Chlamydial Protein Research Articles

Evolution and Conservation of Predicted Inclusion Membrane Proteins in Chlamydiae

Chlamydia spp. are obligate intracellular pathogens that replicate within a vacuole termed the inclusion. Chlamydiae extensively modify the inclusion membrane via the insertion of chlamydial inclusion membrane proteins (Incs) which decorate the cytosolic face of the inclusion. We have assessed the overall relatedness and phylogeny of Incs in order to identify potential evolutionary trends. Despite a high degree of conservation among Incs within C. trachomatis serovars, phylogenetic analysis showed that some Incs cluster according to clinical groupings suggesting that certain Incs may contribute to tissue tropism. Bioinformatic predictions identified Incs in five chlamydial species: 55 in C. trachomatis, 68 in C. felis, 92 in C. pneumoniae, 79 in C. caviae, and 54 in C. muridarum. Inc homologues were compared between chlamydial species and 23 core Incs were identified as shared among all species. Genomic expansion of Incs was identified in C. pneumoniae, C. caviae, and C. felis but not C. trachomatis or C. muridarum.

Protective immunity against mouse upper genital tract pathology correlates with high IFNγ but low IL-17 T cell and anti-secretion protein antibody responses induced by replicating chlamydial organisms in the airway

To search for optimal immunization conditions for inducing protective immunity against upper genital tract pathologies caused by chlamydial intravaginal infection, we compared protection efficacy in mice immunized intranasally or intramuscularly with live or inactivated Chlamydia muridarum organisms. Mice immunized intranasally with live organisms developed strong protection against both vaginal shedding of infectious organisms and upper genital tract pathologies. The protection correlated with a robust antigen-specific T cell response with high IFNγ but low IL-17. Although a significant level of IL-5 was also detected, these mice maintained an overall Th1-dorminant immunity following immunization and challenge infection. On the contrary, mice immunized intranasally with inactivated organisms or intramuscularly with live or inactivated organisms produced high levels of IL-17 and still developed significant upper genital tract pathologies. High titers of antibodies against chlamydial secretion antigens were detected only in mice immunized intranasally with live organisms but not mice in other groups, suggesting that the intranasally inoculated live organisms were able to undergo replication and immune responses to the chlamydial secretion proteins may contribute to protective immunity. These observations have provided important information on how to develop subunit vaccines for inducing protective immunity against urogenital infection with Chlamydia trachomatis organisms.

A Chlamydia-Specific C-Terminal Region of the Stress Response Regulator HrcA Modulates Its Repressor Activity

Chlamydial heat shock proteins have important roles in Chlamydia infection and immunopathogenesis. Transcription of chlamydial heat shock genes is controlled by the stress response regulator HrcA, which binds to its cognate operator CIRCE, causing repression by steric hindrance of RNA polymerase. All Chlamydia spp. encode an HrcA protein that is larger than other bacterial orthologs because of an additional, well-conserved C-terminal region. We found that this unique C-terminal tail decreased HrcA binding to CIRCE in vitro as well as HrcA-mediated transcriptional repression in vitro and in vivo. When we isolated HrcA from chlamydiae, we only detected the full-length protein, but we found that endogenous HrcA had a higher binding affinity for CIRCE than recombinant HrcA. To examine this difference further, we tested the effect of the heat shock protein GroEL on the function of HrcA since endogenous chlamydial HrcA has been previously shown to associate with GroEL as a complex. GroEL enhanced the ability of HrcA to bind CIRCE and to repress transcription in vitro, but this stimulatory effect was greater on full-length HrcA than HrcA lacking the C-terminal tail. These findings demonstrate that the novel C-terminal tail of chlamydial HrcA is an inhibitory region and provide evidence that its negative effect on repressor function can be counteracted by GroEL. These results support a model in which GroEL functions as a corepressor that interacts with HrcA to regulate chlamydial heat shock genes.

Altered protein secretion of Chlamydia trachomatis in persistently infected human endocervical epithelial cells

Chlamydia trachomatis is the most common bacterial infection of the human reproductive tract globally; however, the mechanisms underlying the adaptation of the organism to its natural target cells, human endocervical epithelial cells, are not clearly understood. To secure its intracellular niche, C. trachomatis must modulate the host cellular machinery by secreting virulence factors into the host cytosol to facilitate bacterial growth and survival. Here we used primary human endocervical epithelial cells and HeLa cells infected with C. trachomatis to examine the secretion of bacterial proteins during productive growth and persistent growth induced by ampicillin. Specifically, we observed a decrease in secretable chlamydial protease-like activity factor (CPAF) in the cytosol of host epithelial cells exposed to ampicillin with no evident reduction of CPAF product by C. trachomatis. In contrast, the expression of CopN and Tarp was downregulated, suggesting that C. trachomatis responds to ampicillin exposure by selectively altering the expression of secretable proteins. In addition, we observed a greater accumulation of outer-membrane vesicles from C. trachomatis in persistently infected cells. Taken together, these results suggest that the regulation of both gene expression and the secretion of chlamydial virulence proteins is involved in the adaptation of the bacteria to a persistent infection state in human genital epithelial cells.

