Project description:The aim of this study was to perform a microarray analysis of the response pattern of EEC from both large and small bowel to infection in vitro, using Chlamydia trachomatis infection as a model. Two human EEC lines: LCC-18, derived from a neuroendocrine colonic tumour, and CNDT-2, derived from a small intestinal carcinoid, were infected with C. trachomatis serovar LGV II strain 434 (ATCC VR-902B). Penicillin G was used to induce persistent infection. Gene expression levels in infected and persistently infected EEC cells were investigated by microarray analysis
Project description:Chlamydia trachomatis is a prevalent bacterial cause of urogenital and ocular infections. The pathogen uses the effector CpoS to suppress a host defense response that aborts intracellular bacterial growth by inducing host cell death. We conducted a CRISPR knockout screen to identify host genes contributing to this response, thereby revealing modulators of C. trachomatis parasitophorous vacuole stability. In brief, we transduced HeLa cells, a human cervical epithelial cell line, with a genome-wide knockout library. More specifically, we used the Brunello sgRNA library (which targets 19,114 genes and comprises a total of 77,441 sgRNAs, including about four sgRNAs per gene and 1000 non-targeting control sgRNAs). An aliquot of the transduced cells was collected to determine the composition of the pre-selection cell population (= sample “Pre”). In the selection procedure, we infected transduced cells with C. trachomatis L2/434/Bu, either wildtype (CTL2) or a strain carrying an insertional disruption of cpoS (CTL2-cpoS::cat). Later, we collected cells resistant to infection-mediated killing, that is, cells resistant to late-stage lytic death in the case of CTL2 or premature death in the case of CTL2-cpoS::cat. Hence, we included four distinct conditions: uninfected cells and cells infected with CTL2-cpoS::cat collected at 30 hours post infection (samples “UI30h” and “KO30h”), and uninfected cells and cells infected with CTL2 collected at 60 hours post infection (samples “UI60h” and “WT60h”). The screen was performed in two independent replicates (R1+R2).
Project description:Trachoma, a preventable blinding eye disease, is initiated by ocular infection with Chlamydia trachomatis (Ct). MicroRNA (miR) are post-transcriptional regulators of gene expression and play a major role in health and disease. We have investigated the miR profile during C. trachomatis infection of epithelial cells in vitro and in vivo during follicular trachoma with current C. trachomatis infection. Small RNA sequencing was carried out on human epithelial cells infected in vitro and on samples from five children with follicular trachoma with current Ct infection and five children with no evidence of clinical trachoma or infection. In vitro two strains of ocular Ct that differ in virulence, A2497 and isogenic plasmid-free A2497 were used to infect epithelial cell lines. RNAseq results were confirmed by qPCR in six in vitro biological replicates and in 163 clinical samples. Differential miR expression was not detected in isolated epithelial cells infected in vitro at 48 hours post infection. HCjE cells, a conjunctival epithelial cell line, have markedly different miR background expression compared to Hep2 cells. The differing miR profiles of Hep2c and HCjE suggest caution should be used when extrapolating data from Hep2 cells to a tissue-specific clinical scenario. In follicular trachoma, miR-155, miR-150, miR-142, miR-181b, miR-181a, miR-342 and miR-132 were up-regulated during current Ct infection. MiR-4728 and miR-184 were down-regulated in follicular trachoma independent of Ct infection. In follicular trachoma, miR expression reflects development and regulation of the immune response during current Ct infection and a prolonged period of wound healing following Ct clearance.