Project description:M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using microarray-based analysis of the HeLa cell transcriptome: 0 h to monitor the baseline of transcription, 4 h to examine host reactions to mycoplasma attachment, 48 h to capture in addition the initiation of invasion, and 2 weeks post infection to examine a chronically infected host cell. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen. To elucidate the host-reactions at the different stages of M. hominis infection, four time points post infection (0h, 4h, 48h, 2 weeks) were chosen for microarray gene expression analyses. Total RNA was prepared from infection assays (MOI 100) in parallel with uninfected HeLa cells and the host cell transcriptomes were determined by comparative microarray analyses.
Project description:For a comprehensive characterisation of the M. hominis-action in infection a customized Mho microarray, which was based on two genome sequences (PG21 and LBD-4), was newly designed and validated for M. hominis strain FBG, to analyze the dynamics of the mycoplasma transcriptome during infection. RNA-preparation was evaluated and adopted for highest recovery of mycoplasmal mRNAs from in vitro HeLa cell infection assays. Following cRNA hybridization, the read out strategy of the hybridization results was optimized and confirmed by RT-PCR. A statistically robust infection assay with M. hominis strain FBG enabled the identification of regulated key effector molecules such as critical cytoadhesins (4 h post infection (pI)), invasins (48h pI) and proteins associated with establishing chronic infection of the host (336h pI). Of the 304 differentially regulated genes 130 (43%) encoded hypothetical proteins, including lipoproteins that seem to play a central role as virulence factors at each stage of infection: P75 as novel cytoadhesin candidate, which is also upregulated in the chronic infection, the MHO_2100-protein as postulated invasin and the MHO_730-protein as novel ecto-nuclease and domain of a novel ABC transporter, whose function in chronic infection has to be elucidated in the future. Implementation of the M. hominis microarray strategy led to a comprehensive identification of to date unknown candidates for virulence-factors at relevant stages of host cell infection.
Project description:M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using microarray-based analysis of the HeLa cell transcriptome: 0 h to monitor the baseline of transcription, 4 h to examine host reactions to mycoplasma attachment, 48 h to capture in addition the initiation of invasion, and 2 weeks post infection to examine a chronically infected host cell. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen.
Project description:The sexually transmitted parasite Trichomonas vaginalis is often found in symbiosis with the obligate intracellular pathogen Mycoplasma hominis. M. hominis is itself an opportunistic pathogen of the female reproductive tract associated with bacterial vaginosis. The goal of this experiment was to identify the effects of each pathogen individually and in symbiosis on host cell gene expression.