Chlamydia trachomatis GlgA Is Secreted into Host Cell Cytoplasm
Citation: Lu C, Lei L, Peng B, Tang L, Ding H, et al. (
Chlamydia trachomatis GlgA Is Secreted into Host Cell Cytoplasm
Chunxue Lu 0
Lei Lei 0
Bo Peng 0
Lingli Tang 0
Honglei Ding 0
Siqi Gong 0
Zhongyu Li 0
Yimou Wu 0
Guangming Zhong 0
0 1 Department of Pathogen Biology, University of South China , Hengyang, Hunan , China , 2 Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio , San Antonio, Texas , United States of America
Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms.
Urogenital tract infection with Chlamydia trachomatis is a
leading cause of sexually transmitted bacterial diseases .
However, the molecular mechanisms of C. trachomatis
pathogenicity remain unclear. Nevertheless, the intracellular
survival and replication of C. trachomatis organisms are
thought to contribute significantly to inflammatory pathologies
induced by C. trachomatis infection . The C. trachomatis
organisms have evolved a unique intracellular growth cycle
with distinct biphasic stages . The organisms invade
epithelial cells via induced endocytosis in the form of
elementary bodies (EBs). An endocytosed EB rapidly develops
into a noninfectious but metabolically active reticulate body
(RB) that is able to undergo biosynthesis and multiplication
within the initially established cytoplasmic vacuole called
chlamydial inclusion. The accumulation of progeny RBs in the
inclusions triggers the differentiation of RBs back into EBs for
exiting infected cells and spreading to new cells. The question
is how chlamydial organisms are able to establish and maintain
such a successful intracellular infection. The C. trachomatis
organisms have evolved the ability to secrete proteins into both
the inclusion membranes and host cell cytosol and the
secreted proteins have been hypothesized to play important
roles in modifying host cellular processes for facilitating
chlamydial invasion, intracellular survival/replication and
spreading to new cells [5,922]. Thus, searching for new
Chlamydia trachomatis-secreted proteins (CtSPs) has been a
most promising/productive approach for understanding
chlamydial pathogenic mechanisms.
The C. trachomatis inclusions are known to contain glycogen
that is detectable with iodine [23,24], which is consistent with
the fact that the C. trachomatis genome encodes all necessary
open reading frames (ORFs) required for both glycogen
biosynthesis and catabolism . Thus, the C. trachomatis
organisms can both synthesize and utilize glycogen, which
seems to be important in chlamydial pathogenesis since the
plasmidless chlamydial organisms that are unable to produce a
large amount of glycogen are no longer able to induce
pathology in mouse oviduct . Surprisingly, under electron
microscopy, most glycogen particles were observed in the
lumen of chlamydial inclusion [27,28], suggesting that most
chlamydial glycogens are released out of the chlamydial
organisms after/during synthesis or even synthesized outside
of the organisms. It will be interesting to know whether the
glycogen metabolism enzymes are also released into the
In the current study, we found that of the 6 glycogen
metabolism-related enzymes investigated in the current study,
only GlgA was detected outside of chlamydial organisms. An
anti-GlgA antibody detected signals both inside and outside of
chlamydial inclusions. Confocal microscopic analyses revealed
that some intra-inclusion GlgA signals were completely
independent of chlamydial organisms, suggesting that a portion
of GlgA is secreted out of the organisms into the inclusion
lumen. The extra-inclusion GlgA signal displayed a pattern
similar to that of the C. trachomatis-secreted protease CPAF,
suggesting that GlgA is also secreted into host cell cytosol.
Efforts were made to ensure the antibody labeling specificities.
GlgA secretion into host cell cytosol is highly conserved among
all C. trachomatis serovars tested and may take place during
infection in humans since GlgA is immunogenic in women
urogenitally infected with C. trachomatis but not in rabbits
immunized with dead C. trachomatis organisms, which is
consistent with the concept that secretion of chlamydial
proteins into host cell cytosol is often accompanied with
enhanced immunogenicity. The above observations together
have provided new information and tool for mapping the
molecular basis of C. trachomatis pathogenesis.