P4-S1.01 Role of Chlamydia trachomatis heat shock proteins 60 and 10 in induction of apoptosis in endocervical epithelial cells

BackgroundChlamydiae are known to modulate host cell to escape from immune response and prolong their persistence to cause fallopian tube damage, ectopic pregnancy and infertility. In addition, Chlamydiae have been...

O4-S2.02 Seroprevalence of novel immunogens of chlamydia trachomatis and their cytokine response in PBMC Cells under in vitro conditions

BackgroundChlamydia trachomatis (CT) is an obligate intracellular parasite which causes STD and trachoma. Despite major research into Chlamydial pathogenesis and host immune responses, immuno-protection has been hampered by the incomplete...

Influence of theChlamydia pneumoniaeAR39 bacteriophage φCPAR39 on chlamydial inclusion morphology

The human respiratory tract pathogen Chlamydia pneumoniae AR39 is naturally infected by the bacteriophage ϕCPAR39. The phage genome encodes six ORFs, [ORF8, ORF4, ORF5, and viral protein (VP) 1, VP2 and VP3]. To study the growth of the phage, antibodies were generated to VP1 and used to investigate the ϕCPAR39 infection. Using immunofluorescence laser confocal microscopy and two-dimensional gel electrophoresis, we investigated the ϕCPAR39 infection of C. pneumoniae AR39. It was observed that ϕCPAR39 infection differentially suppressed the C. pneumoniae protein synthesis as the polymorphic membrane protein 10 and the secreted chlamydial protein Cpn0796 was hardly expressed while the secreted chlamydial protein Cpaf was expressed, but not secreted. The inclusion membrane protein, IncA, was demonstrated to surround the phage-infected abnormal reticulate bodies (RB) as well as being located in the inclusion membrane. As IncA is secreted by the type 3 secretion (T3S) system, it is likely that the T3S is disrupted in the phage-infected chlamydiae such that it accumulates around the infected RB.

Unity in Variety--The Pan-Genome of the Chlamydiae

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

Vaccination with DNA vector expressing chlamydial low calcium response protein E (LcrE) against Chlamydophila pneumoniae infection

Chlamydophila pneumoniae is an obligate intracellular human pathogen, which causes acute respiratory tract infections and can also cause chronic infections. C. pneumoniae possess type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and the host cell cytosol. Low calcium response protein E (LcrE) is a part of TTSS. The gene of LcrE in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin, this gene was also cloned into a eukaryotic expression vector (pΔRC). One group of BALB/c mice received an intramuscular pΔRC inoculation then the mice were immunized with purified LcrE protein; the second group of mice was immunized two times with the recombinant plasmid (pΔRCLcrE), and the third group was primed with pΔRCLcrE inoculation then boosted with LcrE protein. LcrE-specific antibody response was induced by DNA immunization with a shift towards Th1 isotype pattern compared to protein-immunization, this shifting pattern was observed in plasmid primed then protein-boosted animals. DNA immunization given as a priming and followed by a protein booster significantly reduced the number of viable bacteria in the lungs after challenge with C. pneumoniae. These results confirm that immunization with pΔRCLcrE can be an effective part of a vaccination schedule against C. pneumoniae.

The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol

BackgroundThe 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.ResultsThe 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.ConclusionSince 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.

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.

Multi-genome identification and characterization of chlamydiae-specific type III secretion substrates: the Inc proteins

BackgroundChlamydiae are obligate intracellular bacteria that multiply in a vacuolar compartment, the inclusion. Several chlamydial proteins containing a bilobal hydrophobic domain are translocated by a type III secretion (TTS) mechanism into the inclusion membrane. They form the family of Inc proteins, which is specific to this phylum. Based on their localization, Inc proteins likely play important roles in the interactions between the microbe and the host. In this paper we sought to identify and analyze, using bioinformatics tools, all putative Inc proteins in published chlamydial genomes, including an environmental species.ResultsInc proteins contain at least one bilobal hydrophobic domain made of two transmembrane helices separated by a loop of less than 30 amino acids. Using bioinformatics tools we identified 537 putative Inc proteins across seven chlamydial proteomes. The amino-terminal segment of the putative Inc proteins was recognized as a functional TTS signal in 90% of the C. trachomatis and C. pneumoniae sequences tested, validating the data obtained in silico. We identified a macro domain in several putative Inc proteins, and observed that Inc proteins are enriched in segments predicted to form coiled coils. A surprisingly large proportion of the putative Inc proteins are not constitutively translocated to the inclusion membrane in culture conditions.ConclusionsThe Inc proteins represent 7 to 10% of each proteome and show a great degree of sequence diversity between species. The abundance of segments with a high probability for coiled coil conformation in Inc proteins support the hypothesis that they interact with host proteins. While the large majority of Inc proteins possess a functional TTS signal, less than half may be constitutively translocated to the inclusion surface in some species. This suggests the novel finding that translocation of Inc proteins may be regulated by as-yet undetermined mechanisms.

