Chlamydia trachomatis Plasmid Gene Protein 3 Is Essential for the Establishment of Persistent Infection and Associated Immunopathology.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease afflicting hundreds of millions of people globally. A fundamental but poorly understood pathophysiological characteristic of chlamydial infection is the propensity to cause persistent infection that drives damaging inflammatory disease. The chlamydial plasmid is a virulence factor, but its role in the pathogenesis of persistent infection capable of driving immunopathology is unknown. Here, we show by using mouse and nonhuman primate infection models that the secreted plasmid gene protein 3 (Pgp3) is essential for establishing persistent infection. Ppg3-dependent persistent genital tract infection resulted in a severe endometritis caused by an intense infiltration of endometrial submucosal macrophages. Pgp3 released from the cytosol of lysed infected oviduct epithelial cells, not organism outer membrane-associated Pgp3, inhibited the chlamydial killing activity of antimicrobial peptides. Genetic Pgp3 rescue experiments in cathelin-related antimicrobial peptide (CRAMP)-deficient mice showed Pgp3-targeted antimicrobial peptides to subvert innate immunity as a pathogenic strategy to establish persistent infection. These findings provide important advances in understanding the role of Pgp3 in the pathogenesis of persistent chlamydial infection and associated immunopathology.IMPORTANCEChlamydia trachomatis can cause persistent infection that drives damaging inflammatory responses resulting in infertility and blindness. Little is known about chlamydial genes that cause persistence or factors that drive damaging pathology. In this work, we show that the C. trachomatis plasmid protein gene 3 (Pgp3) is the essential virulence factor for establishing persistent female genital tract infection and provide supportive evidence that Pgp3 functions similarly in a nonhuman primate trachoma model. We further show that persistent Ppg3-dependent infection drives damaging immunopathology. These results are important advances in understanding the pathophysiology of chlamydial persistence.

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

Chlamydia 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.IMPORTANCE The Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

Infection of Hysterectomized Mice with Chlamydia muridarum and Chlamydia trachomatis.

We studied infection and immunity of hysterectomized mice infected with Chlamydia muridarum and Chlamydia trachomatis to determine if there were differences between these species in their ability to infect vaginal squamous epithelial cells in vivo independently of proximal upper genital tract tissues. We found that C. muridarum readily colonized and infected vaginal squamous epithelial cells, whereas C. trachomatis did not. Primary infection of the vaginal epithelium with C. muridarum produced infections of a duration longer than that reported for normal mice. Infection resulted in an inflammatory response in the vagina characterized by neutrophils and infiltrating submucosal plasma cells consisting primarily of T cells. Despite the delayed clearance, rechallenged C. muridarum-infected mice were highly immune. Mice vaginally infected with C. muridarum produced serum and vaginal wash antibodies and an antigen-specific gamma interferon-dominated Th1-biased T cell response. By comparison, mice vaginally infected with C. trachomatis exhibited transient low-burden infections, produced no detectable tissue inflammatory response, and failed to seroconvert. We discuss how these marked differences in the biology of vaginal infection between these otherwise genetically similar species are possibly linked to pathogen-specific virulence genes and how they may influence pathology and immunity in the upper genital tract.

Chlamydia trachomatis ChxR is a transcriptional regulator of virulence factors that function in in vivo host-pathogen interactions.

Chlamydia trachomatis is an obligate intracellular pathogen characterized by a unique biphasic developmental cycle that alternates between infectious and non-infectious organisms. Chlamydial ChxR is a transcriptional activator that has been implicated in the regulation of the development cycle. We used a reverse genetics approach to generate three chxR null mutants. All three mutants grew normally in cultured mammalian cells. Whole genome sequencing identified SNPs in other genes; however, none of the mutated genes were common to all three ChxR null mutants arguing against a genetic compensatory mechanism that would explain the non-essential in vitro growth phenotype. Comparative proteomics identified five proteins, CT005, CT214, CT565, CT694 and CT695, that were significantly downregulated in all ChxR null mutants. This group includes established inclusion membrane and type III secreted proteins. ChxR transcriptional regulation of these genes was confirmed by qRT-PCR. Importantly, while ChxR null mutants exhibited no growth deficiencies in in vitro, they did show significant differences in in vivo growth using a mouse genital tract model. Collectively, our findings demonstrated that ChxR is a transcriptional activator that regulates the expression of virulence genes whose functions are restricted to in vivo infection.

Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated.

UNLABELLED: Chlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene. IMPORTANCE: Chlamydia's obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

Transcriptional profiling of human epithelial cells infected with plasmid-bearing and plasmid-deficient Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular epitheliotropic bacterial pathogen of humans. Infection of the eye can result in trachoma, the leading cause of preventable blindness in the world. The pathophysiology of blinding trachoma is driven by multiple episodes of reinfection of conjunctival epithelial cells, producing an intense chronic inflammatory response resulting in submucosal tissue remodeling and scarring. Recent reports have shown that infection with trachoma organisms lacking the cryptic chlamydial plasmid is highly attenuated in macaque eyes, a relevant experimental model of human trachoma infection. To better understand the molecular basis of plasmid-mediated infection attenuation and the potential modulation of host immunity, we conducted transcriptional profiling of human epithelial cells infected with C. trachomatis plasmid-bearing (A2497) and plasmid-deficient (A2497P(-)) organisms. Infection of human epithelial cells with either strain increased the expression of host genes coding for proinflammatory (granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage colony-stimulating factor [MCSF], interleukin-6 [IL-6], IL-8, IL-1α, CXCL1, CXCL2, CXCL3, intercellular adhesion molecule 1 [ICAM1]), chemoattraction (CCL20, CCL5, CXCL10), immune suppression (PD-L1, NFKB1B, TNFAIP3, CGB), apoptosis (CASP9, FAS, IL-24), and cell growth and fibrosis (EGR1 and IL-20) proteins. Statistically significant increases in the levels of expression of many of these genes were found in A2497-infected cells compared to the levels of expression in A2497P(-)-infected cells. Our findings suggest that the chlamydial plasmid plays a focal role in the host cell inflammatory response to infection and immune avoidance. These results provide new insights into the role of the chlamydial plasmid as a chlamydial virulence factor and its contributions to trachoma pathogenesis.

CD8+ T cells define an unexpected role in live-attenuated vaccine protective immunity against Chlamydia trachomatis infection in macaques.

Trachoma, caused by the obligate intracellular organism Chlamydia trachomatis, is the world's leading cause of preventable blindness for which a vaccine is needed. We have previously shown that a plasmid-deficient live-attenuated trachoma vaccine delivered ocularly to macaques elicited either solid or partial protective immunity against a virulent ocular challenge. Solidly protected macaques shared the same MHC class II alleles implicating CD4(+) T cells in superior protective immunity. Understandably, we sought to define T cell immune correlates in these animals to potentially improve vaccine efficacy. In this study, following a 2-y resting period, these macaques were boosted i.m. with the live-attenuated trachoma vaccine and their peripheral T cell anamnestic responses studied. Both solidly and partially protected macaques exhibited a CD4(+) and CD8(+) T cell anamnestic response following booster immunization. CD8(+) but not CD4(+) T cells from solidly protected macaques proliferated against soluble chlamydial Ag. We observed a more rapid T cell inflammatory cytokine response in tears of solidly protected animals following ocular rechallenge. Most notably, depletion of CD8(+) T cells in solidly protected macaques completely abrogated protective immunity. Collectively, our findings support the conclusion that CD8(+) T cells play an important but unexpected role in live-attenuated trachoma vaccine-mediated protective immunity.

Chlamydia trachomatis polymorphic membrane protein D is a virulence factor involved in early host-cell interactions.