Materials and Methods
1: Chlamydial infection
The following C. trachomatis organisms were used in the
current study: C. trachomatis serovars A/HAR-13 (ATCC
catalog# VR-571B), B/HAR-36 (VR-573), Ba/Ap-2 (VR-347), C/
UW-1, D/UW-3/Cx (VR-885), E/UW-5/CX, F/IC-Cal-3 (VR-346),
G/UW-57/Cx (VR878), H/UW-43/Cx (VR-879), I/UW-12/Ur
(VR-880), K/UW-31/Cx (VR-887), L1/LGV-440 (VR-901B), L2/
LGV-434/Bu (VR-902B) & L3/LGV-404 (VR-903). These
organisms were either purchased from ATCC (Manassas, VA)
or acquired from Dr. Harlan Caldwell at the Rocky Mountain
Laboratory, NIAID/NIH (Hamilton, MT) or Dr. Ted Kou at the
University of Washington (Seattle, WA) or Dr. Li Shen at the
Louisiana State University. The chlamydial organisms were
propagated, purified, aliquoted and stored as described
previously [17,22]. All chlamydial organisms were routinely
checked for mycoplasma contamination. For infection, HeLa
cells (human cervical carcinoma epithelial cells, ATCC cat#
CCL2) grown in either 24 well plates with coverslips or tissue
flasks containing DMEM (GIBCO BRL, Rockville, MD) with
10% fetal calf serum (FCS; GIBCO BRL) at 37C in an
incubator supplied with 5% CO2 were inoculated with
chlamydial organisms. The infected cultures were processed at
various time points after infection for either
immunofluorescence assays, glycogen measurement or
Western blot analysis as described below.
2: Chlamydial gene cloning, fusion protein expression
and antibody production
The ORFs CT042 (GlgX), CT087 (MalQ), CT248 (GlgP),
CT295 (MrsA), CT489 (GlgC), CT798 (GlgA), CT110 (HSP60),
CT681 (MOMP), CT858 (CPAF; ref :) CT795 (a secreted
protein; ref :), CT813 (an inclusion membrane protein; ref
:) and Pgp3 (a secreted plasmid protein; ref :) from C.
trachomatis serovar D genome (http://
stdgen.northwestern.edu) were cloned into pGEX vectors
(Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and
expressed as glutathione-s-transferase (GST) fusion proteins
as described previously [30,31]. Expression of the fusion
protein was induced with isopropyl-beta-D-thiogalactoside
(IPTG; Invitrogen, Carlsbad, CA) and the fusion proteins were
extracted by lysing bacteria via sonication in a Triton-X100 lysis
buffer (1% Triton X-100, 1mM PMSF, 75 units/ml of Aprotinin,
20 M Leupeptin and 1.6 M Pepstatin, all from Sigma). After a
high-speed centrifugation to remove debris, the fusion
proteincontaining supernatants were used for ELISA in
glutathionecoated microplates or absorbed onto glutathione-conjugated
agarose beads (Pharmacia) for antibody absorption experiment
or further protein purification. The purified fusion proteins were
used to immunize mice for producing antibodies as described
3: Enzyme-linked immunosorbent assay (ELISA)
The fusion protein microplate ELISA was carried out as
described previously . Briefly, the GST-fusion proteins in
the form of bacterial lysates were applied to glutathione-coated
96-well microplates (catalog number 15140B; Pierce, Rockford,
IL) and used to assay antibody reactivities. All primary
antibodies were preabsorbed with a bacterial lysate containing
GST alone before they were assayed on the ELISA plates. The
human and rabbit antisera were obtained and produced as
described previously . The goat anti-human
IgA-IgGIgM or donkey anti-rabbit IgG secondary antibodies conjugated
with horseradish peroxidase (HRP) (catalog numbers
109-035-064 and 711-035-152, respectively; Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA) were
used to probe the primary antibody binding. The soluble
substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS; catalog number A1888-5G; Sigma)
was used to visualize the reactions, and the reactivity was
recorded as the absorbance (optical density [OD]) at 405 nm. A
GST-alone bacterial lysate-coated well in each plate was used
as a negative control. Any wells with an OD equal to or greater
than the mean plus 2 standard deviations were considered
4: Immunofluorescence assay
The immunofluorescence assay was carried out as
described previously [33,34]. HeLa cells with or without C.