Identification of novel markers for uncomplicated lower genital tract infections and upper genital tract pathology due to Chlamydia trachomatis

Untreated Chlamydia trachomatis infections in women can result in disease sequelae such as salpingitis and pelvic inflammatory disease (PID), ultimately culminating in tubal occlusion and infertility. Whilst nucleic acid amplification tests can effectively diagnose uncomplicated lower genital tract (LGT) infections, they are not suitable for diagnosing upper genital tract (UGT) pathological sequelae. As a consequence, this study aimed to identify serological markers that can, with a high degree of sensitivity and specificity, discriminate between LGT infections and UGT pathology. Plasma was collected from 73 women with a history of LGT infection, UGT pathology due to C. trachomatis, or no serological evidence of C. trachomatis infection. Western blotting was used to analyze antibody reactivity against extracted chlamydial proteins. Sensitivity and specificity of differential markers were also calculated. Four antigens (CT157, CT423, CT727 and CT396) were identified and found to be capable of discriminating between the infection and disease sequelae state. Sensitivity and specificity calculations showed that our assay for diagnosing LGT infection had a sensitivity of 75% and specificity of 76%, whilst the assay for identifying UGT pathology demonstrated 80% sensitivity and 86% specificity. The use of these assays could potentially facilitate earlier diagnoses in women suffering UGT pathology due to C. trachomatis.

Structure Prediction and Simulations of the Major Outer Membrane Protein of Chlamydia

Chlamydia trachomatis is an obligate intracellular human pathogen which can cause trachoma, conjunctivitis, infection of urogenital system and lymphogranuloma-venereum in humans. Preventive measures have recently focused on production of vaccine using the variable domain (VD) extracellular loops of the major outer membrane protein (MOMP). MOMP's crystal structure has not been determined, but serious efforts have been made to propose its structure and function experimentally and theoretically. Our objective is to utilize this information to obtain a consistent secondary and tertiary structure. MOMP functions as general diffusion porin having β-barrel structure. We predict 16 β-stranded transmembrane sheets, and variable domains at proper positions, consistent with available experimental data. Having no sequence similarity of MOMP with other porin proteins, a threading technique was utilized for structure prediction with the outer membrane porin protein (2OMF) of E.Coli as a template. Using hand-threading, MOMP sequence was aligned along the structure of 2OMF. The Modeler program was used to build a final structure (pre-refined). We selected a fragment with five β-sheets and loops containing VD-3 and VD-4. This fragment was further refined (post), using simulations, to get full length β-sheets in a realistic Chlamydial membrane. Our current objective is to continue molecular dynamics (MD) simulations to check the consistency of these (pre- and post-refined) fragments. Implicit water simulations of both refined fragments resulted in loss of secondary and tertiary structure, while MD simulations of post-refined fragment in Chlamydial membrane were stable. This implies that vaccine development requires delivery vehicles that are lipid-like to maintain the stability of these protein fragments. Moreover, future MD simulations of both fragments in a Chlamydial membrane will improve our understanding of stable loop conformations and guide experimentalists in developing a peptide-based vaccine.

Immunity and vaccines against sexually transmitted Chlamydia trachomatis infection

The aim is to review recent findings on immunity and vaccine development to Chlamydia trachomatis. There is increasing knowledge on the interactions between C. trachomatis and infected host cells. During genital infection the organism avoids generating protective immunity but immune responses to a number of chlamydial proteins have been associated with reproductive tract pathology. Various vaccine and adjuvant preparations have been tried experimentally. Information generated by proteomics and complex studies of serological and T-lymphocyte immune responses points to novel vaccine candidates. C. trachomatis, an obligate intracellular bacterium, is the commonest sexually transmitted infection worldwide and is associated with reproductive pathology. To develop rational vaccines it is necessary to understand the complex lifecycle of the organism, the host immune response to infection and how these relate to disease. Infection does not prevent re-infection and antibiotic treatment prevents antibody production at a population level. It remains unclear what type of immune response would be sufficient to prevent infection and/or re-infection. Although the prevalence and demographics of infection and the severity of disease associations suggest that it would be desirable, there is no vaccine currently available. A number of studies have identified novel vaccine candidates.

