Spo0A suppressessinlocus expression inClostridioides difficile

Abstract

24Clostridioides difficile is the leading cause of nosocomial infection and is the causative 25 agent of antibiotic-associated diarrhea. The severity of the disease is directly associated 26 with the production of toxins, and spores are responsible for the transmission and 27 persistence of the organism. Previously we characterized sin locus regulators SinR and 28SinR', where SinR is the regulator of toxin production and sporulation, while the SinR' 29 acting as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, 30 regulates SinR, by regulating the expression of its antagonist sinI. However, the role of 31 Spo0A in the expression of sinR and sinR' in C. difficile is not yet reported. In this study, 32we tested spo0A mutants in three different C. difficile strains R20291, UK1, and JIR8094, 33to understand the role of Spo0A in sin locus expression. Western blot analysis revealed 34 that spo0A mutants had increased SinR levels. The qRT-PCR analysis for its expression 35 further supported this data. By carrying out genetic and biochemical assays, we have 36shown that Spo0A can bind to the upstream region of this locus to regulates its 37 expression. This study provides vital information that Spo0A regulates sin locus, which 38 controls critical pathogenic traits such as sporulation, toxin production, and motility in C. 39 difficile. 40 41 42 43 44 45 46 IMPORTANCE 47 Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the 48 United States. During infection, C. difficile spores germinate, and the vegetative bacterial 49cells produce toxins that damage host tissue. In C. difficile, sin locus is known to regulate 50 both sporulation and toxin production. In this study, we have shown that Spo0A, the 51 master regulator of sporulation to control the sin locus expression. We performed various 52 genetic and biochemical experiments to show that Spo0A directly regulates the 53 expression of this locus by binding to its upstream DNA region. This observation adds 54 new detail to the gene regulatory network that connects sporulation and toxin production 55 in this pathogen. 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70Clostridioides difficile is a Gram-positive, anaerobic bacillus and is the principal causative 71 agent of antibiotic-associated diarrhea and pseudomembranous colitis (1-3). Antibiotic 72 use is the primary risk factor for the development of C. difficile associated disease 73 because it disrupts normal protective gut flora and provides a favorable environment for 74 C. difficile to colonize the colon. Two major pathogenic traits of C. difficile are toxin (toxin 75 A and B) and spores (3-5). Deaths related to C. difficile increased by 400% between 2000 76 and 2007, in part because of the emergence of more aggressive C. difficile strains (6, 7). 77Robust sporulation and toxin production were suspected of contributing to the widespread 78 of the C. difficile infections associated with these highly virulent strains (8)(9)(10)(11)(12)(13)(14). How C...

The regulatory role of anti-sigma factor, RsbW, in Clostridioides difficile stress response, persistence and infection

“…SinR and SinR' are transcribed as a single transcript and has not been associated with the control s B expression, nor has a s B promoter been identified upstream of the locus. The sinRR' expression can be supressed by Spo0A, as a phosphorylated Spo0A (Spo0A~P) can bind to the only promoter sequence upstream of this locus 53 . While this locus and s B may contribute to DrsbW phenotypic responses, SinR regulates the transcription of other pleiotropic regulators such as s D , Spo0A and CodY, which can regulate many other genes in C. difficile 39,[54][55][56] .…”

“…In B. subtilis , Spo0A is negatively controlled by Spo0E through dephosphorylation, furthermore Spo0E is positively controlled by a σ B promoter 49 . Additionally, it has been suggested that SinR is able to regulate spo0A expression through an unknown mechanism 53 . The SinR homolog in B. subtilis functions as a repressor for the spo0A promoter 57 .…”

Clostridioides difficile infection: traversing host–pathogen interactions in the gut

“…While Spo0A also regulates biofilm formation, flagella production, expression of cell-wall proteins [178] and indirect regulation with the sin locus [176,181,182], modulators like RstA control sporulation and toxin production through the phosphorylation of Spo0A [183]. Three of five orphan kinases (PtpA, PtpB and PtpC) have been reported to negatively regulate sporulation [184,185].…”