trachomatis infection grown on coverslips were processed for
immunofluorescence assay. For some experiments, HeLa cells
were transfected with the mammalian expression vector
pFLAG-CMV4 (cat#E7158, Sigma) alone or recombinant
pFLAG plasmid that encodes chlamydial GlgA gene. The GlgA
gene from C. trachomatis serovar D was cloned into
pFLAGFigure 1. Localization of GlgA in C. trachomatis-infected cells. HeLa cells infected with C. trachomatis serovar L2 at an MOI
0.5 were processed either 24h or 40h post infection for co-staining with mouse antibodies recognizing individual chlamydial proteins
visualized with a goat anti-mouse IgG conjugated with Cy3 (red), a rabbit antibody to chlamydial organisms visualized with a
Cy2conjugated goat anti-rabbit IgG (green) and DNA dye Hoechst (blue). (A) The mouse antisera raised with 6 GST fusion proteins as
indicated on top of each panel were used to localize the corresponding endogenous chlamydial proteins after 1:1000 dilution. Note
that only the anti-GST-CT798 (GlgA; panels f & l) detected signals that were free of chlamydial organisms. (B) The anti-GlgA
antiserum labeling was repeated at multiple dilutions: 1:100 (panel a), 1:1000 (b) and 1:5000 (c). Note that the antibody detected
both intra-inclusion (yellow & red arrowheads) and extra-inclusion (red arrow) signals under a conventional fluorescence microscope
at all dilutions. (C) The mouse anti-GlgA antiserum labeling at 1:1000 dilution was further observed under a confocal microscope.
Note that the intra-inclusion labeling with anti-GlgA polyclonal antibody displayed two distinct patterns [overlapping with chlamydial
organisms (yellow arrowheads) and free of the organisms (red arrowheads)] while all anti-HSP60 antibody labelings overlapped with
the organisms (panels d-f). The images were taken under an objective lens of either 100x (conventional fluorescence microscopy)
or 60x (confocal microscopy).
Figure 2. Antibody specificity validation. The mouse anti-GlgA (polycolonal antibody, pAb at 1:1000), anti-CPAF (monoclonal
antibody, clone# 100a, IgG1) and anti-Pgp3 (clone# 2H4, IgG2a) antibodies were preabsorbed without (panels a, e & i) or with
GSTGlgA (b, f & j), GST-CPAF (c, g & k) and GST-Pgp3 (d, h & l) fusion proteins prior to reacting with C. trachomatis-infected cultures
as described in Figure 1 legend. Note that the antibody labelings were removed by the corresponding (f, g & k) but not irrelevant
fusion proteins. (B) HeLa cell lysates, Chlamydia trachomatis (Ct)-infected HeLa cell lysates, GST-GlgA or GST-CPAF fusion
proteins were resolved in SDS-polyacrylamide gel and transferred onto nitrocellulose membrane for Western blot detection with
mouse anti-GlgA (GlgA at 1:4000 dilution, panel a), CPAF (mAb 100a, b), MOMP (mAb MC22, c) and human HSP70 (mAb
W27, d). Note that these antibodies only detected their corresponding antigens without any significant cross-reactivity.
Figure 3. Western blot detection of GlgA in fractions of C. trachomatis-infected cells. Normal HeLa lysates (lane 1) or HeLa
cells with Chlamydia trachomatis (Ct) serovar L2 infection (lane 2) were fractionated into pellets (lane 3) and supernatants (S100,
lane 4) along with purified chlamydial reticulate bodies (RB, lane 5) or elementary body (EB, lane 6) were resolved in SDS
polyacrylamide gel and the resolved protein bands were transferred onto nitrocellulose membrane for Western blot detection with
anti-GlgA (panel a), anti-CPAF (b), anti-CT813 (c), anti-MOMP (d) and anti-human HSP70 (e) antibodies. Note that CPAF was
detected only in either Ct-HeLa lysates (lane 2) or Ct-HeLa S100 (lane 4) while GlgA was detected in all samples containing
chlamydial organisms or derived from Chlamydia trachomatis-infected cells.
