlamydial effects of ST-669 in the presence of Cell Cycle Inhibitors
These studies were based on the observation that when C. caviae were
inhibited by ST-669, while the majority of the inclusions appeared to have a
single lobe, some inclusions in a well appeared to have a normal, lobed
morphology (Figure 4). Therefore, we hypothesized that the state of the host cell
was involved in the anti-chlamydial effects of ST-669. Since the mechanism of
action of ST-669 is unknown, it is possible that by determining the interactions
with different states of the cell cycle that the target of the drug will be discovered.
When C. caviae is grown in the presence of ST-669, the anti-chlamydial
properties of the compound are detectable by microscopy (Figure 3) and
measurable by real-time PCR (qPCR). Vero cells were inhibited with a variety of
14
cell cycle inhibitors, infected with C. caviae then treated with ST-669. The
activity of ST-669 was then determined.
Colchicine treatment. The first experiment involved treatment of cells
with colchicne, an inhibitor of mitotic spindle formation that freezes cells in M
phase/G1. When C. caviae were grown in Vero cells the presence of ST-669,
there was no change in the number of C. caviae in the cells when colchicine was
added to the culture (Figure 5). Similarly, when C. caviae were grown in Vero
cells the absence of ST-669, there was no change in the number of C. caviae in
the cells when colchicine was added to the culture (Figure 5). Similar to the
results of the qPCR, when C. caviae was gown in the presence of ST-669 the
bacteria had a single-lobed inclusion morphology and when grown in the absence
of ST-669 had a typical lobed inclusion morphology, regardless of colchicine
treatment (Figure 6). These results show that when Vero cells were inhibited with
colchicine, the anti-chlamydial properties of ST-669 were similar to the anti-
chlamydial properties of ST-669 when Vero cells were un-inhibited.
Thymidine treatment. We next treated cells with thymidine, a compound
that disrupts the metabolic synthesis of compounds necessary for DNA replication
that freezes the cells in S phase. When thymidine was added to the culture, the
number of C. caviae in each condition was similar to when the Vero cells were
treated with colchicine (Figure 7). The number of C. caviae in cells treated with
DMSO were similar in the presence and absence of thymidine and number of C.
caviae in cells that were treated with ST-669 were markedly lower than those
treated with DMSO, although were similar in the presence and absence of
15
thymidine. Similar to the results of the qPCR and the microscopy of colchicine
treated Vero cells, C. caviae, when gown in the presence of ST-669, regardless of
thymidine treatment, had a single-lobed inclusion morphology and when grown in
the absence of ST-669, regardless of thymidine treatment, had a typical lobed
inclusion morphology (Figure 8). When Vero cells were inhibited with thymidine,
the anti-chlamydial properties of ST-669 were similar to the anti-chlamydial
properties of ST-669 when Vero cells were un-inhibited. These results indicate
that the addition of thymidine to culture did not affect the anti-chlamydial
properties of ST-669.
Growth in low serum. Serum is a source of nutrients and chemokines, and
reducing serum levels in medium will freeze cells in the G1 stage of the cell
cycle. In contrast to treatment with colchicine or thymidine, Vero cells that were
grown in the absence of ST-669 contained fewer C. caviae when the Vero cells
were grown in 1% FBS compared to 10% FBS (Figure 9). This trend was also
observed when the cells were treated with ST-669, although both ST-669
conditions were lower than their comparative DMSO conditions. The trends of
qPCR differed between the low serum compared to the thymidine or colchicine
conditions; however, the trends of inclusion morphology were similar between the
conditions. When grown in ST-669, C. caviae exhibited a single lobed inclusion,
regardless of serum concentration and when grown in the absence of ST-669, C.
caviae exhibited lobed inclusion morphology, regardless of serum concentration
(Figure 10). When Vero cells were grown in low serum, the anti-chlamydial
properties of ST-669 were similar to the anti-chlamydial properties of ST-669
16
when Vero cells were grown in MEM-10. Therefore, growth in low serum did not
lower the anti-chlamydial abilities of ST-669.
Vincristine treatment. A different trend was observed when the activity of
ST-669 was tested in the presence of vincristine, a compound that freezes cells in
M phase. The number of C. caviae in Vero cells grown in the absence of ST-669
was similar in the presence and absence of vincristine. There was an increase in
the number of C. caviae when Vero cells were treated with both ST-669 and
vincristine compared to ST-669 alone (Figure 11). When grown in the absence of
ST-669, C. caviae exhibited lobed inclusion morphology regardless of vincristine
treatment. When C. caviae was grown in Vero cells in the presence of both ST-
669 and vincristine, inclusions had similar structures to C. caviae grown in the
absence of ST-669 (Figure 12). However, C. caviae were grown in ST-669 alone,
a single lobed inclusion morphology was seen. When Vero cells were inhibited
with vincristine and ST-669, the anti-chlamydial properties of ST-669 were
lessened.
17
Figure 3: The inclusion morphology of ST-669 treated C. caviae through an infection Fluorescent microscopy of C. caviae infected Vero cells in the presence of ST-669, fixed with methanol A: 11 hours post infection, B: 14 hours post infection, or C: 30 hours post infection. D: C. caviae infected Vero cells in the presence of DMSO, fixed 30 hours post infection. Chlamydial LPS is labeled with red. C. caviae GPIC IncA is labeled with green. Total DNA is stained blue with DAPI.
