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Differential gene expression in C. difficile rough and smooth colony variants is partially determined by CmrRST regulation

ABSTRACT: Clostridioides difficile introduces phenotypic heterogeneity into populations via phase variation. C. difficile modulates phase variation through invertible DNA sequences containing regulatory information, termed switches. Inversion of a switch controls expression of the adjacent genes in an ON/OFF manner, resulting in phenotypic switching. The C. difficile cmr switch (for ‘colony morphology regulator’) coordinates several phenotypes. When cmr is ON, C. difficile displays a rough colony morphology, surface motility, and cell chaining and elongation. When cmr is OFF, colonies are smooth, and high levels of swimming motility and biofilm formation are observed. Prior work has also linked colony morphology and cmr switch orientation to virulence in the hanster model of infection (Garrett et al., PLoS Biology, 2019). The cmr-ON orientation results in the expression of cmrRST, an operon encoding a signal transduction system. CmrS is a predicted histidine kinase, and CmrR and CmrT are response regulators. We have shown that CmrR and CmrT are responsible for the observed cmr-associated phenotypes (Garrett et al., PLoS Biology, 2019). The goal of this study is to identify the genes that are regulated by CmrR and CmrT and determine how these genes contribute to colony morphology and the other cmr-associated phenotypes. To identify genes important for C. difficile colony morphology, we conducted a multi-pronged RNA-Seq study comparing the following transcriptomes: (1) Rough vs. smooth colony variants of wild-type, (2) cmr-ON vs. cmr-OFF phase-locked strains, (3) Rough, smooth, cmr-ON, and cmr-OFF vs. a ΔcmrRΔcmrT mutant, and (4) Wild-type overexpressing either cmrR or cmrT vs. a no-overexpression condition. Transcription analyses revealed that CmrRST accounts for some, but not all, transcriptional differences between rough and smooth colony morphotypes. Fewer than 20 genes were differentially expressed by CmrR and/or CmrT (≥4-fold change, p<0.05). Most of those genes appeared in the rough vs. smooth and cmr-ON vs. -OFF datasets as well, and further phenotypic analyses of CmrRST-regulated genes revealed multiple genes that mediated rough colony development.

ORGANISM(S): Clostridioides difficile

PROVIDER: GSE305463 | GEO | 2025/11/25

REPOSITORIES: GEO

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Project description:Members of the Mycobacterium (M.) abscessus complex (MABC) are rapidly growing mycobacteria showing smooth and/or rough colony morphotype. While not as virulent as M. tuberculosis, they can cause soft tissue infection and fatal pulmonary disease, especially in patients with cystic fibrosis. Diagnosing MABC pulmonary disease is challenging since the isolation of M. abscessus from respiratory samples is in itself not diagnostic and the clinical features are often non-specific. Immunologic assays, which could aid in the understanding and diagnosis of the disease, are not available. In this study eight rough and six smooth colony morphotype isolates were collected from seven clinical MABC strains and the M. abscessus reference strain ATCC19977, as six strains showed both morphotypes simultaneously and two strains only showed a rough morphotype. Clinical isolates were submitted to whole genome sequencing. Quantitative proteomic analysis was performed on bacterial lysates and the culture supernatant of all 14 isolates. Supernatant proteins present in all isolates were compared in a BLAST search against other clinically significant mycobacterial species to determine species-specific proteins of MABC. In silico B- and T-cell epitope prediction was performed for species-specific proteins. All clinical strains were found to be M. abscessus ssp. abscessus. Six of seven rough colony clinical isolates contained genetic changes in the MAB_4099c gene, which is a likely genetic basis for the rough morphotype. Proteomic analysis detected 3 137 different proteins in total of which 79 proteins were found in the culture supernatants of all isolates. BLAST analyses of these 79 proteins identified 12 of those exclusively encoded by all members of MABC plus M. immunogenum. In silico prediction of epitopes predicted B- and T-cell epitopes in all these 12 species-specific proteins, rendering them promising candidates for future studies on immune pathogenesis and immune diagnostic tools for MABC disease.

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Differential gene expression in C. difficile rough and smooth colony variants is partially determined by CmrRST regulation

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