CMV4 using following primers: forward primer
5AAGGAAAAAA-GCGGCCGCG-(NotI)ATGAAAATTATTCACACAGCTATC-3 and back primer
5-CCGATATC-(ECoRV)TTATTGTTTATAAATTTCTAAATATTTAT-3. The transfection
was carried out using the Lipofectamine 2000 following the
manufacturers protocol (Invitrogen). Twenty-four hours after
transfection, Flag-CT798 fusion protein was detected using an
immunofluorescence assay or the transfected cultures were
further infected with Chlamydia trachomatis organisms. For
immunofluorescence assay, all cell samples were fixed with 2%
paraformaldehyde (Sigma, St. Luis, MO) dissolved in PBS for
30 min at room temperature, followed by permeabilization with
2% saponin (Sigma) for an additional 30 min. After washing
and blocking, the cell samples were subjected to antibody and
chemical staining. Hoechst (blue, Sigma) was used to visualize
DNA. A rabbit anti-chlamydial organism antibody (R1L2, raised
with C. trachomatis L2 organisms, unpublished data) plus a
goat anti-rabbit IgG secondary antibody conjugated with Cy2
(green; Jackson ImmunoResearch Laboratories, Inc) was used
to visualize chlamydial organisms. The various mouse
antibodies plus a goat anti-mouse IgG conjugated with Cy3
(red; Jackson ImmunoResearch) were used to visualize the
corresponding antigens. The mouse antibodies included:
polyclonal antibodies (pAbs) made against GST-GlgA, mAb
100a against CPAF , mAb clone 2H4 against Pgp3  and
mAb M1 against FLAG tag. All primary antibodies were
preabsorbed with a bacterial lysate containing GST alone before
use. In addition, for some experiments, the primary antibodies
were further absorbed with either the corresponding or
heterologous fusion proteins immobilized onto
glutathioneconjugated agarose beads prior to staining, which was meant
to prove the antibody binding specificities. The absorption was
carried out by incubating the antibodies with bead-immobilized
antigens for 1h at room temperature (RT) or overnight at 4oC
followed by pelleting the beads. The remaining supernatants
were used for immunostaining. The immunofluorescence
images were acquired using an Olympus AX-70 fluorescence
microscope equipped with multiple filter sets and Simple PCI
imaging software (Olympus, Melville, NY) as described
previously . For some experiments, the immunostaining
was analyzed with confocal microscopy at UTHSCSA imaging
core facility. The images were processed using Adobe
Photoshop (Adobe Systems, San Jose, CA).
5: Western blot assay
The Western blot assay was carried out as described
elsewhere . Briefly, HeLa cells with or without C.
trachomatis infection and with or without fractionation along
with GST fusion proteins were resolved in SDS-polyacrylamide
gels. The resolved protein bands were transferred to
nitrocellulose membranes for antibody detection. The primary
antibodies were: mouse polyclonal antiserum against GlgA
(current study), mAb 100a against CPAF , mAb MC22
against chlamydial major outer membrane protein (MOMP; ref:
), mAb AF1 against the chlamydial inclusion membrane
protein CT813  and mAb W27 against host cell HSP70
(cat#Sc-24, Santa Cruz Biotechnology, CA), The anti-MOMP
antibody was used to ensure that all lanes with chlamydial
organism-containing samples had equivalent amounts of the
organisms loaded while the lanes without chlamydial organism
samples should be negative for MOMP. The anti-HSP70
antibody was used to make sure that an equal amount of
normal HeLa and chlamydia-infected HeLa samples were
loaded. The primary antibody binding was probed with an HRP
(horse radish peroxidase)-conjugated goat anti-mouse IgG
secondary antibody (Jackson ImmunoResearch) and visualized
with an enhanced chemiluminescence (ECL) kit (Santa Cruz
6: Glycogen quantification
Glycogen was quantified using the EnzyChromTM Glycogen
Assay kit (cat#: E2GN-100, BioAssay systems, Hayward, CA)
following a protocol recommended by the manufacturer. Briefly,
HeLa cells grown in 6-well plates were infected with C.
