The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol
The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol
Xiang Wu 0
Lei Lei 0
Siqi Gong 0
Ding Chen 0
Rhonda Flores 0
Guangming Zhong 0
0 Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio , 7703 Floyd Curl Drive, San Antonio, TX 78229 , USA
Background: The periplasmic High Temperature Requirement protein A (HtrA) plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA) in chlamydial pathogenesis is not clear. Results: The cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anticHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor. Conclusion: Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.
Chlamydia trachomatis cHtrA; serine protease; secreted protein
The genus Chlamydia consists of multiple obligate
intracellular bacterial species that infect both humans and
animals. The C. trachomatis organisms infect human ocular
(serovars A to C) and urogenital/colorectal (serovars D to
K & L1 to L3) epithelial tissues, causing trachoma  and
sexually transmitted diseases [2-4] respectively; The C.
pneumoniae organisms invade human respiratory system,
not only causing respiratory diseases but also exacerbating
pathologies in cardiovascular system [5-7]; C. muridarum
(formerly known as C. trachomatis mouse pneumonitis
agent, designated as MoPn; ref: ), although causing no
known diseases in humans, has been used as a model
pathogen for studying chlamydial pathogenesis and
immune responses; The C. psittaci 6BC organisms that
naturally infect birds can cause severe pneumonia in
humans  while the C. caviae GPIC organisms can infect
ocular and urogenital tissues in guinea pig . Despite the
differences in host range, tissue tropism, disease processes,
all chlamydial species share similar genome sequences
[8,10,11] and possess a common intracellular growth cycle
with distinct biphasic stages . A chlamydial infection
starts with the invasion of an epithelial cell by an infectious
elementary body (EB). The internalized EB rapidly develops
into a noninfectious but metabolically active reticulate
body (RB) that undergoes multiplication. The progeny RBs
then differentiate back into EBs for spreading to new cells.
All chlamydial biosynthesis activities are restricted within a
cytoplasmic vacuole known as inclusion .
During the intravacoular developmental cycle,
chlamydial organisms have to take up nutrients and energy from
host cells [13-16] and maintain the integrity of the host
cells . To achieve these goals, chlamydial organisms
have evolved the ability to secrete proteins into the
inclusion membrane [18,19] and host cell cytoplasm [17,20,21].
Identifying the chlamydial secretion proteins has greatly
facilitated the understanding of chlamydial pathogenic
mechanisms [20,22-31]. CPAF, a chlamydial protease/
proteasome-like activity factor that is now known as a
serine protease [32,33], was found to secrete into host cell
cytosol more than a decade ago . CPAF can degrade a
wide array of host proteins including cytokeratins for
facilitating chlamydial inclusion expansion [34-36],
transcriptional factors required for MHC antigen expression for
evading immune detection [37,38] and BH3-only domain
proteins for blocking apoptosis [39,40]. Another example
of chlamydia-secreted proteins is the chlamydial
tail-specific protease that has been found to dampen the
inflammatory responses by cleaving host NF- B molecules [41,42].
These observations have led to the hypothesis that
Chlamydia may have evolved a proteolysis strategy for
manipulating host cell signaling pathways .
Among the several dozens of putative proteases encoded
by chlamydial genomes [11,43], the chlamydial HtrA
(cHtrA) is a most conserved protease. HtrA from
eukaryotic and prokaryotic species exhibits both chaperone and
proteolytic activities [44,45] with a broad proteolytic
substrate specificity [44,45]. HtrA is a hexamer formed by
staggered association of trimeric rings and access to the
proteolytic sites in central cavity is controlled by 12 PDZ
domains in the sidewall [46,47]. In eukaryotic cells, HtrA
responds to unfolded proteins in the endoplasmic
reticulum (ER) by cleaving and releasing the ER
membraneanchored transcription factors ATF6 and SREBP into
nucleus to activate the expression of proteins required for
the unfolded protein response and cholesterol biosynthesis
[48,49]. In bacteria, the periplasmic HtrA, in response to
the binding of C-terminal peptides from unfolded/reduced
outer membrane proteins, cleaves and releases the
sE-factor to activate stress response genes . Since HtrA is
required for bacterial survival under high temperature, it is
called High Temperature Requirement (Htr) protein .
Although both the tertiary structure and the function of
HtrA are well known, the role of cHtrA in chlamydial
pathogenesis remains unclear. In the current study, we
have localized cHtrA both in the chlamydial inclusions
and the host cell cytosol. The specificity of the antibody
labeling and cytosolic localization of cHtrA were
confirmed in independent assays. The secretion of the
periplasmic cHtrA into host cell cytosol appeared to be an
active/selective process since no other chlamydial
periplasmic proteins were detected outside the chlamydial
inclusions. Thus, the chlamydial periplasmic cHtrA may also
contribute to the chlamydial proteolysis strategies for
manipulating host cell signaling pathways.
