Chlamydial Protease-like Activity Factor Research Articles

A TriAdj-Adjuvanted Chlamydia trachomatis CPAF Protein Vaccine Is Highly Immunogenic in Pigs.

Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options-twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.

The role of an enzymatically inactive CPAF mutant vaccination in Chlamydia muridarum genital tract infection

Chlamydia trachomatis urogenital tract infection causes pelvic inflammatory disease and infertility, increases the risk of co-infection with HPV and HIV. Chlamydial vaccination is considered the most promising approach to prevent and control its infection. Among various chlamydial vaccine candidates, chlamydial protease-like activity factor (CPAF) have been reported to provide robust protective immunity against genital chlamydial infection in mice with reduced vaginal shedding and oviduct pathology. However, CPAF is a serine protease which has enzymatical activity to degrade a large number of substrates. In order to increase the safety of CPAF vaccine, in this study, we used a mutant CPAF that is deficient in enzymatical activity to determine whether proteolytic activity of CPAF affect its vaccine efficacy. The wild type or mutant CPAF immunization causes a significant lower chlamydial shedding from the vaginal and resolve the infection as early as day 20, compared to day 28 in adjuvant control mice. More important, reduced upper reproductive tract pathology were also observed in these two groups. The mutant or wild type CPAF immunization induced not only robust splenic IFN-γ and serum IgG2a but also sIgA secretion in the vaginal fluids. Furthermore, neutralization of chlamydia with immune sera did not provide protection against oviduct pathology. However, adoptive transfer of CD4+ splenocytes isolated from the mutant or wild type CPAF immunized mice resulted in a significant and comparable reduced oviduct pathology. Our results indicate mutant CPAF vaccination is as same efficacy as wild type, and the protection relies on CD4+ T cells, which will further promote the development of CPAF as clinical chlamydial vaccine.

Insights into the Autoproteolytic Processing and Catalytic Mechanism of the Chlamydia trachomatis Virulence-Associated Protease CPAF.

CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (kcat = 45 ± 0.6 s-1; kcat/Km = 0.37 ± 0.02 μM-1 s-1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s-1; kcat/Km = 0.29 ± 0.03 μM-1 s-1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (kacyl = 110 ± 2 s-1) followed by slower, partially rate-limiting deacylation (kdeacyl = 57 ± 1 s-1). Both kcat- and kcat/Km-pH profiles showed single acidic limb ionizations with pKa values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.

Combining individual Chlamydia trachomatis IgG antibodies MOMP, TARP, CPAF, OMP2, and HSP60 for tubal factor infertility prediction.

ProblemTubal factor infertility (TFI) is a severe complication of genital Chlamydia trachomatis infections. In fertility workup, chlamydia antibody test (CAT) is used to predict TFI. The predictive value for TFI of most commonly used CAT is moderate.Method of studyA total of 183 infertile Dutch Caucasian women were included in this study. All underwent tubal patency testing (hysterosalpingography [HSG] or laparoscopy). Cases had TFI, and controls had no TFI (ie normal findings during HSG or laparoscopy). TFI was categorized based on severity (TFI 1‐TFI 4). This study investigated the predictive values of major outer membrane protein (MOMP), translocated actin‐recruiting phosphoprotein (TARP), chlamydial protease‐like activity factor (CPAF), heat shock protein‐60 (HSP60) and outer membrane protein 2 (OMP2) for TFI. A predictive algorithm is developed to detect TFI with a high certainty based on combinations of antibody titres. Serum was tested with the Mikrogen recomLine immunoblot and quantified with the recomScan. A greedy algorithm that explores all possible antibody combinations was developed.ResultsSignificant differences in the distributions of antigen titres between cases and controls were observed for CPAF (P = 0.0021), HSP60 (P = 0.0061), MOMP (P = 0.0497) and OMP2 (P = 0.0016). Single antibodies could not discriminate between TFI and controls by themselves. The greedy algorithm performs better in specificity, positive predictive value (PPV), accuracy and clinical utility index than the original Mikrogen algorithm. CPAF combined with HSP60 identified 18.2% of TFI cases with 100% certainty. Most of the TFI 4 cases were identified with cut‐offs of CPAF > 10.7 or OMP2 > 3.9.ConclusionThis proof‐of‐principle study shows that combinations of antibodies in serum are predictive for TFI. A commercially available test can be adapted to predict TFI with a 100% specificity.

Infection of HeLa cells with Chlamydia trachomatis inhibits protein synthesis and causes multiple changes to host cell pathways.