trachomatis L2 or transfected with pFLAG-CMV4 vector alone
or recombinant pFLAG-CMV4-GlgA. 40h after infection or 24h
after transfection, the cells were washed with ice-cold PBS and
harvested in 0.5ml 10% KOH in 1.5ml microcentrifuge tube.
After boiled at 100C for 20 minutes and cooled to room
temperature, Tricholoroacetic acid (TCA) was added to a final
concentration of 10%. After centrifuging at 10,000g for 10min,
the supernatant was transferred to a new tube and mix with
1ml ethanol and centrifuge at 4,000 g for 15min. The pellet was
washed with 70% ethanol and air-dried. The precipitate was
resuspended in 100 l of distilled water. The concentration of
glycogen in suspension was determined using the Glycogen
Assay Kit. The final results were expressed as the total amount
of glycogen per sample or well.
Figure 5. Secretion of GlgA into host cytosol by different biovars/serovars of Chlamydia trachomatis. HeLa cells were
infected with different serovars of chlamydial organisms representing 3 major biovars of Chlamydia trachomatis as indicated on top
of each panel. The infected cultures were subjected to immunofluorescence labelings as described in Figure 4 legend. Note that
GlgA secretion into host cell cytosol was detected in all Chlamydia trachomatis serovars assayed.
1: GlgA is detected in both chlamydial inclusion lumen
and host cell cytosol
Mouse antisera made against 6 glycogen metabolism
enzyme GST fusion proteins were used to label C.
trachomatisinfected HeLa cells in an immunofluorescence assay. We
found that only the endogenous GlgA was detected both inside
and outside of chlamydial inclusions when the infected cultures
were observed either 24h or 40h after infection (Figure 1A),
suggesting that a portion of GlgA is secreted into host cell
cytosol. When the anti-GST-GlgA antiserum was carefully
titrated (B) and analyzed using confocal microscopy (C), we
found that GlgA detected inside chlamydial inclusions
displayed two distinct patterns with or without overlapping with
chlamydial organisms, suggesting that a portion of GlgA is
secreted out of chlamydial organisms into the lumen of
chlamydial inclusions prior to accessing to host cell cytosol.
The host cytosolic labeling pattern of GlgA was similar to those
of CPAF, a known Chlamydia-secreted protease, and Pgp3, a
known Chlamydia-secreted plasmid protein, respectively
Both the granular staining inside inclusion and the diffused
staining in host cell cytosol by anti-GlgA antibody were
removed by pre-absorption with GST-GlgA but not GST-CPAF
or Pgp3 fusion proteins (Figure 2A). The same were true for
anti-CPAF and Pgp3 stainings, demonstrating that the
antiGlgA, anti-CPAF and anti-Pgp3 antibodies specifically labeled
the corresponding endogenous proteins without cross-reacting
with each other. On a Western blot (Figure 2B), the mouse
anti-GlgA antiserum only reacted with the endogenous GlgA
and the GST-GlgA fusion protein without cross-reacting with
any other proteins from C. trachomatis-infected cells or
unrelated fusion proteins. The anti-CPAF mAb 100a detected
both the GST-CPAF fusion protein and the C-terminal fragment
of activated CPAF (CPAFc) in Chlamydia-infected cells as
demonstrated previously . As loading controls, the antibody
specifically recognizing the chlamydial major outer membrane
protein (MOMP) detected MOMP in the infected cell sample
while the anti-human HSP70 antibody recognized HSP70 in
both normal and infected HeLa samples. These results further
confirmed that both anti-GlgA and anti-CPAF antibodies only
specifically detected the corresponding endogenous proteins
without cross-reacting with each other or any unrelated
chlamydial or host cell proteins. Thus, we can conclude that the
signals labeled by the anti-GlgA and anti-CPAF antibodies
revealed under immunofluorescence microscope represent the
corresponding endogenous proteins.