1. Chlamydial infection
The following chlamydial organisms were used in the
current study: C. trachomatis serovars A/HAR-13, B/HAR-36,
Ba/Ap-2, C/UW-1, D/UW-3/Cx, E/UW-5/CX),
F/IC-Cal3, H/UW-43/Cx, I/UW-12/Ur, K/UW-31/Cx,
L1/LGV440, L2/LGV-434/Bu & L3/LGV-404, C. muridarum
(Nigg), C. pneumoniae (AR39), C. caviae (GPIC) &
C. psittaci (6BC). All chlamydial 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). The chlamydial
organisms were propagated, purified, aliquoted and stored
as described previously . 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 or
Western blot analysis as described below. In some
experiments, at 6 hours after infection, the cultures were treated
with a C1 compound
[N-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, cat#5113023, ChemBridge,
San Diego, CA], a small molecule known to inhibit
Yersinia type III secretion system (T3SS) and block chlamydial
growth . The treated cultures were processed for
immunofluorescence microscopy analysis at 36 hours after
infection. The C1 compound was dissolved in dimethyl
sulfoxide (DMSO; Sigma, St Luis, MO) at a stock
concentration of 50 mM and diluted into culture medium at a
final concentration of 50 M with 0.1% DMSO.
2. Chlamydial gene cloning, fusion protein expression and antibody production
The ORF CT823 (cHtrA) from C. trachomatis serovar D
organisms was cloned into pGEX vectors (Amersham
Pharmacia Biotech, Inc., Piscataway, NJ). The following
primers were used for cloning the ORF: cHtrA forward
primer, 5-CGC-GGATCC (BamHI)-ATGATGAAAAGAT
TATTATGTGTG-3, cHtrA back primer, 5-TTTT
CCTTTT-GCGGCCGC(NotI)-CTACTCGTCTGATTTCAAGAC-3. The ORF was expressed as a fusion protein
with glutathione-S-transferase (GST) fused to the
Nterminus as previously described . Expression of the
fusion protein was induced with
isopropyl-beta-D-thiogalactoside (IPTG; Invitrogen, Carlsbad, CA) and the fusion
proteins were extracted by lysing the bacteria via sonication
in a Triton-X100 lysis buffer (1%TritonX-100, 1 mM
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 protein was purified
using glutathione-conjugated agarose beads (Pharmacia)
and the purified protein was used to immunize mice for
producing antibodies, including monoclonal antibodies
(mAbs), as described previously [53-55]. The mouse
antibodies against GST-CT067, GST-CT539 and GST-CT783
were produced similarly. The fusion protein-specific
antibodies were used to localize endogenous proteins in
C. trachomatis-infected cells via an indirect
immunofluorescence assay and to detect endogenous proteins using
a Western blot assay. All mouse anti-GST fusion protein
antibodies were preabsorbed with bacterial lysates
containing GST alone before any applications. In some
experiments, the GST fusion proteins bound onto the
glutathione-agarose beads were also used to absorb the
mouse antibodies to confirm antibody specificities.
3. Immunofluorescence assay
The immunofluorescence assay was carried out as
described previously . Briefly, HeLa cells grown on
coverslips were fixed with 2% paraformaldehyde (Sigma,
St. Luis, MO) 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) or
antiIncA from C. trachomatis [kindly provided by Ted
Hackstadt. Laboratory of Intracellular Parasites, Rocky
Mountain Laboratories, NIAID, NIH, Hamilton, Montana;
], C. pneumoniae or C. psittaci (both current study)
plus a goat anti-rabbit IgG secondary antibody
conjugated with Cy2 (green; Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA) was used to visualize
chlamydial organisms or inclusion membrane. The
various mouse antibodies plus a goat anti-mouse IgG
conjugated with Cy3 (red; Jackson ImmunoResearch, West
Grove, PA) were used to visualize the corresponding
antigens. The mouse antibodies used included: polyclonal
antibodies (pAbs) made against GST-CT823 (HtrA),
GST-CT783, GST-CT621, GST-CT539, GST-CT067 (all
current study) and mAbs 6A2 against HtrA (current
study), 100a against CPAF , BB2 against IncA
(CT119) & 1L11C3 against chlamydial HSP60
(unpublished data). All primary antibodies were preabsorbed
with a bacterial lysate containing GST alone before use.
In addition, for some experiments, the primary antibodies
were absorbed with either the corresponding or
heterologous fusion proteins immobilized onto
glutathioneconjugated agarose beads (Pharmacia). The absorption
was carried out by incubating the antibodies with
beadimmobilized antigens for 1 h at room temperature (RT)
or overnight at 4C 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 . An
Olympus FluoView laser confocal microscope (Olympus,
Center Valley, PA) was used to further analyze some of
the immunofluorescence samples at the University of
Texas Health Science Center at San Antonio institutional core facility as described previously . The images were processed using Adobe Photoshop (Adobe Systems, San Jose, CA).
