Inhibition of proteolytic activity of a chlamydial proteasome/protease-like activity factor by antibodies from humans infected with Chlamydia trachomatis

We have previously shown that individuals infected with Chlamydia trachomatis can develop a robust antibody response to a chlamydia-secreted protein (designated chlamydial proteasome/protease-like activity factor [CPAF]). We now report that sera collected from these infected individuals neutralized the proteolytic activity of CPAF. Depletion of the serum sample with CPAF proteins to remove the CPAF-specific antibodies effectively blocked the neutralization, whereas similar depletion with the HSP60 proteins failed to do so. We further demonstrated that the CPAF central region covering residues 200 to 338 was predominantly recognized by the human neutralization antibodies. The significance of the CPAF neutralization antibodies generated in chlamydia-infected individuals is discussed.     

Chlamydial protease-like activity factor induces protective immunity against genital chlamydial infection in transgenic mice that express the human HLA-DR4 allele

There is no licensed vaccine available against Chlamydia trachomatis, the leading cause of bacterial sexually transmitted disease. We have found that intranasal immunization with recombinant chlamydial protease-like activity factor (CPAF) induces CD4(+) T-cell- and gamma interferon (IFN-gamma)-dependent protective immunity against murine genital chlamydial infection, thus making CPAF a viable vaccine candidate for further characterization. HLA-DR4 is the predominant allele involved in chlamydial antigen presentation to CD4(+) T cells in humans. We used engineered mice that lack endogenous major histocompatibility complex class II (MHC-II) alleles but express a human HLA allele (HLA-DR4 transgenic [tg] mice) to examine primary immune and CPAF-mediated responses against genital Chlamydia muridarum challenge. Upon primary bacterial exposure, HLA-DR4 tg mice developed Chlamydia-specific IFN-gamma and antibody production and resolved the infection within 30 days, similar to challenged conventional C57BL/6 animals. Moreover, C. muridarum-challenged HLA-DR4 tg mice exhibited CPAF-specific antibody and IFN-gamma production. Upon CPAF-plus-interleukin-12 (IL-12) vaccination, HLA-DR4 tg animals exhibited robust CPAF-specific IFN-gamma production and elevated titers of anti-CPAF total antibody and immunoglobulin G2a (IgG2a) and lower titers of IgG2b and IgG1 antibodies. HLA-DR4 tg and C57BL/6 mice vaccinated with CPAF plus IL-12 resolved the primary genital chlamydial infection significantly earlier than mock-immunized animals, whereas similarly vaccinated MHC class II-deficient mice displayed minimal antigen-specific immune responses and failed to resolve the infection even at 30 days postchallenge. Together, these results demonstrate the importance of human HLA-DR4 molecules in the recognition and presentation of CPAF epitopes, leading to the generation of protective antichlamydial immunity and making these mice a valuable model for mapping HLA-DR4-restricted chlamydial epitopes.     

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.     

Autoprocessing and self-activation of the secreted protease CPAF in Chlamydia-infected cells

The Chlamydia-secreted protease/proteasome-like activity factor (CPAF) is synthesized as a proenzyme (proCPAF) and requires processing for proteolytic activity. Recent structural studies have further demonstrated that CPAF is a serine protease that can undergo autoprocessing and self-activation in a concentration-dependent manner in vitro. However, it is not known how CPAF is processed and activated during chlamydial infection. In the current study, we used a mutant CPAF designated as CPAF(E558A) that is deficient in processing by itself as a substrate to search for putative CPAF activation factor(s) in Chlamydia-infected cells. CPAF(E558A) was processed by the lysates made from Chlamydia-infected cells and the processing activity correlated with the presence of endogenous active CPAF in the fractionated lysate samples. CPAF produced in the Chlamydia-infected cells is required for processing the mutant CPAF(E558A) since the processing activity was removed by depletion with anti-CPAF but not control antibodies. Furthermore, a purified and activated wild type CPAF alone was sufficient for processing CPAF(E558A) and no other chlamydial proteases are required. Finally, fusion tag-induced oligomerization can lead to autoprocessing and self-activation of the wild type CPAF in mammalian cells. These observations together have demonstrated that CPAF undergoes autoprocessing and self-activation during chlamydial infection.     

