Harlan D. Caldwell

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.

Immunity to Murine Chlamydial Genital Infection

Chlamydia trachomatis sexually transmitted infections cause considerable morbidity and socioeconomic burden worldwide, despite significant advances in our understanding of the biology (29, 31, 57), pathogenesis (11, 83, 117), genomics (94), and epidemiology (91) of this parasite. Chlamydial urogenital tract infections are readily cured with antibiotics, but control measures based on antimicrobial chemotherapy alone are hampered by the frequency of asymptomatic infections and delayed diagnosis (9). Definitive control of C. trachomatis sexually transmitted diseases (STDs) is possible through the development of a safe and efficacious vaccine (24). Progress toward the development of an effective vaccine has been disappointingly modest, as it has been for vaccines to other sexually transmitted pathogens that infect the genital tract mucosae. The strict tropism for mucosal epithelial cells, the complex biology and antigenic structure, and the predilection to cause persistent infection have presented formidable challenges to chlamydial vaccine development. A heightened understanding of protective immunity to C. trachomatis urogenital infection has emerged in the past decade from studies using a mouse model of chlamydial genital tract infection. The insights are of considerable interest because they offer promise for the development of an efficacious chlamydial vaccine. This review focuses on that progress and summarizes the current understanding of protective immune mechanisms that function against murine chlamydial urogenital infection. We also discuss specific requirements for a vaccine to protect against chlamydial STDs and the challenges presently confronting us in achieving that goal.

Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogens tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates - specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma-mediated eradication and thus establish persistent infection.

Immunity to Murine Chlamydia trachomatis Genital Tract Reinfection Involves B Cells and CD4+ T Cells but Not CD8+ T Cells

ABSTRACT CD4+ T-helper type 1 (Th1) responses are essential for the resolution of a primary Chlamydia trachomatis genital tract infection; however, elements of the immune response that function in resistance to reinfection are poorly understood. Defining the mechanisms of immune resistance to reinfection is important because the elements of protective adaptive immunity are distinguished by immunological memory and high-affinity antigen recognition, both of which are crucial to the development of efficacious vaccines. Using in vivo antibody depletion of CD4+ and CD8+ T cells prior to secondary intravaginal challenge, we identified lymphocyte populations that functioned in resistance to secondary chlamydial infection of the genital tract. Depletion of either CD4+ or CD8+ T cells in immune wild-type C57BL/6 mice had a limited effect on resistance to reinfection. However, depletion of CD4+ T cells, but not CD8+ T cells, in immune B-cell-deficient mice profoundly altered the course of secondary infection. CD4-depleted B-cell-deficient mice were unable to resolve a secondary infection, shed high levels of infectious chlamydiae, and did not resolve the infection until 3 to 4 weeks following the discontinuation of anti-CD4 treatment. These findings substantiated a predominant role for CD4+ T cells in host resistance to chlamydial reinfection of the female genital tract and demonstrated that CD8+ T cells are unnecessary for adaptive immune resistance. More importantly, however, this study establishes a previously unrecognized but very significant role for B cells in resistance to chlamydial reinfection and suggests that B cells and CD4+ T cells may function synergistically in providing immunity in this model of chlamydial infection. Whether CD4+ T cells and B cells function independently or dependently is unknown, but definition of those mechanisms is fundamental to understanding optimum protective immunity and to the development of highly efficacious immunotherapies against chlamydial urogenital infections.

Comparative Genomic Analysis of Chlamydia trachomatis Oculotropic and Genitotropic Strains

ABSTRACT Chlamydia trachomatis infection is an important cause of preventable blindness and sexually transmitted disease (STD) in humans. C. trachomatis exists as multiple serovariants that exhibit distinct organotropism for the eye or urogenital tract. We previously reported tissue-tropic correlations with the presence or absence of a functional tryptophan synthase and a putative GTPase-inactivating domain of the chlamydial toxin gene. This suggested that these genes may be the primary factors responsible for chlamydial disease organotropism. To test this hypothesis, the genome of an oculotropic trachoma isolate (A/HAR-13) was sequenced and compared to the genome of a genitotropic (D/UW-3) isolate. Remarkably, the genomes share 99.6% identity, supporting the conclusion that a functional tryptophan synthase enzyme and toxin might be the principal virulence factors underlying disease organotropism. Tarp (translocated actin-recruiting phosphoprotein) was identified to have variable numbers of repeat units within the N and C portions of the protein. A correlation exists between lymphogranuloma venereum serovars and the number of N-terminal repeats. Single-nucleotide polymorphism (SNP) analysis between the two genomes highlighted the minimal genetic variation. A disproportionate number of SNPs were observed within some members of the polymorphic membrane protein (pmp) autotransporter gene family that corresponded to predicted T-cell epitopes that bind HLA class I and II alleles. These results implicate Pmps as novel immune targets, which could advance future chlamydial vaccine strategies. Lastly, a novel target for PCR diagnostics was discovered that can discriminate between ocular and genital strains. This discovery will enhance epidemiological investigations in nations where both trachoma and chlamydial STD are endemic.

Transcriptome analysis of chlamydial growth during IFN-γ-mediated persistence and reactivation

Chlamydia trachomatis is an obligatory intracellular prokaryotic parasite that causes a spectrum of clinically important chronic inflammatory diseases of humans. Persistent infection may play a role in the pathophysiology of chlamydial disease. Here we describe the chlamydial transcriptome in an in vitro model of IFN-γ-mediated persistence and reactivation from persistence. Tryptophan utilization, DNA repair and recombination, phospholipid utilization, protein translation, and general stress genes were up-regulated during persistence. Down-regulated genes included chlamydial late genes and genes involved in proteolysis, peptide transport, and cell division. Persistence was characterized by altered but active biosynthetic processes and continued replication of the chromosome. On removal of IFN-γ, chlamydiae rapidly reentered the normal developmental cycle and reversed transcriptional changes associated with cytokine treatment. The coordinated transcriptional response to IFN-γ implies that a chlamydial response stimulon has evolved to control the transition between acute and persistent growth of the pathogen. In contrast to the paradigm of persistence as a general stress response, our findings suggest that persistence is an alternative life cycle used by chlamydiae to avoid the host immune response.

Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes

Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified. The C. trachomatis genome contains genes that encode proteins with significant homology to large clostridial cytotoxins. Here we show that C. trachomatis makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells. The protein was present transiently in infected cells during the period of cytotoxicity. Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis diseases.

The Chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor.

ABSTRACT Chlamydia trachomatis possesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless human C. trachomatis strain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic of C. trachomatis strains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 × 106 inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 × 104 IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes was glgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmids ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.

In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses.

We report a strategy for high through‐put sequence analyses of large MHC class II‐bound peptide repertoires which combines automated electrospray ionization tandem mass‐spectrometry with computer‐assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I‐Abin activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H‐2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen‐presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II‐bound self peptidesderived from cytosolic proteins exists in vivo.

Chlamydia trachomatis Plasmid-Encoded Pgp4 Is a Transcriptional Regulator of Virulence-Associated Genes

ABSTRACT 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 plasmids role in chlamydial pathogenesis at the molecular level.