4. Western blot assay
The Western blot assay was carried out as described
elsewhere [38,55]. Briefly, HeLa cells with or without
C. trachomatis infection and with or without fractionation
(into pellet and S100 fractions), purified chlamydial RB
and EB organisms, GST fusion proteins or fractionated
bacterial periplasmic or cytosolic samples were resolved in
SDS polyacrylamide gels. The resolved protein bands were
transferred to nitrocellulose membranes for antibody
detection. The primary antibodies used included: mouse
pAb and mAb 6A2 against cHtrA, mouse pAb against
CT067 (all current study), mAb 100a against CPAF ,
mAb MC22 against chlamydial major outer membrane
protein [MOMP; ref ], mAb W27 against host cell
HSP70 (cat#Sc-24, Santa Cruz Biotechnology, CA), mAb
against FLAG tag (cat#F3165, Sigma, St. Luis, MO) and
rabbit polyclonal antibody against bacterial GroEL
(cat#G6532, Sigma, St. Luis, MO). The anti-MOMP
antibody was used to ensure that all lanes with chlamydial
organism-containing samples were loaded with equivalent
amounts of the organisms while the lanes without
chlamydial organism samples should be negative for MOMP. The
anti-HSP70 antibody was used to make sure that equal
amounts of normal HeLa and Chlamydia-infected HeLa
samples were loaded and to also check whether the
cytosolic fractions are contaminated with components from
the pellet fractions during cellular fractionation (see
below). All primary antibodies used in the current study
were pre-absorbed with an excess amount of bacterial
lysates containing the GST alone. The primary antibody
binding was probed with an HRP (horse radish
peroxidase)-conjugated goat anti-mouse IgG secondary antibody
(Jackson ImmunoResearch, West Grove, PA) and
visualized with an enhanced chemiluminescence (ECL) kit
(Santa Cruz Biotech). Some of the C. trachomatis-infected
HeLa cell (Ct-HeLa) samples were fractionated into pellet
(containing host cell nuclei and chlamydial inclusions) and
cytosolic fraction (S100, containing Chlamydia-secreted
proteins) as described previously [26,29]. Briefly, cell
samples were collected by centrifugation at 600 g for 10 min
at 4C. The cell pellets were washed once with ice-cold
PBS and resuspended with five volumes of buffer A (20
mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2,
1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM
dithiothreitol, and 0.1 mM phenylmethylsulfoyl fluoride)
containing 250 mM sucrose on ice for 15 min. The cells were
homogenized with 10 to 15 strokes using a number 22
kontes douncer with the B pestle (Kontes Glass Company,
Vineland, NJ) to break cytoplasmic membrane but without
breaking inclusion/nuclear membrane. The integrity
of cytoplasmic and inclusion/nuclear membranes was
monitored microscopically by smearing an aliquot of the
homogenates on a slide. The final homogenates were
centrifuged twice at 750 g for 10 min at 4C to pellet
inclusions/nuclei. The pellets from both centrifugations were
combined and washed once with cold PBS and stored as
pellet fraction. The supernatants were centrifuged at
10,000 g for 15 min at 4C followed by a further
centrifugation at 100,000 g for 1 h at 4C. The resulting
supernatants were designated as S100 or cytosolic fraction. The
chlamydial organisms were purified as described
previously . The RB organisms were purified from 24 h
cultures while the EB organisms from 40 to 50 h cultures.
The bacterial cell fraction samples were prepared as the
following: a pellet from 10 ml bacteria culture was washed
with ice-cold PBS once and pelleted again by
centrifugation at 3000 rmp 10 min at 4C. The pelleted bacterial
cells were resuspended in 0.5 ml of a Periplasting buffer
containing 20 mM Tris-HCl (pH7.5), 20% sucrose
(cat#SX1075-1, EMD Chemicals Inc., Gibbstown, NJ),
1 mM EDTA (cat#E5134, Sigma), 3 mg/ml lysozyme
(cat#100834, MP biomedicals, Solon, Ohio). After
incubating on ice for 5 min, 0.5 ml ice-cold distilled water was
added to the suspension and mixed by pipetting up and
down. After incubating on ice for another 5 min, the
mixture was pelleted by centrifugation at 12,000 g for 2 min at
4C. The periplasmic fraction (per) in the supernatant was
collected to a new tube while the cytoplasmic proteins
(cyt) in the remaining pellet were resuspended in 1 ml
Periplasting buffer. Both per & cyt fractions were used on
the Western blot assay.
5. BCIP Assay
To construct the plasmid pFLAG-CTC-cHtrAss-PhoA, a
69 bp DNA sequence coding for the HtrA signal peptide
(M1-S23, designated as cHtrAss, with restriction enzyme
sites of XhoI/BamHI) was amplified from Chlamydia
trachomatis serovar D genome and 1400 bp DNA sequence
coding for PhoA (BamHI/KpnI) was amplified from
pFLAG-CTC-CPAFss-PhoA plasmid, both 69 bp HtrA
and 1400 bp PhoA were inserted into the XhoI/KpnI
sites of the plasmid pFLAG-CTC (cat#E8408, sigma;
PhoA stands for mature PhoA without the signal
peptide). The DH5a bacterial strain (Invitrogen, Carlsbad,
CA) was used to express the plasmids. The products
from all the three plasmids (pFLAG-PhoA,
pFLAGPhoA & pFLAG-HtrAss-PhoA) contain a FLAG tag
fused to the C-terminus of PhoA. For BCIP assay,
bacterial cells were grown in LB supplemented with the
corresponding selection antibiotics at 37C overnight. The
overnight cultures were streaked onto LB agar containing
the same selection antibiotics and 50 g/ml
5-bromo-4chloro-3-indolyl phosphate (BCIP, cat# B6149, Sigma)
and the plates were incubated at 30C for 2 days. The
bacterial colonies that are capable of exporting mature
PhoA into periplasm turn blue while the colonies
incapable of doing so remain white.