A B
C D
18
Figure 4: Lobed inclusions in the presence of ST-669 Fluorescent microscopy of C. caviae infected Vero cells in the presence of ST-669. And fixed with methanol 30 hours post infection. The inclusion on the right appears to have a normal lobed inclusion while other inclusions are inhibited by ST-669. Chlamydial LPS is labeled with red. C. caviae GPIC IncA is labeled with green. Total DNA is stained blue with DAPI.
19
Figure 5: qPCR to determine the effects of colchicine and ST-669 C. caviae infected Vero cells, treated with colchicine (black) or DMSO (white) in the presence or absence of ST-669, were lysed by freezing and genome copy number was determined by qPCR for each cell lysate.
20
Figure 6: Visualization of the effects of colchicine and ST-669 Fluorescent microscopy of C. caviae infected Vero cells, fixed with methanol 30 hours post infection, in the presence of A: Colchicine and ST-669 B: ST-669 and DMSO C: Colchicine and DMSO D: DMSO. Chlamydial LPS is labeled with red. C. caviae GPIC IncA is labeled with green. Total DNA is stained blue with DAPI.
A B
C D
21
Figure 7: qPCR to determine the effects of thymidine and ST-669 C. caviae infected Vero cells, treated with thymidine (black) or DMSO (white) in the presence or absence of ST-669, were lysed by freezing and genome copy number was determined by qPCR for each cell lysate.
22
Figure 8: Visualization of the effects of thymidine and ST-669 Fluorescent microscopy of C. caviae infected Vero cells, fixed with methanol 30 hours post infection, in the presence of A: Thymidine and ST-669 B: ST-669 and DMSO C: Thymidine and DMSO D: DMSO. Chlamydial LPS is labeled with red. C. caviae GPIC IncA is labeled with green. Total DNA is stained blue with DAPI.
A B
C D
23
Figure 9: qPCR to determine the effects of low serum and ST-669 C. caviae infected Vero cells, treated with 1% FBS (black) or 10% FBS (white) in the presence or absence of ST-669, were lysed by freezing and genome copy number was determined by qPCR for each cell lysate.
24
Figure 10: Visualization of the effects of low serum and ST-669 Fluorescent microscopy of C. caviae infected Vero cells, fixed with methanol 30 hours post infection, in the presence of A: 1% FBS and ST-669 B: 10% FBS and ST-669 C: 1% FBS and DMSO D: 10% FBS and DMSO. Chlamydial LPS is labeled with red. C. caviae GPIC IncA is labeled with green. Total DNA is stained blue with DAPI.
A B
C D
25
Figure 11: qPCR to determine the effects of vincristine and ST-669 C. caviae infected Vero cells, treated with vincristine (black) or PBS (white) in the presence or absence of ST-669, were lysed by freezing and genome copy number was determined by qPCR for each cell lysate.
26
=
Figure 12: Visualization of the effects of vincristine and ST-669 Fluorescent microscopy of C. caviae infected Vero cells, fixed with methanol 30 hours post infection, in the presence of A: Vincristine and ST-669 B: PBS and ST-669 C: Vincristine and DMSO D: PBS and DMSO. Chlamydial LPS is labeled with red. C. caviae GPIC IncA is labeled with green. Total DNA is stained blue with DAPI.
A B
C D
27
Discussion
Since the mechanism of action of ST-669 is unknown, it is possible that by
determining the interactions with different states of the cell cycle that the target of
the drug will be discovered. Of the cell cycle inhibitors tested, only vincristine
lessened the anti-chlamydial effects of ST-669. Interestingly, both vincristine and
colchicine act on microtubules and cause the eukaryotic cells to freeze in M phase
or G1 phase; however colchicine treated cells appeared similar to controls. These
results could be due to inadequate inhibition by either of the compounds or could
be due to a differing effect of vincristine compared to colchicine. To determine
that the cell cycle inhibitors are working properly in the model, flow cytometery
could be used to determine chromosome copy number in the cells to determine
the step in the cell cycle that the Vero cells are in.
If both vincristine and colchicine cause the Vero cells to be inhibited in
the same step of the cell cycle then it is likely that vincristine is acting in some
way to affect the state of the host cells that colchicine is not, which then has an
effect on ST-669. ST-669 is likely interacting with a host protein that, when the
host cell is treated with vincristine, is either up or down regulated which causes
ST-669 to lose its anti-chlamydial effects. To determine if this is the case, the
proteome of Vero cells inhibited with vincristine should be compared to Vero
cells inhibited with colchicine. Differences should be examined as a potential
target of ST-669.
When Vero cells are treated with ST-669 an inclusion with a single lobe is
observed late in the infection, regardless of the initial multiplicity of infection
28
(MOI). Despite this, when cells are infected with a high MOI of bacteria, C.
caviae will initially grow in multiple vacuoles in the presence of ST-669. We
hypothesize that fusion of the vacuoles takes place between the initial growth in
the lobed inclusion and the final inclusion with a single lobe. This fusion may be
due to the host protein that is the target of ST-669: a host protein could be up
regulated that is involved with membrane fusion and the higher concentrations of
this protein cause the chlamydial inclusions to fuse together. This fusion may
also due to biology of C. caviae: ST-669 could cause inclusion membranes to be
loosely associated with the RBs early in infection, preventing division of the
inclusions and causing the inclusions to fuse through the processes used by other
species of Chlamydia, which are always functioning in C. caviae but the division
of the inclusion membranes is normally faster than the fusion. Either ST-669 will
likely be a useful tool in the future to study why C. caviae grows in a lobed
inclusion unlike most other Chlamydia, or C. caviae will continue to provide
insights to how the novel anti-viral drug functions.
29
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