The intracellular distribution of GlgA is also confirmed using
a cell fractionation plus Western blot detection approach
(Figure 3). CPAF was only detected in either the C.
Figure 6. Effect of chlamydial GlgA expression on glycogen synthesis and subsequent chlamydial infection. HeLa cells
were transfected with pFlag plasmid alone or pFlag plasmid encoding chlamydial GlgA gene. Twenty-four hours after transfection,
the cell samples were processed for immunofluorescence labelings with mouse anti-FLAG or anti-GlgA (A), harvested for glycogen
detection (B) or further infected with C. trachomatis L2 (C). (A) The images were taken 24h after transfection under a 60X objective
lens using a conventional fluorescence microscope. (B) HeLa cells with or without transfection with pFLAG or pFLAG-GlgA or
infection with L2 were harvested 24h post-transfection or 48h post-infection for glycogen measurements. The results coming from 5
independent experiments were expressed as g per ml of samples as shown along the Y-axis. Note that both exogenous GlgA
overexpression and chlamydial infection significantly enhanced the levels of glycogen synthesis in HeLa cells (Student t-test). (C)
Some of the transfected cells were further infected with chlamydial organisms and 40h after chlamydial infection, the cultures were
processed for immunofluorescence labelings with mouse anti-FLAG to identify positive transfection cells and rabbit anti-chlamydial
EBs to identify chlamydial inclusions. Number of total and C. trachomatis-infected cells was counted from 5 random views of each
coverslip. The infection rate was calculated for anti-Flag positive (solid bar) and negative (open bar) cells respectively and
expressed as mean plus/minus standard deviation along the Y-axis. Note that the infection rates displayed no statistically significant
differences between cells with or without transfection from either pFLAG vector alone or pFLAG/GlgA recombinant
plasmidtransfected cultures (p>0.05, Fisher Exact).
Figure 7. Reactivity of GlgA with antisera from women infected and rabbits immunized with Chlamydia trachomatis
organisms. (A) Chlamydial GST fusion proteins or GST alone were resolved in a SDS polyacrylamide gel and stained with a
Coomassie blue dye. All GST fusion proteins along with their gene names/ORF numbers and fusion protein molecular weights were
listed on top of the gel image. Note that besides the GST-containing degradation fragments, the major bands are full length fusion
proteins (marked with circles) for all GST fusion proteins. (B) The above fusion proteins listed along the X-axis were reacted with
antisera from women diagnosed with acute Chlamydia trachomatis infection (STI patients, n=20, panel a) or tubal factor infertility
(TFI patients, n=24, b) or from normal women without C. trachomatis infection (n=10, c) or from rabbits intramuscularly immunized
with dead Chlamydia trachomatis organisms (Rabbits, n=7, c) in ELISA assays. The OD values obtained at the wavelength of
405nm in the format of mean plus/minus standard deviation were displayed along the Y-axis. Any reactivity with an OD value equal
to or above the mean plus 2 standard deviations is defined as positive (+ve), which is noted on top of each bar. Note that 12 of the
20 STI, 7 of the 24 TFI and 0 of the 10 normal women or 7 rabbit antisera positively reacted with GlgA.
trachomatis-infected whole cell lysate (Ct-HeLa) or cytosolic
fraction (Ct-HeLa S100) samples but not other samples
including the purified C. trachomatis RB and EB organisms
(pane b), which is consistent with what has been described
previously . However, GlgA was detected in all but normal
HeLa cell samples (panel a), which is consistent with the
observation that GlgA is associated with chlamydial organisms
and secreted into both the inclusion lumen and host cell
cytosol. To monitor the quality of the fractionation, the
antiCT813 (a known inclusion membrane protein; ref :) and
anti-MOMP antibodies were used to indicate the pellet fraction
containing the chlamydial inclusions while an anti-human
HSP70 antibody was used to indicate the host cell cytosolic
fraction containing the C. trachomatis-secreted proteins.
