Project description:Chlamydia trachomatis serovariants are responsible for either Trachoma, the leading cause of infectious blindness or sexually transmitted disease, wherein the endocervix is the most frequently infected site in women. Disease caused by Chlamydia typically involves chronic inflammation and scarring. Recent work with a live-attenuated A2497 plasmid deficient vaccine strain (A2497-) demonstrated protection in nonhuman primates against trachoma and a lack of measurable ocular pathology in A2497- infected monkeys. We therefore performed host cell transcriptome analysis of Hela cells infected with A2497 plasmid-containing (A2497) and A2497- Chlamydia over time. Our results indicate that relative to wild type A2497, the A2497- variant illicits a transcriptome response indicative of lowered inflammation response a delayed apoptosis response, a reduction in immune cell recruitement cytokine expression and a reduction in genes involved in cell proliferation and or fibrosis-like activities. The data provided here suggests a model that may explain how plasmid deficient chlamydia may provide an immuno-protective response without the pathology normally seen with plasmid-containing bacteria.
Project description:Chlamydia trachomatis serovariants are responsible for either Trachoma, the leading cause of infectious blindness or sexually transmitted disease, wherein the endocervix is the most frequently infected site in women. Disease caused by Chlamydia typically involves chronic inflammation and scarring. Recent work with a live-attenuated A2497 plasmid deficient vaccine strain (A2497-) demonstrated protection in nonhuman primates against trachoma and a lack of measurable ocular pathology in A2497- infected monkeys. We therefore performed host cell transcriptome analysis of Hela cells infected with A2497 plasmid-containing (A2497) and A2497- Chlamydia over time. Our results indicate that relative to wild type A2497, the A2497- variant illicits a transcriptome response indicative of lowered inflammation response a delayed apoptosis response, a reduction in immune cell recruitement cytokine expression and a reduction in genes involved in cell proliferation and or fibrosis-like activities. The data provided here suggests a model that may explain how plasmid deficient chlamydia may provide an immuno-protective response without the pathology normally seen with plasmid-containing bacteria. Ct infection with and without plasmid time series
Project description:Experimental screening of a compound library identified a molecule that potently inhibits the growth of the obligate intracellular bacterial pathogen Chlamydia trachomatis in human cells. To identify the molecular target of the compound, three mutant bacterial strains resistant to its inhibitory action were generated by long-term passage in the presence of initially low but increasing concentrations of the molecule. Subsequently, genomic DNA of the three mutant and the wildtype bacteria was isolated and subjected to whole genome sequencing to identify resistance-promoting mutations.
Project description:To determine the role that GrgA plays in chlamydial physiology, we constructed a Chlamydia trachomatis mutant that we term L/cgad-peig, in which the chromosomal grgA (ctl0766 or ct504) has been disrupted by Targetron mutagenesis, and the plasmid carries an inducible grgA under the control of anhydrotetracycline (ATC). RNA-Seq analysis was performed for L2/cgad-peig grown with and without ATC.
