Pleiotropic roles of Clostridium difficile sin locus

Brintha Parasumanna Girinathan,Bruno Dupuy,Junjun Ou,Revathi Govind,Theresa M Koehler

doi:10.1371/journal.ppat.1006940

Brintha Parasumanna Girinathan, Bruno Dupuy + Show 3 more

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https://doi.org/10.1371/journal.ppat.1006940

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Abstract

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.

Highlights

Clostridium difficile, a major nosocomial pathogen, is the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis [1, 2]
This study identified that mutation of the sin locus in C. difficile could affect toxin production and sporulation along with motility and reports a new regulatory element of this network
B. subtilis SinR (BsSinR) is a DNA-binding protein that binds to a conserved DNA sequence upstream of the translational start site of target genes to negatively control their transcription

Summary

Introduction

Clostridium difficile, a major nosocomial pathogen, is the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis [1, 2]. Nearly half a million cases of C. difficile infections (CDI) occur in the United States and result in approximately 14,000 deaths [3]. C. difficile toxins damage the colonic epithelium, which results in moderate to severe diarrhea [4]. Recent studies have shown that these toxins are essential for C. difficile pathogenesis [4,5,6,7]. Due to the strictly anaerobic nature of the vegetative cell, C. difficile survives outside the host in the form of dormant spores, which are highly resilient and resistant to most disinfectants. C. difficile spores are critical for its host to host transmission and persistence in the hospital environment [8]

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