Detection with these antibodies revealed no cross
contamination between the pellet and cytosolic fractions. In
addition, detection with the anti-MOMP antibody showed that
the amounts of chlamydial organisms in the C.
trachomatisinfected HeLa whole cell lysate, the pellet fraction and purified
EB and RB samples were equivalent. These results together
have independently confirmed that chlamydial GlgA has a wide
distribution in the infected cells.
2: Characterization of GlgA secretion
We further used the specific anti-GlgA antibody to monitor
biosynthesis and secretion of GlgA at single cell level (Figure
4). As shown in panel A, GlgA was first detected 12h post
infection. Clear secretion into host cell cytosol was detected
24h after infection. These expression and distribution patterns
were similar to those of CPAF with the exception that some
GlgA molecules were obviously accumulated in organism-free
granules in the lumen of chlamydial inclusions throughout the
infection course. CPAF secretion into host cell cytosol was
more complete without any significant accumulation in the
lumen of inclusions. We further found that about 75% of the
infected cells secreted GlgA or CPAF (Figure 4B & C).
Secretion of GlgA into the chlamydial inclusion lumen and host
cell cytosol was detected in all C. trachomatis biovar-infected
cells (Figure 5), suggesting that GlgA secretion may represent
an essential process required by all C. trachomatis organisms.
To test whether GlgA secretion into host cell cytosol can impact
chlamydial intracellular infection, we expressed GlgA via a
transgene in HeLa cells and examined the effect of the
preexisting GlgA in the host cell cytosol on subsequent
chlamydial infection (Figure 6). Expression of chlamydial GlgA
in HeLa cells (A) resulted in elevated levels of glycogen while
the vector plasmid alone transfection failed to do so (B),
suggesting that chlamydial GlgA expressed in the host cell
cytosol is functional. However, HeLa cells with or without GlgA
expression were similarly susceptible to the subsequent
chlamydial infection (C), suggesting that neither the expressed
GlgA nor the elevated level of glycogen in HeLa cells has any
impact on chlamydial infection.
3: Chlamydial GlgA is immunogenic during chlamydial
infection in women
Since it is known that C. trachomatis-secreted proteins such
as CPAF [30,35], Pgp3  and CT795  are highly
immunogenic during chlamydial infection, we compared the
immunogenicity of GlgA and other glycogen
metabolismrelated chlamydial proteins along with various control
chlamydial proteins in both women urogenitally infected with C.
trachomatis and rabbits intramuscularly immunized with dead
C. trachomatis organisms (Figure 7). These fusion proteins
were monitored for quality on a Coomassie blue-staining gel
(A) prior to reacting with serum samples (B). GlgA was
recognized by antisera from women diagnosed with either
acute C. trachomatis infection (STI patients) or tubal factor
infertility (TFI patients) with a recognition frequency of 60% or
29% respectively, suggesting that anti-GlgA antibodies are
associated with acute C. trachomatis infection. However, the
secreted proteins CPAF, Pgp3 and CT795 did not display such
a preference with a recognition frequency of 100%, 95% & 35%
by STI antisera and 92%, 96% & 50% by TFI antisera
respectively. None of the 10 normal women reacted
significantly with any of the chlamydial fusion proteins. When
the chlamydial fusion proteins were reacted with 7 rabbits
antisera, 100% recognition frequency was detected for
chlamydial HSP60, MOMP and Pgp3 while no reactivity was
detected for GlgA, CPAF or CT795. Since Pgp3 is also
localized in chlamydial outer membrane, it is not surprising that
Pgp3, like MOMP, is highly immunogenic when the dead EBs
were used to immunize rabbits. However, GlgA is also
associated with the chlamydial organisms but it failed to induce
any antibody responses in rabbits. The observation that none
of the other glycogen metabolism-related proteins were
immunogenic following either chlamydial infection in humans or
immunization in rabbits suggests that the organism-associated
glycogen metabolism proteins are not immunogenic.