The obligate intracellular bacterium Chlamydia trachomatis replicates in a cytosolic vacuole in human epithelial cells. Infection of human cells with C.trachomatis causes substantial changes to many host cell-signalling pathways, but the molecular basis of such influence is not well understood. Studies of gene transcription of the infected cell have shown altered transcription of many host cell genes, indicating a transcriptional response of the host cell to the infection. We here describe that infection of HeLa cells with C.trachomatis as well as infection of murine cells with Chlamydia muridarum substantially inhibits protein synthesis of the infected host cell. This inhibition was accompanied by changes to the ribosomal profile of the infected cell indicative of a block of translation initiation, most likely as part of a stress response. The Chlamydia protease-like activity factor (CPAF) also reduced protein synthesis in uninfected cells, although CPAF-deficient C.trachomatis showed no defect in this respect. Analysis of polysomal mRNA as a proxy of actively transcribed mRNA identified a number of biological processes differentially affected by chlamydial infection. Mapping of differentially regulated genes onto a protein interaction network identified nodes of up- and down-regulated networks during chlamydial infection. Proteomic analysis of protein synthesis further suggested translational regulation of host cell functions by chlamydial infection. These results demonstrate reprogramming of the host cell during chlamydial infection through the alteration of protein synthesis.

Intranasal immunization with recombinant chlamydial protease-like activity factor attenuates atherosclerotic pathology following Chlamydia pneumoniae infection in mice.

We have shown previously that intranasal vaccination with recombinant chlamydial protease-like activity factor (rCPAF: antigen) and interleukin-12 (IL-12) as an adjuvant induces robust protection against pathological consequences of female genital tract infection with Chlamydia muridarum, a closely related species and a rodent model for the human pathogen Chlamydia trachomatis. Another related species Chlamydia pneumoniae, a human respiratory pathogen, has been associated with exacerbation of atherosclerotic pathology. CPAF is highly conserved among Chlamydia spp. leading us to hypothesize that immunization with rCPAF with IL-12 will protect against high-fat diet (HFD) and C.pneumoniae-induced acceleration of atherosclerosis. rCPAF±IL-12 immunization induced robust splenic antigen (Ag)-specific IFN-γ and TNF-α production and significantly elevated serum total anti-CPAF Ab, IgG2c, and IgG1 antibody levels compared to mock or IL-12 alone groups. The addition of IL-12 to rCPAF significantly elevated splenic Ag-specific IFN-γ production and IgG2c/IgG1 anti-CPAF antibody ratio. Following intranasal C.pneumoniae challenge and HFD feeding, rCPAF±IL-12-immunized mice displayed significantly enhanced splenic IFN-γ, not TNF-α, response on days 6 and 9 after challenge, and significantly reduced lung chlamydial burden on day 9 post-challenge compared to mock- or IL-12-immunized mice. Importantly, rCPAF±IL-12-immunized mice displayed significantly reduced atherosclerotic pathology in the aortas after C.pneumoniae challenge. Serum cholesterol levels were comparable between the groups suggesting that the observed differences in pathology were due to protective immunity against the infection. Together, these results confirm and extend our previous observations that CPAF is a promising candidate antigen for a multisubunit vaccine regimen to protect against Chlamydia-induced pathologies, including atherosclerosis.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars.

ABSTRACTChlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars—lymphogranuloma venereum (LGV) and trachoma—which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.

Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared to day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high level 4 weeks post challenge. Upregulation of antigen specific IFN-γ gene expression was observed in rCPAF/CpG vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared to CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation.

ABSTRACTThe chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; however, challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets. Here, we use a genetic and proteomic approach to identify authentic CPAF targets. Human epithelial cells infected with CPAF-sufficient and CPAF-deficient chlamydiae were lysed using known CPAF-denaturing conditions. Their protein profiles were analyzed using isobaric mass tags and liquid chromatography-tandem mass spectrometry. Comparative analysis of CPAF-sufficient and CPAF-deficient infections identified a limited number of CPAF host and chlamydial protein targets. Host targets were primarily interferon-stimulated gene products, whereas chlamydial targets were type III secreted proteins. We provide evidence supporting a cooperative role for CPAF and type III secreted effectors in blocking NF-κB p65 nuclear translocation, resulting in decreased beta interferon and proinflammatory cytokine synthesis. Genetic complementation of null organisms with CPAF restored p65 nuclear translocation inhibition and proteolysis of chlamydial type III secreted effector proteins (T3SEs). We propose that CPAF and T3SEs cooperate in the inhibition of host innate immunity.

Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics

We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins.

The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106–212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins.