1. Chlamydial HtrA is localized in both chlamydial inclusion and host cell cytosol
A mouse antiserum raised with GST-cHtrA fusion protein
detected the endogenous cHtrA protein both inside and
outside of the chlamydial inclusions in C.
trachomatisinfected HeLa cells (Figure 1A). The amount of
intrainclusion labeling appeared to be greater since the labeling
in the host cell cytosol (outside inclusions) disappeared
first as the dilution of the antiserum increased.
Interestingly, some of the cHtrA-positive intra-inclusion granules
appeared to be distinct from C. trachomatis organisms,
suggesting that a portion of cHtrA may be secreted out of
the organisms but still trapped inside the inclusions. Both
the intra-inclusion and cytosolic distribution of cHtrA
were confirmed with a mAb against cHtrA (Figure 1B).
Similar intra-inclusion stainings that are free of organisms
were reported previously [15,57,58]. In contrast, most
CPAF molecules were secreted out of the inclusions
without obvious intra-inclusion accumulation. As expected,
most of the chlamydial HSP60 molecules co-localized with
the chlamydial organisms. The secretion of cHtrA into
host cell cytosol became more obvious when the
chlamydial inclusion membrane was counter-labeled using an
anti-inclusion membrane protein antibody (Figure 1C).
The cHtrA molecules were detected both inside and
outside the inclusion membrane. The above observations
together suggested that cHtrA might be secreted into both
intra-inclusion space and the host cell cytosol.
We next confirmed the antibody binding specificity by
using an absorption procedure (Figure 2A). Both the
intrainclusion and host cell cytosolic signals detected by the
anti-cHtrA antiserum or anti-cHtrA mAb 6A2 were
removed by absorption with GST-cHtrA but not
GSTCPAF fusion proteins. Similarly, the cytosolic signal
detected with the anti-CPAF antibody was removed by
absorption with the GST-CPAF but not GST-cHtrA fusion
proteins, demonstrating that the anti-cHtrA and anti-CPAF
antibodies specifically labeled the corresponding
endogenous proteins without cross-reacting with each other. In a
Western blot assay (Figure 2B), the anti-cHtrA antibodies
recognized both the GST-cHtrA fusion protein and the
endogenous cHtrA from the C. trachomatis-infected HeLa
cells (Ct-HeLa) while the various control antibodies
recognized the corresponding antigens without any significant
cross-reactivity with each other. The anti-CPAF antibody
detected the GST-CPAF fusion protein and also the
Cterminal fragment (CPAFc) of the endogenous CPAF from
Figure 1 Detection of cHtrA protease in the cytosol of C. trachomatis-infected cells. HeLa cells infected with C. trachomatis L2 organisms
were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies
visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue). The mouse antibodies included an anti-cHtrA
(CT823) antiserum (raised with GST-cHtrA fusion protein) at various dilutions (A), the anti-cHtrA antiserum at 1:1000 dilution (B, panels a & f),
mAb 6A2 (b & g, also raised with the GST-cHtrA fusion protein), mAb (100a) against CPAF (c & h), mAb (BB2) against IncA (d & i) and mAb
(1L11C3) against HSP60 (e & j). The mouse anti-cHtrA staining (red) was also co-labeled with a rabbit anti-IncA antibody (green; C). Note that the
anti-cHtrA antibodies detected signals both inside the chlamydial inclusions with (yellow arrowheads) or without (red arrowheads) overlapping
with the chlamydial organisms and in the host cell cytosol (red arrows) while the anti-CPAF antibody mainly detected signals in the host cell
Figure 2 The anti-GST-cHtrA fusion protein antibodies specifically detected the endogenous cHtrA produced by chlamydial organisms.
The anti-cHtrA antibodies with or without absorption with GST fusion proteins were used to detect the endogenous proteins in C.
trachomatisinfected cells (A) and on nitrocellulose membranes (B). (A) C. trachomatis-infected cells were processed for immunostaining as described in
Figure 1A legend. Note that the antibody labeling of endogenous antigens was blocked only by corresponding but not unrelated control fusion
proteins. (B) In a Western blot assay, HeLa alone or HeLa infected with C. trachomatis (Ct-HeLa), GST-CPAF & GST-cHtrA fusion proteins were
used as antigens as indicated on top of the figure. The antigens blotted onto nitrocellulose membrane were detected with mouse antibodies as
displayed at the bottom of the figure. The anti-CPAF mAb 100a is specific to the C-terminal fragment of CPAF (CPAFc) and the full length CPAF
is rapidly processed into the N- and C-terminal fragments to form intramolecular dimmers for activity during chlamydial infection. The control
antibodies anti-MOMP and anti-human HSP70 were used to indicate that the Ct-HeLa samples contain chlamydial organisms and both HeLa and
Ct-HeLa samples were loaded with similar amounts. Note that each antibody only detected a major protein band migrated at the molecular
weight that matched the corresponding chlamydial or host proteins as indicated on the right side of the figure.
