The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol
© Wu et al; licensee BioMed Central Ltd. 2011
Received: 31 October 2010
Accepted: 28 April 2011
Published: 28 April 2011
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.
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 anti-cHtrA 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.
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.
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-κB 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 membrane-anchored 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 σE-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-Cal-3, H/UW-43/Cx, I/UW-12/Ur, K/UW-31/Cx, L1/LGV-440, 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 37°C 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 μM 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)-ATGATGAAAAGATTATTATGTGTG-3', cHtrA back primer, 5'-TTTTCCTTTT-GCGGCCGC(NotI)-CTACTCGTCTGATTTCAAGAC-3'. The ORF was expressed as a fusion protein with glutathione-S-transferase (GST) fused to the N-terminus 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 μM Leupeptin and 1.6 μM 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 anti-IncA 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 glutathione-conjugated agarose beads (Pharmacia). The absorption was carried out by incubating the antibodies with bead-immobilized antigens for 1 h at room temperature (RT) or overnight at 4°C 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 4°C. 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 4°C 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 4°C followed by a further centrifugation at 100,000 g for 1 h at 4°C. 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 4°C. 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 4°C. 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, pFLAG-'PhoA & 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 37°C overnight. The overnight cultures were streaked onto LB agar containing the same selection antibiotics and 50 μg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP, cat# B6149, Sigma) and the plates were incubated at 30°C 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
2. Secretion of cHtrA but not other chlamydial periplasmic proteins into host cell cytosol
3. Expression and secretion of cHtrA during chlamydial infection
4. The secretion of chlamydial HtrA may require a type II but not type III 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 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 Chlamydia-infected 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 pathogenesis.
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 questions.
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 pathways.
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. 11:798-804.
This work was supported in part by grants (to G. Zhong) from the US National Institutes of Health.
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