Chlamydia trachomatis is an obligate intracellular mucosotropic pathogen of significant medical importance. It is the etiological agent of blinding trachoma and bacterial sexually transmitted diseases, infections that afflict hundreds of millions of people globally. The C. trachomatis polymorphic membrane protein D (PmpD) is a highly conserved autotransporter and the target of broadly cross-reactive neutralizing antibodies; however, its role in host-pathogen interactions is unknown. Here we employed a targeted reverse genetics approach to generate a pmpD null mutant that was used to define the role of PmpD in the pathogenesis of chlamydial infection. We show that pmpD is not an essential chlamydial gene and the pmpD null mutant has no detectable deficiency in cultured murine cells or in a murine mucosal infection model. Notably, however, the pmpD null mutant was significantly attenuated for macaque eyes and cultured human cells. A reduction in pmpD null infection of human endocervical cells was associated with a deficiency in chlamydial attachment to cells. Collectively, our results show that PmpD is a chlamydial virulence factor that functions in early host-cell interactions. This study is the first of its kind using reverse genetics to evaluate the contribution of a C. trachomatis gene to disease pathogenesis.

Plasmid-mediated transformation tropism of chlamydial biovars.

Chlamydia trachomatis and C. muridarum are human and mouse pathogens, respectively, which show high conservation of gene order and content. Both species contain a common 7.5-kb plasmid that is an important virulence factor. Recently described transformation systems have been used to characterize C. trachomatis L2 plasmid gene functions; however, similar studies have not been reported for C. trachomatis ocular tropic serovar A or the mouse strain, C. muridarum. Here, we have conducted genetic experiments with C. trachomatis serovar A and C. muridarum and report the following: (1) successful transformation of C. muridarum and C. trachomatis serovar A is restricted to a shuttle vector with a C. muridarum or C. trachomatis serovar A plasmid backbone, respectively; (2) transformation of plasmid-deficient C. muridarum with the C. muridarum-based shuttle vector complement glycogen accumulation and inclusion morphology; and (3) C. muridarum plasmid-encoded Pgp4 is a regulator of chromosomal (glgA) and plasmid (pgp3) virulence genes. In summary, our findings show a previously unrecognized and unexpected role for the chlamydial plasmid in its transformation tropism and confirm the plasmids regulatory role of virulence genes in C. muridarum.

Infectivity of urogenital Chlamydia trachomatis plasmid-deficient, CT135-null, and double-deficient strains in female mice.

Chlamydia trachomatis is the most common cause of human bacterial sexually transmitted infections and is the world's leading cause of infectious preventable blindness. The chlamydial 7.5-kb plasmid and chromosomal gene CT135 have been shown to be important virulence factors in both nonhuman primate and mouse infection models. Chlamydia trachomatis plasmid-deficient urogenital isolates and a predicted CT135 null mutant have been evaluated independently in the female mouse genital tract model and both have been shown to reduce infectivity and virulence. However, these attenuating phenotypes have not been evaluated collectively in the murine model. Here, we test the infectivity of C. trachomatis serovar D strains in the mouse model that are plasmid-deficient, CT135 disrupted, or possess a combination of these attenuating genotypes. We find that the presence of the plasmid results in infections with higher infectious burdens, whereas CT135 facilitates a more protracted or chronic infection. Not unexpectedly, a combination of these genetic deficiencies resulted in a strain with enhanced infection attenuation characteristics.

Antibody signature of spontaneous clearance of Chlamydia trachomatis ocular infection and partial resistance against re-challenge in a nonhuman primate trachoma model.