the Ct-HeLa sample. CPAF is rapidly processed into the
Nand C-terminal fragments during chlamydial infection and
the mAb 100a is specific to the 35 kDa C-terminal fragment
. The anti-MOMP antibody detected MOMP from
CtHeLa, confirming the presence of whole chlamydial
organisms in the sample while the anti-human HSP70 antibody
detected similar amounts of HSP70 in the HeLa alone and
Ct-HeLa samples, indicating that an equivalent amount of
whole cell lysates was loaded in both samples. These
observations together have demonstrated that the anti-cHtrA
antibodies only recognized cHtrA without cross-reacting
with any other chlamydial or host cell proteins, suggesting
that the cellular signals detected with the anti-HtrA fusion
protein antibodies in the immunofluorescence assay were
specific to the endogenous cHtrA produced by chlamydial
2. Secretion of cHtrA but not other chlamydial
periplasmic proteins into host cell cytosol
Since cHtrA is a periplasmic protein, we next tested
whether localization in the host cell cytosol is a common
characteristic of all chlamydial periplasmic proteins. The
intracellular distributions of two periplasmic proteins
involved in disulfide bond formation (CT539, TrxA or
thioredoxin) and isomerization (CT783, PDI or protein
disulfide bond isomerase; http://stdgen.northwestern.edu/)
respectively and one periplasmic iron binding protein
(CT067, YtgA, an ABC transporter system component;
ref: [59,60]) were compared with that of cHtrA (Figure 3).
Under a conventional fluorescence microscope (A), only
cHtrA but not the other periplasmic proteins including
CT067, CT539 & CT783 was detected outside of the
chlamydial inclusions. This observation was confirmed under
a confocal microscope (B). The Z-axis serial section
images showed that cHtrA was clearly detected both
inside and outside the inclusion membrane but CT067
was only detected inside the inclusion membrane.
To directly visualize the molecular basis of the
anticHtrA antibody-labeled cytosolic signals in
Chlamydiainfected cells, the infected cells were fractionated into
cytosolic (S100) and nuclear/inclusion (pellet) fractions.
The distribution of cHtrA and CT067 in different fractions
was compared in a Western blot (Figure 4). As a control
for chlamydial proteins that are secreted into the host cell
cytosol, CPAF was only detected in either the
Chlamydiainfected whole cell lysate (Ct-HeLa) or cytosolic fraction
(Ct-HeLa S100) samples but not other samples, which is
consistent with what has been described previously .
Interestingly, cHtrA and its cleavage fragments but not
CT067 was also detected in the cytosolic fraction,
suggesting that cHtrA but not CT067 is secreted into host cell
cytosol although both are periplasmic proteins. The cHtrA
degradation fragments are likely generated during in vitro
sample processing as HtrA is a powerful serine protease
that is known to cleave itself . To monitor the quality
of the fractionation, the anti-MOMP antibody was used to
indicate the pellet fraction that contains the chlamydial
inclusions while an anti-human HSP70 antibody was used
to indicate the host cell cytosolic fraction that contains the
Chlamydia-secreted proteins. Detection with these
antibodies revealed no cross contamination between the pellet
and cytosolic fractions. In addition, detection with the
anti-MOMP antibody also showed that the amounts of
chlamydial organisms in the infected HeLa whole cell
lysate, the pellet fraction and purified EB and RB samples
were equivalent. These results together have
independently confirmed that cHtrA is secreted into cytoplasm of
Chlamydia-infected cells although it is also associated
with the chlamydial RB and EB organisms.
3. Expression and secretion of cHtrA during chlamydial infection
We further used the specific anti-cHtrA antibodies to
characterize the endogenous cHtrA. As shown in Figure 5,
cHtrA protein was detected inside the inclusions as early
as 12 h after infection and secretion of cHtrA into host
cell cytosol became apparent by 24 h post infection.
Although CPAF was also detectable at 12 h, the secretion
of CPAF was more robust and became very obvious as
early as 16 h after infection. The cHtrA protein was
detected both within the chlamydial inclusions and in the
host cell cytosol while CPAF mainly accumulated in the
host cell cytosol as infection progressed. Although both
CPAF and cHtrA are serine proteases secreted by C.
trachomatis organisms, their distinct secretion kinetics and
intracellular distribution patterns suggest that they may
fulfill different functions during chlamydial infection. To
further evaluate whether cHtrA secretion is common to
all chlamydial organisms, we monitored the cHtrA protein
distribution in cells infected with various serovars and
strains from different chlamydial species, including 13 C.
trachomatis serovars and also isolates representing species
of C. muridarum, C. caviae, C. pneumoniae and C. psittaci
(Figure 6). The cHtrA protein was consistently detected in
both the lumen of chlamydial inclusion and cytosol of
host cells infected with all serovars of C. trachomatis
organisms and isolates of C. muridarum, C. caviae and C.
pneumoniae but not C. psittaci. Although secretion of
cHtrA into the inclusion lumen and further into the
cytosol of the infected cells seems to be a common feature of
most chlamydial organisms tested, it is not known at this
moment why the species C. psittaci, which primarily infect
birds, failed to secrete cHtrA into host cytosol.
