Guangming Zhong

Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis

Chlamydia trachomatis is one of the most common bacterial pathogens and is the etiological agent of debilitating sexually transmitted and ocular diseases in humans. The organism is an obligate intracellular prokaryote characterized by a highly specialized biphasic developmental cycle. We have performed genomic transcriptional analysis of the chlamydial developmental cycle. This approach has led to the identification of a small subset of genes that control the primary (immediate-early genes) and secondary (late genes) differentiation stages of the cycle. Immediate-early gene products initiate bacterial metabolism and potentially modify the bacterial phagosome to escape fusion with lysosomes. One immediate early gene (CT147) is a homolog of the human early endosomal antigen-1 that is localized to the chlamydial phagosome; suggesting a functional role for CT147 in establishing the parasitophorous vacuole in a nonfusogenic pathway. Late gene products terminate bacterial cell division and constitute structural components and remodeling activities involved in the formation of the highly disulfide cross-linked outer-membrane complex that functions in attachment and invasion of new host cells. Many of the genes expressed during the immediate-early and late differentiation stages are Chlamydia-specific and have evolutionary origins in eukaryotic lineages.

The atherogenic effects of chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae

Epidemiological investigations have linked Chlamydia pneumoniae infection to atherosclerosis. It is not clear, however, whether C. pneumoniae infection plays a causal role in the development of atherosclerosis. Mice with low-density lipoprotein receptor deficiency were induced to develop atherosclerotic lesions in aorta with a cholesterol-enriched diet that increased serum cholesterol by two- to threefold. Using this mouse model, we found that the chlamydial infection alone with either the C. pneumoniae AR39 or the C. trachomatis MoPn strain failed to induce any significant atherosclerotic lesions in aorta over a period of nine months. However, in the presence of a high-cholesterol diet, infection with the C. pneumoniae AR39 strain significantly exacerbated the hypercholesterolemia-induced atherosclerosis, demonstrating that a hypercholesterolemic condition is required for the C. pneumoniae to aggravate the development of atherosclerosis. Although both AR39 and MoPn antigens were detected in aorta of mice infected with the corresponding strains, only mice infected with the C. pneumoniae strain AR39 displayed enhanced atherosclerotic lesions, suggesting that the C. pneumoniae species may possess a unique atherogenic property. This study may provide a model for further understanding the mechanisms of C. pneumoniae atherogenesis and evaluating chlamydial intervention strategies for preventing the advancement of atherosclerotic lesions enhanced by C. pneumoniae infection.

Activation of Raf/MEK/ERK/cPLA2 Signaling Pathway Is Essential for Chlamydial Acquisition of Host Glycerophospholipids

Chlamydiae, a diverse group of obligate intracellular pathogens replicating within cytoplasmic vacuoles of eukaryotic cells, are able to acquire lipids from host cells. Here we report that activation of the host Raf-MEK-ERK-cPLA2 signaling cascade is required for the chlamydial uptake of host glycerophospholipids. Both the MAP kinase pathway (Ras/Raf/MEK/ERK) and Ca2+-dependent cytosolic phospholipase A2 (cPLA2) were activated in chlamydia-infected cells. The inhibition of cPLA2 activity resulted in the blockade of the chlamydial uptake of host glycerophospholipids and impairment in chlamydial growth. Blocking either c-Raf-1 or MEK1/2 activity prevented the chlamydial activation of ERK1/2, leading to the suppression of both chlamydial activation of the host cPLA2 and uptake of glycerophospholipids from the host cells. The chlamydia-induced phosphorylation of cPLA2 was also blocked by a dominant negative ERK2. Furthermore, activation of both ERK1/2 and cPLA2 was dependent on chlamydial growth and restricted within chlamydia-infected cells, suggesting an active manipulation of the host ERK-cPLA2 signaling pathway by chlamydiae.

ANTIGEN PRESENTATION BY MHC CLASS II MOLECULES : INVARIANT CHAIN FUNCTION,PROTEIN TRAFFICKING, AND THE MOLECULAR BASIS OF DIVERSE DETERMINANT CAPTURE

Major histocompatibility complex class II molecules are heterodimeric integral membrane proteins whose primary function is the presentation of antigenic peptides derived from proteins entering the endocytic pathway to CD4+ T lymphocytes. To accomplish this physiologic function, class II molecules must assemble in the secretory pathway without undergoing irreversible ligand association at that site, traffic efficiently to the endocytic pathway, and productively interact with protein ligands in these organelles before their ultimate expression on the plasma membrane. Here we review our work describing how invariant chain promoters the assembly and transport process, the complex itinerary of class II-invariant chain complexes through the endocytic pathway, the role of large protein fragments as substrates for class II binding, and the existence of a second pathway for antigen capture by mature class II molecules that complements that involving newly synthesized dimers. We integrate these observations into a coherent model for the operation of a class II-dependent antigen processing and presentation system able to capture diverse antigenic determinants present in proteins of varying structure.