The chlamydial plasmid-encoded protein pgp3 is secreted into the cytosol of Chlamydia-infected cells

The chlamydial cryptic plasmid encodes eight putative open reading frames (ORFs), designated pORF1 to -8. Antibodies raised against these ORF proteins were used to localize the endogenous proteins during chlamydial infection. We found that the pORF5 protein (also known as pgp3) was detected mainly in the cytosol of Chlamydia-infected cells, while the remaining seven proteins were found inside the chlamydial inclusions only. The pgp3 distribution pattern in the host cell cytosol is similar to but not overlapping with that of chlamydial protease/proteasome-like activity factor (CPAF), a chlamydial genome-encoded protein known to be secreted from chlamydial inclusions into the host cell cytosol. The anti-pgp3 labeling was removed by preabsorption with pgp3 but not CPAF fusion proteins and vice versa, demonstrating that pgp3 is a unique secretion protein. This conclusion is further supported by the observation that pgp3 was highly enriched in cytosolic fractions and had a minimal presence in the inclusion-containing nuclear fractions prepared from Chlamydia-infected cells. The pgp3 protein was detected as early as 12 h after infection and was secreted by all chlamydial species that carry the cryptic plasmid, suggesting that there is a selection pressure for maintaining pgp3 secretion during chlamydial infection. Although expression of pgp3 in the host cell cytosol via a transgene did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection, purified pgp3 protein stimulated macrophages to release inflammatory cytokines, suggesting that pgp3 may contribute to chlamydial pathogenesis.     

Vaccination against Chlamydia genital infection utilizing the murine C. muridarum model

Chlamydia trachomatis genital infection is a worldwide public health problem, and considerable effort has been expended on developing an efficacious vaccine. The murine model of C. muridarum genital infection has been extremely useful for identification of protective immune responses and in vaccine development. Although a number of immunogenic antigens have been assessed for their ability to induce protection, the majority of studies have utilized the whole organism, the major outer membrane protein (MOMP), or the chlamydial protease-like activity factor (CPAF). These antigens, alone and in combination with a variety of immunostimulatory adjuvants, have induced various levels of protection against infectious challenge, ranging from minimal to nearly sterilizing immunity. Understanding of the mechanisms of natural infection-based immunity and advances in adjuvant biology have resulted in studies that are increasingly successful, but a vaccine licensed for use in humans has not yet been brought to fruition. Here we review immunity to chlamydial genital infection and vaccine development using the C. muridarum model.     

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.     

Localization of Chlamydia trachomatis hypothetical protein CT311 in host cell cytoplasm

The chlamydia-specific hypothetical protein CT311 was detected both inside and outside of the chlamydial inclusions in Chlamydia trachomatis-infected cells. The extra-inclusion CT311 molecules were distributed in the host cell cytoplasm with a pattern similar to that of CPAF, a known Chlamydia-secreted protease. The detection of CT311 was specific since the anti-CT311 antibody labeling was only removed by absorption with CT311 but not CPAF fusion proteins. In addition, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Although both CT311 and CPAF proteins were first detected 12 h after infection, localization of CT311 into host cell cytosol was delayed until 24 h while CPAF secretion into host cell cytosol was already obvious by 18 h after infection. The host cell cytosolic localization of CT311 was further confirmed in human primary cells. CT311 was predicted to contain an N-terminal secretion signal sequence and the CT311 signal sequence directed secretion of PhoA into bacterial periplasmic region in a heterologous assay system, suggesting that a sec-dependent pathway may play a role in the secretion of CT311 into host cell cytosol. This hypothesis is further supported by the observation that secretion of CT311 in Chlamydia-infected cells was blocked by a C16 compound known to inhibit signal peptidase I. These findings have provided important molecular information for further understanding the C. trachomatis pathogenic mechanisms.     

Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR.

A nested PCR for genus-specific amplification of the Chlamydia omp1 locus was established. This PCR detected single template molecules in 200-microl specimen aliquots. Amplified chlamydial omp1 alleles were typed by heminested species PCRs and allele PCRs. We applied this method to 407 specimens from several host animals with various clinical conditions, and we detected prevalences of chlamydiae from 6 to 50%. Amplicons from peacock enteritis and equine infertility specimens were not typeable according to present omp1 allelic criteria for the chlamydial species. DNA sequencing revealed novel omp1 alleles which were 29.9 and 47.6% divergent in the deduced peptide sequences from the most closely related chlamydiae. Phylogenetic reconstruction indicated segregation of these alleles from the current four chlamydial species (90 and 97% bootstrap support), thus strongly suggesting the existence of additional chlamydial species. Allele typing of amplicons from swine with intestinal, urogenital, and respiratory infections demonstrated several unique omp1 allelic variants of Chlamydia trachomatis. These novel alleles had deduced peptide sequences which were 11.6 to 19% divergent from porcine C. trachomatis S45. Mutations were clustered in the C-terminal region of variable segment IV of the omp1 locus encoding subspecies and serovar determinants of the chlamydial major outer membrane protein, thus implying that there are numerous serovars of porcine C. trachomatis. These results demonstrate the need for routine application of sensitive genus-specific detection of chlamydiae in animal specimens and suggest a more prominent role than anticipated for chlamydiae in animal diseases.     

TestPack Chlamydia, a new rapid assay for the direct detection of Chlamydia trachomatis.