4. The secretion of chlamydial HtrA may require a type II but not type III secretion pathway
To determine the secretion pathway that chlamydial
organisms may use to secrete cHtrA, we analyzed the
Figure 3 The cHtrA but not other chlamydial periplasmic proteins are secreted into host cell cytosol. HeLa cells infected with
C. trachomatis organisms were processed and co-labeled with mouse antibodies against various periplasmic proteins (red) and a rabbit antibody
against IncA (green) as described in Figure 1 legend. The Hoechst dye was used to visualize DNA (blue). The triple labeling was analyzed under
a conventional fluorescence microscope (A) and confocal microscope (B). Under the confocal microscope, a series of four images were taken
along the Z-axis by varying 1 M between each. Note that cHtrA (red arrows) but none of the other periplasmic proteins including CT067,
CT539 & CT783 was detected outside of the inclusion membrane (green arrows) by either immunofluorescence microscopy or confocal
Figure 4 The cHtrA but not CT067 is detected in the cytosolic fraction of the chlamydia-infected HeLa cells. HeLa cells infected with
C. trachomatis organisms (Ct-HeLa) were fractionated into nuclear (Ct-HeLa pellet, containing chlamydial inclusions, lane 3) and cytosolic
(CtHeLa S100, containing chlamydia-secreted proteins, lane 4) fractions. The cellular fractions along with total cell lysates (normal HeLa, lane 1 &
CtHeLa, lane 2) and purified chlamydial RB (lane 5) and EB (lane 6) organisms as listed at the top were resolved in SDS-polyacrylamide gels. The
resolved protein bands were blotted onto nitrocellulose membrane for reacting with antibodies (listed on the left) against cHtrA (panel a), CT067
(b, a periplasmic iron binding protein), CPAF (c, a chlamydia-secreted protein), MOMP (d, a chlamydial outer membrane protein) and human
HSP70 (e, a host cell cytosolic protein). All antibodies detected their corresponding proteins in the HeLa-L2 whole-cell lysate sample (lane 2) and
other corresponding samples (as indicated on the right). Note that both cHtrA and CPAF but not CT067 or MOMP were detected in the
cytosolic fraction (lane 4). CPAFc represents the C-terminal fragment of CPAF processed during chlamydial infection. The cHtrA degradation
fragments (likely produced during in vitro sample processing) can always be detected with varying levels as HtrA is a powerful serine protease
known to cleave itself  under certain conditions.
Figure 5 Time course of cHtrA expression during C. trachomatis infection. The C. trachomatis-infected culture samples were processed at
various times after infection (as indicated on the top) for immunofluorescence staining as described in Figure 1 legend. The mouse anti-cHtrA (a
to h) and anti-CPAF (mAb 100a; i to p) were visualized with a goat anti-mouse IgG conjugated with Cy3 (red) while the chlamydial organisms
were visualized with a rabbit anti-chlamydia antibody plus a goat anti-rabbit IgG-Cy2 conjugate (green). Note that cHtrA was first detected inside
the chlamydial inclusions at 12 hours after infection [panel d, yellow (overlapping with organisms) & red (free of chlamydial organisms)
arrowheads], similar to the detection of CPAF. However, cHtrA secretion into host cell cytosol was only detected 24 h after infection while
secretion of CPAF was already obvious by 16 h post infection.
amino acid sequence of cHtrA for secretion signal
sequences using the program SignalP version 3.0 with
NN (neural network) and HMM (hidden markov model)
algorithms http://www.expasy.ch. Both NN and HMM
algorithms predict an N-terminal signal peptide in cHtrA
but with different cleavage sites. NN predicts a cleavage
between S16 and S17 while HMM predicts the cleavage
site between S23 and A24 (Figure 7A). We then tested
the functionality of the cHtrA N-terminal sequence
M1S23 using a bacterium-based phoA gene fusion system
(Figure 7B &7C). This assay system takes advantage of
two characteristics of PhoA: the enzyme is only active
after translocation into the bacterial periplasm, and the
phosphatase activity can be conveniently monitored with
the chromogenic substrate BCIP. DNA coding for the
cHtrA N-terminal signal sequence covering residues M1
to S23 (designated as cHtrAss) was fused to the DNA
sequence coding for mature PhoA (designated as PhoA).
The fusion construct was expressed in pFLAG-CTC
vector which adds a Flag epitope to the C-terminus of
PhoA. The mature PhoA alone construct was used as a
negative control while the precursor full-length PhoA
(with its native N-terminal signal peptide) served as a
positive control. As shown in Figure 7B, in the presence
of BCIP, bacteria expressing either the precursor PhoA
or the cHtrAss-PhoA fusion constructs turned blue
whereas bacteria expressing the mature PhoA alone
(PhoA) remained white, indicating that both the native
PhoA and cHtrA signal peptides directed the
translocation of PhoA into periplasm. We further used a Western
blot analysis to monitor the distribution of PhoA protein
in periplasmic (per) and cytosolic (cyto) fractions (Figure
7C). Mature PhoA was detected in the periplasm of
bacteria expressing either the precursor PhoA or
HtrAssPhoA fusion constructs while mature PhoA was only
detected in the cytoplasm of the bacteria expressing the
leaderless PhoA. Thus, the cHtrA N-terminal signal
peptide is sufficient for directing PhoA across the bacterial
inner membrane. We further found that the secretion of
cHtrA was not inhibited by the C1 compound, an
inhibitor known to inhibit chlamydial type III secretion system
. As positive controls, C1 inhibited the secretion of
both IncA and CT621, two known chlamydial type III
secretion substrates [30,52]. Consistently, the secretion of
CPAF was not affected by C1. This is because secretion
of CPAF is dependent on type II secretion pathway .
The obligate intracellular growth of Chlamydia requires
the organisms to intimately interact with host cells.