Characterization of Fifty Putative Inclusion Membrane Proteins Encoded in the Chlamydia trachomatis Genome

ABSTRACT 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 Chlamydia-infected 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 Secreted Protease Factor CPAF Is Responsible for Degrading Pro-apoptotic BH3-only Proteins in Chlamydia trachomatis-infected Cells

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 Pumaα 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.

The Chlamydial Plasmid-Encoded Protein pgp3 Is Secreted into the Cytosol of Chlamydia-Infected Cells

ABSTRACT 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.

Caspase-1 Contributes to Chlamydia trachomatis-Induced Upper Urogenital Tract Inflammatory Pathologies without Affecting the Course of Infection

ABSTRACT Chlamydia trachomatis infection induces inflammatory pathologies in the upper genital tract, potentially leading to ectopic pregnancy and infertility in the affected women. Caspase-1 is required for processing and release of the inflammatory cytokines interleukin-1β (IL-1β), IL-18, and possibly IL-33. In the present study, we evaluated the role of caspase-1 in chlamydial infection and pathogenesis. Although chlamydial infection induced caspase-1 activation and processing of IL-1β, mice competent and mice deficient in caspase-1 experienced similar courses of chlamydial infection in their urogenital tracts, suggesting that Chlamydia-activated caspase-1 did not play a significant role in resolution of chlamydial infection. However, when genital tract tissue pathologies were examined, the caspase-1-deficient mice displayed much reduced inflammatory damage. The reduction in inflammation was most obvious in the fallopian tube tissue. These observations demonstrated that although caspase-1 is not required for controlling chlamydial infection, caspase-1-mediated responses can exacerbate the Chlamydia-induced inflammatory pathologies in the upper genital tract, suggesting that the host caspase-1 may be targeted for selectively attenuating chlamydial pathogenicity without affecting the host defense against chlamydial infection.

Profiling of Human Antibody Responses to Chlamydia trachomatis Urogenital Tract Infection Using Microplates Arrayed with 156 Chlamydial Fusion Proteins

ABSTRACT 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.

Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production.

ABSTRACT There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of sexually transmitted bacterial disease worldwide. Conventional vaccination attempts using surface-exposed chlamydial antigens have achieved only partial success. We have employed a novel vaccination strategy using a secreted protein, chlamydial protease-like activity factor (CPAF), which has been shown to degrade host major histocompatibility complex transcription factors and keratin-8 and therefore may allow immune evasion and establishment of a productive infection. Intranasal immunization using recombinant CPAF (rCPAF) plus interleukin-12 (IL-12) (rCPAF+IL-12 immunization) was used to assess the protective immunity against genital Chlamydia muridarum infection in BALB/c mice. rCPAF+IL-12 immunization induced robust gamma interferon (IFN-γ) production and minimal IL-4 production by splenocytes upon in vitro recall with rCPAF. The total and immunoglobulin G2a (IgG2a) anti-rCPAF antibody levels in serum were significantly elevated after rCPAF+IL-12 vaccination, as were the total antibody, IgG2a, and IgA levels in bronchoalveolar lavage and vaginal fluids when the animals were compared to animals that received rCPAF alone. rCPAF+IL-12-vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and accelerated resolution of infection compared to mock-immunized (phosphate-buffered saline) animals. Moreover, rCPAF+IL-12-immunized animals exhibited protection against pathological consequences of chlamydial infection, including the development of hydrosalpinx and oviduct dilatation. This vaccination regimen also reduced the development of fibrosis and the influx of neutrophils into the upper genital tract when the animals were compared to mock-immunized (phosphate-buffered saline) animals after bacterial challenge. rCPAF+IL-12-mediated resolution of the bacterial infection and protection against Chlamydia-induced inflammatory disease were highly dependent on endogenous IFN-γ production. Together, these results demonstrate that secreted chlamydial antigens may be novel vaccine candidates to induce protective immunity.