BACKGROUND: Chlamydia trachomatis is the etiological agent of trachoma the world's leading cause of infectious blindness. Here, we investigate whether protracted clearance of a primary infection in nonhuman primates is attributable to antigenic variation or related to the maturation of the anti-chlamydial humoral immune response specific to chlamydial antigens. METHODOLOGY/PRINCIPAL FINDINGS: Genomic sequencing of organisms isolated throughout the protracted primary infection revealed that antigenic variation was not related to the inability of monkeys to efficiently resolve their infection. To explore the maturation of the humoral immune response as a possible reason for delayed clearance, sera were analyzed by radioimmunoprecipitation using intrinsically radio-labeled antigens prepared under non-denaturing conditions. Antibody recognition was restricted to the antigenically variable major outer membrane protein (MOMP) and a few antigenically conserved antigens. Recognition of MOMP occurred early post-infection and correlated with reduction in infectious ocular burdens but not with infection eradication. In contrast, antibody recognition of conserved antigens, identified as PmpD, Hsp60, CPAF and Pgp3, appeared late and correlated with infection eradication. Partial immunity to re-challenge was associated with a discernible antibody recall response against all antigens. Antibody recognition of PmpD and CPAF was destroyed by heat treatment while MOMP and Pgp3 were partially affected, indicating that antibody specific to conformational epitopes on these proteins may be important to protective immunity. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that delayed clearance of chlamydial infection in NHP is not the result of antigenic variation but rather a consequence of the gradual maturation of the C. trachomatis antigen-specific humoral immune response. However, we cannot conclude that antibodies specific for these proteins play the primary role in host protective immunity as they could be surrogate markers of T cell immunity. Collectively, our results argue that an efficacious subunit trachoma vaccine might require a combination of these antigens delivered in their native conformation.

Chlamydia trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence-associated genes.

Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.

A live-attenuated chlamydial vaccine protects against trachoma in nonhuman primates.

Blinding trachoma is an ancient neglected tropical disease caused by Chlamydia trachomatis for which a vaccine is needed. We describe a live-attenuated vaccine that is safe and efficacious in preventing trachoma in nonhuman primates, a model with excellent predictive value for humans. Cynomolgus macaques infected ocularly with a trachoma strain deficient for the 7.5-kb conserved plasmid presented with short-lived infections that resolved spontaneously without ocular pathology. Multiple infections with the attenuated plasmid-deficient strain produced no inflammatory ocular pathology but induced an anti-chlamydial immune response. Macaques vaccinated with the attenuated strain were either solidly or partially protected after challenge with virulent plasmid-bearing organisms. Partially protected macaques shed markedly less infectious organisms than controls. Immune correlates of protective immunity were not identified, but we did detect a correlation between MHC class II alleles and solid versus partial protection. Epidemiological models of trachoma control indicate that a vaccine with this degree of efficacy would significantly reduce the prevalence of infection and rates of reinfection, known risk factors which drive blinding disease.

Chlamydia pneumoniae inhibits activated human T lymphocyte proliferation by the induction of apoptotic and pyroptotic pathways.

Chlamydia pneumoniae is an omnipresent obligate intracellular bacterial pathogen that infects numerous host species. C. pneumoniae infections of humans are a common cause of community acquired pneumonia but have also been linked to chronic diseases such as atherosclerosis, Alzheimer's disease, and asthma. Persistent infection and immune avoidance are believed to play important roles in the pathophysiology of C. pneumoniae disease. We found that C. pneumoniae organisms inhibited activated but not nonactivated human T cell proliferation. Inhibition of proliferation was pathogen specific, heat sensitive, and multiplicity of infection dependent and required chlamydial entry but not de novo protein synthesis. Activated CD4(+) and CD8(+) T cells were equally sensitive to C. pneumoniae antiproliferative effectors. The C. pneumoniae antiproliferative effect was linked to T cell death associated with caspase 1, 8, 9, and IL-1ß production, indicating that both apoptotic and pyroptotic cellular death pathways were activated after pathogen-T cell interactions. Collectively, these findings are consistent with the conclusion that C. pneumoniae could induce a local T cell immunosuppression and inflammatory response revealing a possible host-pathogen scenario that would support both persistence and inflammation.