Characterization of CPAF Critical Residues and Secretion during Chlamydia trachomatis Infection

CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were defined in cell-free systems. Complementation of a CPAF-deficient chlamydial organism with a plasmid-encoded CPAF has enabled us to characterize CPAF during infection. The transformants expressing CPAF mutated at intermolecular dimerization, catalytic, or cleavage residues still produced active CPAF, although at a lower efficiency, indicating that CPAF can tolerate more mutations inside Chlamydia-infected cells than in cell-free systems. Only by simultaneously mutating both intermolecular dimerization and catalytic residues was CPAF activation completely blocked during infection, both indicating the importance of the critical residues identified in the cell-free systems and exploring the limit of CPAF's tolerance for mutations in the intracellular environment. We further found that active CPAF was always detected in the host cell cytoplasm while nonactive CPAF was restricted to within the chlamydial inclusions, regardless of how the infected cell samples were treated. Thus, CPAF translocation into the host cell cytoplasm correlates with CPAF enzymatic activity and is not altered by sample treatment conditions. These observations have provided new evidence for CPAF activation and translocation, which should encourage continued investigation of CPAF in chlamydial pathogenesis.

Preparation and evaluation of monoclonal antibodies against chlamydial protease-like activity factor to detect Chlamydia pneumoniae antigen in early pediatric pneumonia.

Chlamydia pneumoniae causes diseases in humans, including community-acquired pneumonia, bronchitis, and sinusitis. It is also associated with atherosclerosis, coronary heart disease, and hyperlipidemia. In this study, we investigated novel materials with which to develop a sensitive and specific method to identify early C. pneumoniae infection, to allow more effective clinical treatment and prevention. We prepared novel monoclonal antibodies (mAbs) against a recombinant protein equivalent to the immunodominant region of chlamydial protease-like activity factor (CPAF) from C. pneumoniae. The mAbs specifically reacted with the endogenous CPAF antigen of the C. pneumoniae type strain in immunoblotting and indirect immunofluorescence (IIF) assays, but did not react with C. trachomatis type strains or genital secretions from patients with acute C. trachomatis infection. The mAb with the highest titer was used to develop a new IIF assay and enzyme-linked immunosorbent assay (ELISA) to detect the C. pneumoniae antigen in clinical specimens from child patients suspected of pneumonia. The sensitivity, specificity, and concordance rate of the mAb-based IIF and ELISA tests were compared with those of polymerase chain reaction (PCR). Our results show that these mAbs have excellent specificity and may be used to develop new screening tools for the diagnosis of early pediatric pneumonia.

Screening of immunodominant proteins for sensitive detection of Chlamydia trachomatis infection by serological test

Objective To detect the antibodies against recombinant chlamydial plasmid-encoded protein 3（rPgp3）,chlamydial protease-like activity factor（rCPAF）,Ct143 encoded protein（rCT143）, Ct101 encoded protein（rCT101）,Ct694 encoded protein（rCT694）,Ct813 encoded protein（rCT813）, Chlamydia membrane protein A（rIncA）,Ct875 encoded protein（rCT875）,major outer membrane protein （rMOMP）and heat shock protein 60（ rHsp60）in serum samples collected form patients with urogenital Chlamydia trachomatis（Ct）infection and to evaluate the antigenicity of those proteins. Methods The recombinant plasmids expressing the 10 proteins and a blank plasmid were transformed into E. coli BL21 strains,respectively. The transformed E. coli BL21 strains were induced by isopropyl β-D-1-thiogalactopyr-anoside（IPTG）to express recombinant proteins. The glutathione pre-coated 96-well ELISA plates were coa-ted with lysates. Serum samples were collected from 50 patients with Ct infection and 10 patients without Ct infection. ELISA was performed to detect the antibodies against 10 recombinant proteins. Results The anti-bodies against rPgp3,rCPAF,rCT143,rCT101,rCT694,rCT875,rCT813,rMOMP,rIncA and rHsp60 proteins were respectively detected in 44 cases（88% ）,38 cases（76% ）,37 cases（74% ）,36 cases （72% ）,33 cases（66% ）,31 cases（62% ）,30 cases（60% ）,26 cases（52% ）,24 cases（48% ）and 17 cases（34% ）out of 50 serum samples. No antibodies against 10 recombinant proteins were detected in the serum samples collected from patients without Ct infection. Conclusion The rPgp3 protein showed the strongest antigenicity among all of the studied proteins,followed by rCPAF and rCT143 proteins. The rHsp60 protein showed the lowest antigenicity. Key words: Chlamydia trachomatis; Immunodominant protein; Serological test

Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections.

Immunoglobulin A is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intra-epithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant secretory IgA (SIgA) we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra- and intra-epithelial stages of infection. We developed an in vitro model using polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model using pIgR(-/-) mice. Secretory IgA targeting the extra-epithelial chlamydial antigen, the major outer membrane protein, significantly reduced infection in vitro by 24% and in vivo by 44%. Conversely, pIgR-mediated delivery of IgA targeting the intra-epithelial inclusion membrane protein A bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intra-epithelial IgA targeting the secreted protease Chlamydia protease-like activity factor also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra-epithelial, but not intra-epithelial, chlamydial antigens for protection against a genital tract infection.

