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Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection.

ABSTRACT: Mycoplasma hominis is the second smallest facultative pathogen of the human urogenital tract. With less than 600 protein-encoding genes, it represents an ideal model organism for the study of host-pathogen interactions. 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 designed to analyze the dynamics of the mycoplasma transcriptome during infection and validated for M. hominis strain FBG. RNA preparation was evaluated and adapted to ensure the 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 differentially regulated key effector molecules such as critical cytoadhesins (4 h post infection (pI)), invasins (48 h pI) and proteins associated with establishing chronic infection of the host (336 h pI). Of the 294 differentially regulated genes (>2-fold) 128 (43.5%) encoded hypothetical proteins, including lipoproteins that seem to play a central role as virulence factors at each stage of infection: P75 as a novel cytoadhesin candidate, which is also differentially upregulated in chronic infection; the MHO_2100 protein, a postulated invasin and the MHO_730-protein, a novel ecto-nuclease and domain of an ABC transporter, the function of which in chronic infection has still to be elucidated. 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.

SUBMITTER: Henrich B

PROVIDER: S-EPMC5533444 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection.

Henrich Birgit B Kretzmer Freya F Deenen René R Köhrer Karl K

PloS one 20170728 7

Mycoplasma hominis is the second smallest facultative pathogen of the human urogenital tract. With less than 600 protein-encoding genes, it represents an ideal model organism for the study of host-pathogen interactions. 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 designed to analyze the dynamics of the mycoplasma transcriptome during infection and validated for M. hominis str ...[more]

PMID: 28753664

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Project description:Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. 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), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. 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:Recent studies have suggested that the vaginal microenvironment plays a role in persistence of high-risk human papillomavirus (hrHPV) infection and thus cervical carcinogenesis. Furthermore, it has been shown that some mycoplasmas are efficient methylators and may facilitate carcinogenesis through methylation of hrHPV and cervical somatic cells. We examined associations between prevalence and persistence of Mycoplasma spp. in the vaginal microbiota, and prevalent as well as persistent hrHPV infections.We examined 194 Nigerian women who were tested for hrHPV infection using SPF25/LiPA10 and we identified Mycoplasma genitalium and Mycoplasma hominis in their vaginal microbiota established by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. We defined the prevalence of M. genitalium, M. hominis, and hrHPV based on positive result of baseline tests, while persistence was defined as positive results from two consecutive tests. We used exact logistic regression models to estimate associations between Mycoplasma spp. and hrHPV infections.The mean (SD) age of the study participants was 38 (8) years, 71% were HIV positive, 30% M. genitalium positive, 45% M. hominis positive, and 40% hrHPV positive at baseline. At follow-up, 16% of the women remained positive for M. genitalium, 30% for M. hominis, and 31% for hrHPV. There was a significant association between persistent M. hominis and persistent hrHPV (OR 8.78, 95% CI 1.49-51.6, p 0.01). Women who were positive for HIV and had persistent M. hominis had threefold increase in the odds of having persistent hrHPV infection (OR 3.28, 95% CI 1.31-8.74, p 0.008), compared to women who were negative for both.We found significant association between persistent M. hominis in the vaginal microbiota and persistent hrHPV in this study, but we could not rule out reverse causation. Our findings need to be replicated in larger, longitudinal studies and if confirmed, could have important diagnostic and therapeutic implications.

Project description:Introduction.Mycoplasma hominis is a bacterium belonging to the class Mollicutes. It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Dataset Information

Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection.

Publications

Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection.

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