Secretion of chlamydial proteins into host cells is necessary for
chlamydial organisms to ensure a safe intracellular niche
for completing biosynthesis and producing progenies.
Identifying chlamydial proteins that are secreted into host
cell cytoplasm has been a productive approach for
understanding chlamydial pathogenic mechanisms [20,22-31]. In
the current study, we characterized the chlamydial serine
protease cHtrA by localizing its intracellular distribution.
We have presented convincing evidence that cHtrA is
secreted out of the chlamydial organisms into both
chlamydial inclusion lumen and cytosol of the infected cells.
First, both the cHtrA fusion protein-specific polyclonal
and monoclonal antibodies detected intracellular secretion
patterns distinct from those of CPAF, another secreted
serine protease by chlamydial organisms. The cytosolic
signals were confirmed using inclusion membrane as a
Figure 6 Secretion of cHtrA into host cell cytosol by most chlamydial organisms tested. HeLa cells infected with C. trachomatis serovars A,
B, Ba, C, D, E, F, H, I, K, L1, L2, L3, C. muridarum Nigg strain, C. caviae GPIC, C. penumonaie AR39 isolate &C. psiitaci 6BC organisms (as indicated in
each panel) were processed at 40 h (all C. trachomatis serovars), 24 h (Nigg, GPIC & 6BC) or 72 h (AR39) after infection. (A) The processed
samples were detected for cHtrA using the mouse anti-cHtrA fusion protein polyclonal antibody (red) in an immunofluorescence assay. The
chlamydial organisms were visualized using a rabbit anti-CT395 fusion protein antibody (green) while the DNA was labeled with Hoechst dye
(blue). Note that cHtrA was consistently detected in both the lumen of chlamydial inclusion (red arrowheads) and cytosol (red arrows) of cells
infected with all C. trachomatis serovars and C. muridarum and C. caviae isolates. However, the cytosolic labeling of cHtrA was not clear in cells
infected with C. pneumoniae AR39 and C. psittaci 6BC organisms which were reexamined by co-staining with either anti-organisms (B, panels a-c)
or anti-IncA (panels d-f) antibodies. Note that cytosolic cHtrA was detected in cells infected with C. pneumoniae AR39 (panels b & e) but not
C. psittaci 6BC organisms (c & f).
Figure 7 cHtrA is secreted via a sec-dependent pathway. (A) The SignalP 3.0 program with both the Neural Networks (NN) and Hidden
Markov Model (HMM) algorithms http://www.expasy.ch was used to analyze the precursor cHtrA sequence from C. trachomatis serovar D http://
stdgen.northwestern.edu/. The NN algorithm predicts a signal peptide from the first methionine residue (M1) to a serine residue at position 16
(S16) while the HMM-predicted signal peptide is M1-S23. (B) The M1-S23 peptide of cHtrA (cHtrAss) directed translocation of PhoA into bacterial
periplasmic space (cHtrAss-PhoA, slot 1, blue). Expression of the positive control full-length PhoA construct also led to the translocation of
mature PhoA (with its intrinsic signal peptide, slot 3, blue) but the negative control mature PhoA construct failed to do so (without a signal
peptide, PhoA, slot 2, white). (C) Bacterial transformants expressing the same three constructs were fractionated into periplasmic (per) and
cytosolic (cyto) fractions and the fractions were detected with antibodies against a FLAG tag (anti-Flag, panel a) and GroEL (anti-GroEL, panel b)
on a Western blot. Mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or HtrAss-PhoA
construct while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct. (D) cHtrA secretion into the
cytosol of chlamydia-infected cells is not inhibited by the type III secretion inhibitor C1 compound. HeLa monolayers infected with C.
trachomatis L2 for 6 hr were treated with DMSO (panels a, c & e) or 50 M C1 (b, d & f). Thirty-six hours after treatment, the cultures were
processed for triply labeling with antibodies against IncA (green) and cHtrA, CT621 or CPAF (red) and DAPI for DNA (blue). C1 inhibited secretion
of IncA and CT621 but not cHtrA or CPAF. Red arrows indicate chlamydia proteins that are secreted into host cell cytosol.
reference and under a confocal microscope. Second, the
antibody labeling of cHtrA was removed by absorption
with the cHtrA but not CPAF fusion proteins while the
labeling of CPAF was removed by CPAF but not cHtrA
fusion proteins, indicating that there was no
cross-reactivity between anti-cHtrA and anti-CPAF antibodies. Third,
in a Western blot with both HeLa alone and
Chlamydiainfected whole cell lysates as antigens, the anti-cHtrA
fusion protein antibodies detected a major protein band
migrated at the molecular position expected for cHtrA,
demonstrating that the anti-cHtrA antibodies specifically
recognized the endogenous cHtrA without cross-reacting
with any other cellular or chlamydial proteins. Fourth, the
cytosolic cHtrA signals are likely due to active secretion
but not passive leaking of cHtrA since various other
abundant periplasmic proteins were not detected in the host
cell cytosol. Finally, secretion of cHtrA into host cell
cytosol was detected 24 h after infection while CPAF secretion
occurred at 16 h after infection. Secretion of cHtrA was
detected in most chlamydial species but not C. psittaci.