Project description:Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract of global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases as plasmid-deficient organisms are highly attenuated. The plasmid encodes both noncoding RNAs and eight conserved ORFs of undefined function. 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 in terms of plasmid biology and transcriptional profiling. 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 plasmid-less strain in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that pgp4 is involved in transcriptional regulation of multiple chromosomal genes; including the glycogen synthase gene glgA. Based on our results, we propose that Pgp1 is a plasmid replicative helicase, Pgp2 is a plasmid replication protein, Pgp4 is a transcriptional regulator of virulence associated chromosomal genes, and Pgp6-8 are plasmid partitioning proteins. These findings have important implications for understanding the plasmidM-bM-^@M-^Ys role in chlamydial pathogenesis and the development of novel antigenically multivalent live-attenuated chlamydial vaccines. Chlamydia trachomatis wild type vs. two deletion mutants, and mock
Project description:Chlamydia trachomatis, an intracellular pathogen, stands as the most prevalent sexually transmitted bacterial infection among women globally. Traditionally recognized as a genital pathogen, recent research indicates that the gastrointestinal tract may also act as a reservoir for its long-term colonization. However, the mechanisms underlying Chlamydia's ability to persist in the gut remain poorly understood. This gap in knowledge limits our ability to develop effective treatments for persistent Chlamydia infections. In this study we utilized single-cell RNA sequencing to analyze the gene expression profiles and cellular heterogeneity of mouse colonic tissues during Chlamydia long- term infection. This approach provided detailed insights into the transcriptional changes and cellular interactions involved in the persistence of Chlamydia in the gut. Our results revealed significant alterations in the gene expression profiles of various intestinal cell populations, with distinct molecular pathways contributing to Chlamydia persistence. Notably, we observed a reduction in the expression of markers associated with epithelial tight junctions, indicating a potential breakdown of the intestinal epithelial barrier. This impairment may facilitate the penetration of Chlamydia into deeper tissues and contribute to the initiation of infection. We also found dysregulation of the transcriptional networks in goblet cells and an imbalance in communication between immune and epithelial cells. These disruptions were linked to the pathogen's ability to establish persistent colonization and infection.
Project description:Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract of global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases as plasmid-deficient organisms are highly attenuated. The plasmid encodes both noncoding RNAs and eight conserved ORFs of undefined function. 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 in terms of plasmid biology and transcriptional profiling. 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 plasmid-less strain in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that pgp4 is involved in transcriptional regulation of multiple chromosomal genes; including the glycogen synthase gene glgA. Based on our results, we propose that Pgp1 is a plasmid replicative helicase, Pgp2 is a plasmid replication protein, Pgp4 is a transcriptional regulator of virulence associated chromosomal genes, and Pgp6-8 are plasmid partitioning proteins. These findings have important implications for understanding the plasmid’s role in chlamydial pathogenesis and the development of novel antigenically multivalent live-attenuated chlamydial vaccines.
Project description:The T cell response to Chlamydia genital tract infections in humans and mice is unusual in that the majority of antigen-specific CD8 T cells are not restricted by HLA/MHC class I and therefore have been referred to as “unrestricted” or “atypical”. We previously reported that a subset of unrestricted murine Chlamydia-specific CD8 T cells had an unusual cytokine polarization pattern that included IFN-ɣ and IL-13. For this report, we investigated the transcriptome of Chlamydia-specific CD8ɣ13 T cells, comparing them to Chlamydia-specific multifunctional Tc1 clones using gene expression micro array analysis. The molecular study revealed that CD8ɣ13 polarization included IL-5 in addition to IFN-γ and IL-13. Adoptive transfer studies were performed with Tc1 clone and CD8ɣ13 T cell clones to determine whether either influenced bacterial clearance or immunopathology during Chlamydia muridarum (Cm) genital tract infections. To our surprise, an adoptively transferred CD8ɣ13 T cell clone was remarkably proficient at preventing chlamydia immunopathology while the multifunctional Tc1 clone did not enhance clearance or significantly protect from immunopathology. Mapping studies with MHC class I- and class II-deficient splenocytes showed our previously published Chlamydia-specific CD8 T cell clones are MHC class II-restricted. MHC class II-restricted CD8 T cells may play important roles in protection from intracellular pathogens that limit class I antigen presentation or deplete the CD4 T cell compartment.
Project description:We applied Formaldehyde-Assisted Isolation of Regulatory Elements enrichment followed by sequencing (FAIRE-Seq) to generate genome-wide temporal chromatin maps of Chlamydia trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal regions of chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions, including several Clusters of Open Regulatory Elements (COREs) and temporally-enriched sets of transcription factors, may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signaling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work will serve as a basis for future functional studies of transcriptional regulation and epigenomic regulatory elements in Chlamydia-infected human cells.
Project description:Transcriptional profiling of human epithelial cell line (HL) infected with Chlamydia penumoniae compared to control cells at time points 12h, 24h, 48h, 72h after the infection. Keywords: Chlamydia peneumoniae infection