Biology subjects

Sekulovic, O.

Publications and source records attributed to Sekulovic, O..

3 recordsLinked to original sources

bioRxiv2021

Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the cmr switch, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCEClostridioides difficile is a leading cause of hospital-acquired intestinal infections in the U.S. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here we define the complex, multi-level regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.

microbiology↗

bioRxiv2019

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies, and that bacteria derived from the isolates display opposing surface and swimming motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein CmrRST, which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of Type IV pili, suggesting a novel mechanism of surface expansion in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration. In a hamster model of acute C. difficile disease, colony morphology correlates with virulence, and the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.\n\nSignificance StatementPhenotypic heterogeneity within a genetically clonal population allows many mucosal pathogens to survive within their hosts, balancing the need to produce factors that promote colonization and persistence with the need to avoid the recognition of those factors by the host immune system. Recent work suggests that the human intestinal pathogen Clostridium difficile employs phase variation during infection to generate a heterogeneous population differing in swimming motility, toxin production, and more. This study identifies a signal transduction system that broadly impacts C. difficile physiology and behavior in vitro and in an animal model. Phase variation of this system is therefore poised to modulate the coordinated expression of multiple mechanisms influencing C. difficile disease development.

microbiology↗

bioRxiv2018

Epigenomic landscape of the human pathogen Clostridium difficile

Clostridioides difficile is a leading cause of health care-associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we performed the first comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity whose corresponding gene is highly conserved across our dataset and in all ~300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacted sporulation, a key step in C. difficile disease transmission, consistently supported by multi-omics data, genetic experiments, and a mouse colonization model. Further experimental and transcriptomic analysis also suggested that epigenetic regulation is associated with cell length, biofilm formation, and host colonization. These findings open up a new epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies.

genomics↗