Question the questions on CPAF.

This is in response to the editorial commentary on CPAF substrates and secretion authored by Bavoil & Byrne (2014). Chlamydial protease like activity factor (CPAF) was first purified in two fragments (Zhong et al ., 2001) that formed intramolecular heterodimers (Dong et al ., 2004a, b) for acquiring a serine protease activity (Huang et al ., 2008; Chen et al ., 2009, 2010a). The crystal structure revealed a proximity dependent super dimerization and sequential processing mechanism for CPAF activation (Huang et al ., 2008). CPAF was characterized as a sec dependent secretion protein (Chen et al ., 2010b …

Profiling Antibody Responses to Infections by Chlamydia abortus Enables Identification of Potential Virulence Factors and Candidates for Serodiagnosis

Enzootic abortion of ewes (EAE) due to infection with the obligate intracellular pathogen Chlamydia (C.) abortus is an important zoonosis leading to considerable economic loss to agriculture worldwide. The pathogen can be transmitted to humans and may lead to serious infection in pregnant women. Knowledge about epidemiology, clinical course and transmission to humans is hampered by the lack of reliable diagnostic tools. Immunoreactive proteins, which are expressed in infected animals and humans, may serve as novel candidates for diagnostic marker proteins and represent putative virulence factors. In order to broaden the spectrum of immunogenic C. abortus proteins we applied 2D immunoblot analysis and screening of an expression library using human and animal sera. We have identified 48 immunoreactive proteins representing potential diagnostic markers and also putative virulence factors, such as CAB080 (homologue of the “macrophage infectivity potentiator”, MIP), CAB167 (homologue of the “translocated actin recruitment protein”, TARP), CAB712 (homologue of the “chlamydial protease-like activity factor”, CPAF), CAB776 (homologue of the “Polymorphic membrane protein D”, PmpD), and the “hypothetical proteins” CAB063, CAB408 and CAB821, which are predicted to be type III secreted. We selected two putative virulence factors for further characterization, i.e. CAB080 (cMIP) and CAB063, and studied their expression profiles at transcript and protein levels. Analysis of the subcellular localization of both proteins throughout the developmental cycle revealed CAB063 being the first C. abortus protein shown to be translocated to the host cell nucleus.

A T cell epitope-based vaccine protects against chlamydial infection in HLA-DR4 transgenic mice

Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4+ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4+ T cells in host defense against Chlamydia. In this study, we identify CD4+ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4+ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4+ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4+ clones could be detected in the mouse spleen following Chlamydia muridarum and a human Chlamydia trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4+ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4+ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.

Protochlamydia Induces Apoptosis of Human HEp-2 Cells through Mitochondrial Dysfunction Mediated by Chlamydial Protease-Like Activity Factor

Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7–1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae.

Persistent Chlamydia trachomatis Infection of HeLa Cells Mediates Apoptosis Resistance through a Chlamydia Protease-Like Activity Factor-Independent Mechanism and Induces High Mobility Group Box 1 Release

Intracellular persistence of Chlamydia trachomatis has been implicated in the development of chronic infection that can result in pelvic inflammatory disease and tubal sterility. By inhibition of host cell apoptosis, chlamydiae have evolved a strategy to maintain the intracellular environment for replication and persistence. Both antiapoptotic host cell-derived factors and the chlamydial protease-like activity factor (CPAF) are involved in Chlamydia-mediated apoptosis resistance. Here, we show that in HeLa cells infected with gamma interferon (IFN-γ)-induced persistent C. trachomatis serovar D, the expression of CPAF is downregulated, and proapoptotic protease substrates are not cleaved. Persistent infection protected HeLa cells from apoptosis when they were exposed to staurosporine. Small-interfering RNA-mediated inhibition of myeloid cell leukemia 1 (Mcl-1) protein upregulation sensitized persistently infected cells for apoptosis. The inhibitor of apoptosis protein 2 (IAP-2) seems not to be relevant in this context because IAP-2 protein was not induced in response to IFN-γ treatment. Although apoptosis was inhibited, persistent infection caused cell membrane disintegration, as measured by the increased release of cytokeratin 18 from HeLa cells. Moreover, persistently infected cells released significantly increased amounts of high mobility group box 1 (HMGB1) protein which represents a proinflammatory damage-associated pattern molecule. The data of this study suggest that cells infected with persistent C. trachomatis are protected from apoptosis independently of CPAF but may promote chronic inflammation through HMGB1 release.