Chlamydia muridarum Major Outer Membrane Protein-Specific Antibodies Inhibit In Vitro Infection but Enhance Pathology In Vivo

Chlamydia trachomatis is a significant worldwide health problem, and the often-asymptomatic disease can result in infertility. To develop a successful vaccine, a complete understanding of the immune response to chlamydial infection and development of genital tract pathology is required. We utilized the murine genital model of chlamydial infection. Mice were immunized with chlamydial major outer membrane protein, and vaginal lavage was assessed for the presence of neutralizing antibodies. These samples were then pre-incubated with Chlamydia muridarum and administered to the vaginal vaults of immune-competent female BALB/c mice to determine the effect on infection. The administration of C. muridarum in conjunction with neutralizing antibodies reduced the numbers of mice infected, but a surprising finding was that this accelerated the development of severe oviduct pathology. Antibodies play an under-recognized role in chlamydial infection and pathology development, which possibly involves interaction with Th1 immunity.

Cytokine gene polymorphism and Chlamydia trachomatis-specific immune responses

Chlamydia trachomatis-induced fallopian tube damage leading to tubal factor infertility (TFI) is linked with TNF, IL-10, and probably IFNG gene polymorphisms. The aim of this study was to clarify the contribution of these cytokine gene polymorphisms to interindividual variation in C trachomatis-specific immune responses and the cross-regulation of secreted cytokines and single nucleotide polymorphisms (SNPs). Cytokine polymorphisms ( IL-10 -1082A/G, -819T/C, and -592A/C, IFNG +874T/A, and TNF -308G/A) were genotyped by polymerase chain reaction in 139 women. C trachomatis-specific immune responses were measured using lymphocyte proliferation (LP) induced by C trachomatis E and F strains and chlamydial heat shock protein 60 antigens. Cytokine secretion was measured in culture supernatants of infected and uninfected mononuclear leukocytes. IL-10 -1082/-819/-592 and IFNG +874 SNPs were associated with the intensity of LP responses to C trachomatis antigens. These cytokines also interact with each other and a cumulative effect of IL-10 -1082 and IFNG +874 genotypes was seen in LP responses to C trachomatis antigens. Our data suggest that interleukin-10 and interferon-γ regulate C trachomatis-specific immune responses in humans and that genetic variation in the expression of their coding genes explains interindividual variation in host immune responses to C trachomatis infection.

Is C. Pneumoniae Research in Peril?

Is C. Pneumoniae Research in Peril?

Chlamydia trachomatisandMycoplasma genitaliumPlasma Antibodies in Relation to Epithelial Ovarian Tumors

Objective. To assess associations of Chlamydia trachomatis and Mycoplasma genitalium antibodies with epithelial ovarian tumors. Methods. Plasma samples from 291 women, undergoing surgery due to suspected ovarian pathology, were analyzed with respect to C. trachomatis IgG and IgA, chlamydial Heat Shock Protein 60-1 (cHSP60-1) IgG and M. genitalium IgG antibodies. Women with borderline tumors (n = 12), ovarian carcinoma (n = 45), or other pelvic malignancies (n = 11) were matched to four healthy controls each. Results. Overall, there were no associations of antibodies with EOC. However, chlamydial HSP60-1 IgG antibodies were associated with type II ovarian cancer (P = .002) in women with plasma samples obtained >1 year prior to diagnosis (n = 7). M. genitalium IgG antibodies were associated with borderline ovarian tumors (P = .01). Conclusion. Chlamydial HSP60-1 IgG and M. genitalium IgG antibodies are in this study associated with epithelial ovarian tumors in some subsets, which support the hypothesis linking upper-genital tract infections and ovarian tumor development.

Antichlamydial Antibodies, Human Fertility, and Pregnancy Wastage

Genital infections with Chlamydia trachomatis (C. trachomatis) continue to be a worldwide epidemic. Immune response to chlamydia is important to both clearance of the disease and disease pathogenesis. Interindividual responses and current chlamydial control programs will have enormous effects on this disease and its control strategies. Humoral immune response to C. trachomatis occurs in humans and persistent antibody levels appear to be most directly correlated with more severe and longstanding disease and with reinfection. There is a close correlation between the presence of antichlamydial antibodies in females and tubal factor infertility; the closest associations have been found for antibodies against chlamydial heat shock proteins. The latter antibodies have also been shown to be useful among infertile patients with prior ectopic pregnancy, and their presence has been correlated with poor IVF outcomes, including early pregnancy loss. We review the existing literature on chlamydial antibody testing in infertile patients and present an algorithm for such testing in the infertile couple.