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 Chlamydiainfected 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.Chlamydia represents a group of obligate intracellular bacterial pathogens consisting of many different species and causing various health problems in both humans and animals. The species C. trachomatis is composed of more than 15 different serovars (including A to L serovars); some infect the human ocular epithelium, potentially leading to preventable blindness (64), while others infect human urogenital tract tissue, which can potentially cause severe complications such as ectopic pregnancy and infertility (56). C. muridarum, formerly known as the murine biovar of C. trachomatis (designated MoPn, with the single strain Nigg), has been extensively used to study C. trachomatis pathogenesis and immunology in mouse models (32,37,38,42,43). Although most isolates of C. pneumoniae organisms often infect the human respiratory system, causing respiratory pathologies and exacerbating lesions in the vascular wall (3, 28), the C. pneumoniae N16 strain has been isolated from equines (60). Other chlamydial species that mainly infect animals include C. caviae (50), C. psittaci (47), C. abortus (66), and C. felis (1). Despite the apparent differences in tissue tropism, all chlamydial species share similar genome sequences (1,29,49,50,66) and possess a common intracellular growth cycle with distinct biphasic stages (24). The chlamydial intracellular infection-induced inflammation is considered a major cause of Chlamydi...

Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydiainfected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.

The available chlamydial genome sequences have made it possible to comprehensively analyze host responses to all chlamydial proteins, which is essential for further understanding of chlamydial pathogenesis and development of effective chlamydial vaccines. Microplates arrayed with 156 Chlamydia trachomatis fusion proteins were used to evaluate antibody responses in women urogenitally infected with C. trachomatis. Based on both the antibody recognition frequency and titer, seven chlamydial antigens encoded by open reading frames (ORFs) CT089, CT147, CT226, CT681, CT694, CT795, and CT858, respectively, were identified as relatively immunodominant; six of these are encoded by hypothetical ORFs. Antibody binding to these chlamydial fusion proteins was blocked by C. trachomatis-infected but not by normal HeLa cell lysates or irrelevant bacterial lysates. These results have revealed novel immune-reactive chlamydial antigens, not only indicating that the hypothetical ORF-encoded proteins are expressed during chlamydial infection in humans but also providing the proof of principle that the fusion protein-based approach can be used to profile human immune responses to chlamydial infection at the whole-genome scale.

Chlamydia trachomatis has evolved a profound anti-apoptotic activity that may aid in chlamydial evasion of host defense. The C. trachomatis anti-apoptotic activity has been correlated with blockade of mitochondrial cytochrome c release, inhibition of Bax and Bak activation, and degradation of BH3-only proteins. This study presents evidence that a chlamydia-secreted protease factor designated CPAF is both necessary and sufficient for degrading the BH3-only proteins. When the C. trachomatis-infected cell cytosolic extracts were fractionated by column chromatography, both the CPAF protein and activity elution peaks overlapped with the BH3-only protein degradation activity peak. Depletion of CPAF with a CPAF-specific antibody removed the BH3-only protein degradation activity from the infected cell cytosolic extracts, whereas depletion with control antibodies failed to do so. Notably, recombinant CPAF expressed in bacteria was able to degrade the BH3-only proteins, whereas CPAF mutants similarly prepared from bacteria failed to do so. Finally, bacterium-expressed CPAF also degraded the human BH3-only protein Puma␣ purified from bacteria. These results demonstrate that CPAF contributes to the chlamydial anti-apoptotic activity by degrading the pro-apoptotic BH3-only Bcl-2 subfamily members.

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We found that IL-17, a signature cytokine of Th17, was produced early in the innate immunity phase after an intranasal infection with the obligate intracellular pathogen Chlamydia muridarum. The airway IL-17, which peaked at 48 h after infection, was dependent on live chlamydial organism replication and MyD88-mediated signaling pathways. Treatment with antibiotics or knockout of the MyD88 gene, but not Toll/IL receptor domain-containing adapter-inducing IFN-β, can block the early IL-17 production. Treatment of mice with an anti-IL-17-neutralizing mAb enhanced growth of chlamydial organisms in the lung, dissemination to other organs, and decreased mouse survival, whereas treatment with an isotype-matched control IgG had no effect. Although IL-17 did not directly affect chlamydial growth in cell culture, it enhanced the production of other inflammatory cytokines and chemokines by Chlamydia-infected cells and promoted neutrophil infiltration in mouse airways during chlamydial infection, which may contribute to the antichlamydial effect of IL-17. These observations suggest that an early IL-17 response as an innate immunity component plays an important role in initiating host defense against infection with intracellular bacterial pathogens in the airway.

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We have previously correlated Chlamydia trachomatis antiapoptotic activity with the blockade of mitochondrial cytochrome c release and the inhibition of Bax and Bak activation. We now report that C. trachomatis infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.