Chlamydia trachomatis has evolved strategies for secreting
proteins into host cell cytosol, which may benefit chlamydial
intracellular living. Identifying C. trachomatis-secreted proteins
(CtSPs) represent an essential first step for uncovering the
mysteries of the chlamydial intracellular life and understanding
the molecular mechanisms of C. trachomatis pathogenesis.
Here, we have presented evidence that the chlamydial
glycogen synthase GlgA is secreted into host cell cytosol
during C. trachomatis infection. First, the anti-GlgA antiserum
detected GlgA outside of the chlamydial inclusions in C.
trachomatis-infected cells. The extra-inclusion GlgA molecules
displayed a cytosolic distribution pattern similar to that of
CPAF, a known Chlamydia-secreted protease. Second, the
detection of the endogenous GlgA was specific as
demonstrated both in fluorescence microscopy and Western
blot assays. Third, GlgA secretion into inclusion lumen and
host cell cytosol was also confirmed using a cell fractionation
plus Western blot detection approach. Finally, like CPAF and
CT795, GlgA also contains a N-terminal signal sequence.
Although we have not characterized the functionality of the
signal sequence, its presence suggests that GlgA, like many
other CtSPs that contain N-terminal secretion sequence, may
use a sec-dependent pathway to gain access to both inclusion
lumen and host cell cytosol.
Although C. trachomatis GlgA and other glycogen-related
enzymes are expected to carry out glycogen biosynthesis and
metabolism in the chlamydial organisms, the role of GlgA
secreted out of the organisms remains unknown. Glycogen is
abundantly detected in the lumen of chlamydial inclusion under
electron microscopy [27,28]. Our current finding that GlgA is
also localized in the lumen suggests that either chlamydial
glycogen along with its synthase is transported into the lumen
from the organisms or a portion of glycogen is synthesized in
the lumen. More careful studies are required for accurately
identifying the sites of glycogen biosynthesis. The next
question is the role of GlgA secreted into host cytosol.
Transient expression of chlamydial GlgA in the host cells
resulted in an elevated level of glycogen. However, neither the
preexisting GlgA nor the elevated glycogen in the host cell
cytosol affected the subsequent chlamydial infection (see
Figure 6), suggesting that Chlamydia-enhanced biosynthesis of
glycogen in host cell cytosol serves no apparent advantage for
chlamydial intracellular life. Efforts are underway to further
elucidate the biological significance of GlgA secretion into host
Among all the glycogen-related enzymes analyzed, only
GlgA is immunogenic during chlamydial infection in humans,
correlating the unique localization of GlgA in host cell cytosol
since secretion of chlamydial proteins into host cell cytosol is
known to enhance the immunogenicity of the secreted proteins
[14,18,20,30,31,35,36]. The lack of immunogenicity of all
analyzed chlamydial glycogen-related enzymes in rabbits when
inactivated chlamydial organisms were used as immunogens
suggests that the chlamydial organism-associated glycogen
enzymes are immune recessive, which may be due to their
inaccessibility to immune system when configured in the
organisms. However, we previously showed that the glycogen
metabolism enzymes were immunogenic when injected into
mice . Immunization with C. muridarum glycogen
phosphorylase (GlgP) but not with the other glycogen
metabolism enzymes induced a significant protection against
intravaginal infection with C. muridarum organisms, suggesting
that GlgP is immunogenic both during immunization and C.
muridarum infection. However, it is not known whether C.
trachomatis GlgP is immunogenic in mice. Nevertheless, the
immunogenicity of C. trachomatis GlgA in women urogenitally
infected with C. trachomatis indicates that GlgA is expressed
and possibly secreted into host cell cytosol during chlamydial
infection. Plasmid-free chlamydial organisms with a
significantly reduced level of GlgA and lack of glycogen
synthesis are highly attenuated in animal models [26,28],
suggesting that the secreted C. trachomatis GlgA may
contribute to C. trachomatis pathogenesis in humans.
Conceived and designed the experiments: GZ CL ZL YW.
Performed the experiments: LL SG BP LT HD. Analyzed the
data: CL LL GZ. Wrote the manuscript: CL GZ.
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