TestPack Chlamydia (Abbott Laboratories) is a rapid enzyme immunoassay for the direct antigen detection of Chlamydia trachomatis in endocervical specimens. The assay is self-contained, requires no specialized equipment, and yields results in less than 30 min. The clinical performance of TestPack Chlamydia versus chlamydial cell culture was evaluated with a total of 1,694 paired endocervical specimens. Discordant samples were further investigated by immunofluorescent staining and by Chlamydiazyme immunoassay, with confirmatory procedures. The sensitivity of TestPack Chlamydia with less-than-48-h-old specimens was 76.5%, while culture sensitivity was 86.7%. TestPack Chlamydia specificity was determined to be 99.5%. These results indicate that TestPack Chlamydia is an accurate test for chlamydial infection, with a positive predictive value of 96.2%. This assay is suitable for low-volume chlamydial testing in physician offices, clinics, and smaller laboratories.     

CPAF,HSP60 and MOMP antigens elicit pro-inflammatory cytokines production in the peripheral blood mononuclear cells from genital Chlamydia trachomatis-infected patients

Background

Persistent inflammation caused by Chlamydia trachomatis in the female genital compartment represents one of the major causes of pelvic inflammatory disease (PID), ectopic pregnancy and infertility in females. Here, we examined the pro-inflammatory cytokine response following stimulation with three different types of C. trachomatis antigens, viz. chlamydial protease-like factor (CPAF), heat shock protein 60 (HSP60) and major outer membrane protein (MOMP).

Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit

The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth.     

A Chlamydia trachomatis OmcB C-terminal fragment is released into the host cell cytoplasm and is immunogenic in humans

The Chlamydia trachomatis outer membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals in C. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed that OmcB was partially processed into OmcBc and OmcBn fragments. The processed OmcBc was released into host cell cytosol, while the OmcBn and remaining full-length OmcB were retained within the chlamydial inclusions. The organism-associated OmcB epitopes became detectable only after the C. trachomatis-infected cells were permeabilized with strong detergents such as SDS. However, the harsh permeabilization conditions also led to the leakage of the already secreted OmcBc and chlamydia-secreted protease (CPAF) out of the host cells. The OmcBc processing and release occurred in all biovars of C. trachomatis. Moreover, the released OmcBc but not the retained OmcBn was highly immunogenic in C. trachomatis-infected women, which is consistent with the concept that exposure of chlamydial proteins to host cell cytosol is accompanied by increased immunogenicity. These observations have provided important information for further exploring/optimizing OmcB as a target for the development of diagnosis methods and vaccines.     

Regulation of chlamydial infection by host autophagy and vacuolar ATPase-bearing organelles

As arguably the most successful parasite, Chlamydia is an obligate intracellular bacterium replicating inside a vacuole of eukaryotic host cells. The chlamydial vacuole does not fuse with the defense cell organelle lysosome. We previously showed that chlamydial infection increases markers of autophagy, an innate antimicrobial activity requiring lysosomal function. However, the work presented here demonstrates that p62, an autophagy protein that is degraded in lysosomes, either remained unchanged or increased in chlamydia-infected human epithelial, mouse fibroblast, and mouse macrophage cell lines. In addition, the activities of three lysosomal enzymes analyzed were diminished in chlamydia-infected macrophages. Bafilomycin A1 (BafA), a specific inhibitor of vacuolar ATPase (vATPase) required for lysosomal function, increased the growth of the human pathogen Chlamydia trachomatis (L2) in wild-type murine fibroblasts and macrophages but inhibited growth in the autophagy-deficient ATG5(-/-) fibroblasts. BafA exhibited only slight inhibition or no effect on L2 growth in multiple human genital epithelial cell lines. In contrast to L2, the mouse pathogen Chlamydia muridarum (MoPn) was consistently inhibited by BafA in all cell lines examined, regardless of species origin and autophagy status. Finally, L2 but not MoPn grew more efficiently in the ATG5(-/-) cells than in wild-type cells. These results suggest that there are two types of vATPase-bearing organelles that regulate chlamydial infection: one supports chlamydial infection, while the other plays a defensive role through autophagy when cells are artificially infected with certain chlamydiae that have not been adapted to the host species.     

Chlamydia trachomatis proteasome protein as a significant pathogenicity factor

Sexually transmitted diseases are a global problem. These infections may lead to acute or chronic diseases. Chlamydia trachomatis is a dangerous and widespread pathogenicity factor that is not sensitive to conventional drugs and has no obvious symptoms. CPAF protein is a leading factor of pathogenesis. This protein inhibits the signaling pathways of the host cell and supports long survival of the pathogen in the host cell. The goal of this work was to review the general properties of the proteosome chlamydia CPAF protein, its functions, and role in pathology. The role of CPAF protein in the antichlamydia immune reaction is discussed. The prospects of the development of a promising antichlamydia vaccine, as well as new effective antichlamydia, drugs are also discussed.