Generation of targeted Chlamydia trachomatis null mutants.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

Murine Chlamydia trachomatis genital infection is unaltered by depletion of CD4+ T cells and diminished adaptive immunity.

Chlamydia muridarum and Chlamydia trachomatis mouse models of genital infection have been used to study chlamydial immunity and vaccine development. To assess the protective role of CD4(+) T cells in resolving C. trachomatis and C. muridarum genital tract infections, we used the female mouse model and evaluated infection in the presence and absence of CD4(+) T cells. In contrast to C. muridarum infection, C. trachomatis infection was unaltered in the absence of CD4(+) T cells. Mice infected with C. trachomatis developed protective immunity to re-challenge, but unlike C. muridarum infection, optimum resistance required multiple infectious challenges, despite the generation of adaptive serum and local chlamydial specific immune responses. Thus, understanding the chlamydial pathogenic and host immunologic factors that result in a diminished protective role for CD4(+) T cells in C. trachomatis murine infection might lead to new insights important to human immunity and vaccine development.

Frameshift mutations in a single novel virulence factor alter the in vivo pathogenicity of Chlamydia trachomatis for the female murine genital tract.

Chlamydia trachomatis is a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model of C. trachomatis infection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivated in vitro prior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiple in vitro passages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrasting in vivo virulence characteristics, the strains exhibited no differences in the results of in vitro infectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.

Biological characterization of Chlamydia trachomatis plasticity zone MACPF domain family protein CT153.

Chlamydia trachomatis strains are obligate intracellular human pathogens that share near genomic synteny but have distinct infection and disease organotropisms. The genetic basis for differences in the pathogen-host relationship among chlamydial strains is linked to a variable region of chlamydial genomes, termed the plasticity zone (PZ). Two groups of PZ-encoded proteins, the membrane attack complex/perforin (MACPF) domain protein (CT153) and members of the phospholipase D-like (PLD) family, are related to proteins that modify membranes and lipids, but the functions of CT153 and the PZ PLDs (pzPLDs) are unknown. Here, we show that full-length CT153 (p91) was present in the elementary bodies (EBs) of 15 C. trachomatis reference strains. CT153 underwent a rapid infection-dependent proteolytic cleavage into polypeptides of 57 and 41 kDa that was independent of de novo chlamydial protein synthesis. Following productive infection, p91 was expressed during the mid-developmental cycle and was similarly processed into p57 and p41 fragments. Infected-cell fractionation studies showed that insoluble fractions contained p91, p57, and p41, whereas only p91 was found in the soluble fraction, indicating that unprocessed CT153 may be secreted. Finally, CT153 localized to a distinct population of reticulate bodies, some of which were in contact with the inclusion membrane.

Chlamydia trachomatis native major outer membrane protein induces partial protection in nonhuman primates: implication for a trachoma transmission-blocking vaccine.

A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immunization with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immunogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a nonhuman primate trachoma model. Cynomolgus monkeys (Macaca fascicularis) were immunized systemically with nMOMP, and monkeys were challenged ocularly. Immunization induced high serum IgG and IgA ELISA Ab titers, with Abs displaying high strain-specific neutralizing activity. The PBMCs of immunized monkeys produced a broadly cross-reactive, Ag-specific IFN-gamma response equivalent to that induced by experimental infection. Immunized monkeys exhibited a significant decrease in infectious burden during the early peak shedding periods (days 3-14). However, at later time points, they exhibited no difference from control animals in either burden or duration of infection. Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is highly immunogenic in nonhuman primates and elicits partially protective immunity against ocular chlamydial challenge. This is the first time a subunit vaccine has shown a significant reduction in ocular shedding in nonhuman primates. A partially protective vaccine, particularly one that reduces infectious burden after primary infection of children, could interrupt the natural trachoma reinfection cycle. This would have a beneficial effect on the transmission between children and sensitized adults which drives blinding inflammatory disease.

Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure.

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.