These results together suggest that cHtrA secretion into
host cell cytosol is a specific process and the secreted
cHtrA may play an important role in chlamydial
HtrA is a highly conserved serine protease present in
the ER of eukaryotic and periplasmic space of bacterial
cells. However, there has been no report on its secretion
outside of eukaryotic or bacterial cells. Secretion of
cHtrA out of chlamydial organisms may represent a
unique feature Chlamydia has evolved during its
interactions with host cells. A sec-dependent pathway may play
an important role in exporting cHtrA into host cell
cytosol since the N-terminal leader peptide of cHtrA is
functional and the secretion is not inhibitable by a type III
secretion inhibitor. However, The sec-dependent
pathway can only translocate cHtrA into the periplasmic
region. It is still unknown how the periplasmic cHtrA
passes through the outer membrane to enter the
chlamydial inclusion lumen and further into host cell cytosol.
The same challenge also applies to the secretion of
CPAF. A sec-dependent pathway is necessary for CPAF
secretion . Similarly, how the periplasmic CPAF
crosses the outer membrane remains unclear. Since
CPAF was detected in granules in the lumen of
inclusions during the early stage of chlamydial intracellular
growth, an outer membrane vesicular budding model has
been proposed for CPAF secretion into host cell cytosol
, which may also be suitable for the secretion of
cHtrA (Figure 8). Evidence for supporting this hypothesis
comes from the observation that cHtrA-laden granules/
vesicles that are free of chlamydial organisms were
readily detected in the chlamydial inclusions. Although it
remains to be determined how exactly cHtrA or CPAF is
secreted out of the organisms and into host cell cytosol,
Figure 8 A proposed model for C. trachomatis secretion of effectors into host cell cytosol. When an infectious and metabolically inactive
elementary body (EB) attaches to an epithelial cell, preexisting effectors such as TARP and CT694 can be injected into host cell cytosol via a
single step type 3 secretion system (T3SS) for facilitating EB invasion. Once the internalized EB is differentiated into a non-infectious but
metabolically active reticulate body (RB), newly synthesized chlamydial proteins can be secreted into host cell cytosol via either the single step
T3SS (for example, secretion of CT847) or multi-step pathways. The C. trachomatis-secreted proteins (CtSPs) with an N-terminal signal sequence
(termed Sec-CtSPs) such as cHtrA & CPAF may be translocated into periplasm via a SecY-dependent pathway while those without any N-terminal
signal sequences (Nonsec-CtSPs) may be translocated into the periplasmic space via a novel translocon or a leaking T3SS pathway. The
periplasmically localized CtSPs may exit the chlamydial organisms via an outer membrane vesicle (OMV) budding mechanism. The CtSP-laden
vesicles in the inclusion lumen can enter host cell cytosol via vesicle fusion with or passing through the inclusion membrane. Thats why CT621
can be visualized in granules in the lumen of inclusion and its secretion can also be inhibited by C1, a small molecule inhibitor known to target
as more effector molecules are identified, more tools will
be available for figuring out the secretion pathways
Chlamydia has evolved for exporting virulence factors.
HtrA is a hexamer formed by two trimeric rings
staggered on top of each other [46,47]. It possesses dual
functions as both a chaperone and a protease .
Whether in eukaryotic ER or prokaryotic periplasmic
space, HtrA can transmit the stress signals from unfold
proteins into stress responses [48-51]. lt appears that
Chlamydia can respond to various stress signals by
regulating the expression levels of cHtrA . Although it
is still unknown how the periplasmic cHtrA works,
these previous observations can at least suggest that
cHtrA is functional during chlamydial infection.
Nevertheless, a more important question relevant to the
current study is what roles cHtrA has after it is secreted
into host cell cytosol and whether the secreted cHtrA
contributes to chlamydial pathogenesis. Can the secreted
cHtrA gain access to host cell ER to regulate host
unfolded protein stress responses? What cellular
proteins the secreted cHtrA molecules target during
chlamydial infection in the presence or absence of stress
stimulation. Efforts are underway to address these
Secretion of chlamydial proteins into host cells is
necessary for chlamydial organisms to establish and complete
intracellular growth. Thus, identifying chlamydial
proteins secreted into host cell cytoplasm has become a hot
subject. Here, we have presented convincing evidence
that the chlamydial periplasmic stress response serine
protease cHtrA is secreted out of the chlamydial
organisms into both chlamydial inclusion lumen and host cell
cytosol. This secretion is specific since various other
abundant chlamydial periplasmic proteins remained
within the organisms. This novel finding suggests that
the highly conserved cHtrA, in addition to its role in
modifying chlamydial proteins in the periplasmic region,
may also target host proteins, which is consistent with
the overall concept that Chlamydia may use proteolysis
as a powerful tool for manipulating host signaling
Note added in proof
During revision of the manuscript, Hoy et al published a
report on Helicobacter pylori HtrA as a new secreted
virulence factor that cleaves E-cadherin to disrupt
intercellular adhesion. Hoy et al. 2010. EMBO reports.
XW carried out most of the immunofluorescence and PhoA experiments; LL
performed the confocal and Western blot assays as well as repeated some
immunofluorescence assays; SG did the inhibitor experiment and carried out
some immunofluorescence assays; RF participated in the
immunofluorescence experiments; DC participated in the design of the
experiments and also provided technical guidance to XW. GZ conceived of
the study, and participated in its design and coordination and drafted the
manuscript. All authors read and approved the final manuscript.
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