Pleiotropic roles of Clostridium difficile sin locus

Girinathan, Brintha P.; Ou, Jiangtao; Dupuy, Bruno; Govind, Revathi

doi:10.1371/journal.ppat.1006940

Cited by 51 publications

(89 citation statements)

References 73 publications

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“…Cecal supernatants from mice at 20h of infection extracted and subjected to SDS-PAGE and transfer to PVDF membrane (PerkinElmer, Waltham, MA) as described (59)…”

Section: Western Blot For Toxin B Integritymentioning

confidence: 99%

In vivocommensal control ofClostridioides difficilevirulence

Girinathan

DiBenedetto

Worley

et al. 2020

Preprint

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We define multiple mechanisms by which commensals protect against or worsen Clostridioides difficile infection.Using a systems-level approach we show how two species of Clostridia with distinct metabolic capabilities modulate the pathogen's virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Clostridium bifermentans survived infection, while colonization with the butyrate-producer, Clostridium sardiniense, more rapidly succumbed. Systematic in vivo analyses revealed how each commensal altered the pathogen's carbon source metabolism, cellular machinery, stress responses, and toxin production. Protective effects were replicated in infected conventional mice receiving C. bifermentans as an oral bacteriotherapeutic that prevented lethal infection. Leveraging a systematic and organism-level approach to host-commensalpathogen interactions in vivo, we lay the groundwork for mechanistically-informed therapies to treat and prevent this disease.Clostridioides difficile, the etiology of pseudomembranous colitis, causes substantantial morbidity, mortality and >$5 billion/year in US healthcare costs. Infections commonly arise after antibiotic disruption of the microbiota, allowing the pathogen to proliferate and release toxins that ADP ribosylate host rho GTPases (1, 2). In patients with recurrent C. difficile infections, fecal microbiota transplant (FMT) has become standard of care to reconstitute the microbiota and prevent recurrence. While intensive efforts to develop defined microbial replacements for FMT have been undertaken, relatively little is known about the molecular, metabolic, and microbiologic mechanisms by which specific members of the microbiota modulate the pathogen's virulence in vivo, information critical for therapeutics development (3,4). Given deaths in immunocompromised patients from drug-resistant pathogens in FMT preparations (5), therapies informed by molecular mechanisms of action among will enable options with improved safety and efficacy (6, 7).C. difficile's pathogenicity locus (PaLoc) contains the tcdA, tcdB and tcdE genes that encode the A and B toxins, and holin involved in toxin export, respectively. tcdR encodes a sigma factor specific for the toxin gene promoters, and the tcdC gene a TcdR anti-sigma factor (8-10). Multiple metabolic regulators influence PaLoc expression (11,12). In particular, C. difficile elaborates toxin under starvation conditions to extract nutrients from the host and promote the shedding of spores.C. difficile, like other cluster XI Clostridia, possesses diverse genetic machinery to utilize different carbon sources for energy and growth. In addition to carbohydrate fermentation, the pathogen uses Stickland fermentations, and Stickland-independent fermentations of other amino acids including threonine and cysteine (13), to extract energy from amino acids. The pathogen can ferment ethanolamine, extract electrons from primary bile salts, and undergo carbon fixation through the Wood-Ljungdhal pathway to generate acetate for metabolism...

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“…Cecal supernatants from mice at 20h of infection extracted and subjected to SDS-PAGE and transfer to PVDF membrane (PerkinElmer, Waltham, MA) as described (59)…”

Section: Western Blot For Toxin B Integritymentioning

confidence: 99%

In vivocommensal control ofClostridioides difficilevirulence

Girinathan

DiBenedetto

Worley

et al. 2020

Preprint

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“…It was suggested that opp and app 542 may be involved in sporulation as a response to nutrient availability. Like csiA, 543 these mutants also show an increase on the expression of the sinRR' operon 544 (Table 3), previously shown to be involved in sporulation, toxin production and 545 motility (Girinathan et al, 2018). It was shown that overexpression of sinR 546 increased sporulation efficiency, while overexpression of sinR' reduced sporulation.…”

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confidence: 86%

A regulatory protein that represses sporulation inClostridioides difficile

Martins

Mendes

Antunes

et al. 2020

Preprint

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Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50 to 60% of those bacteria have the ability to produce resilient spores, important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile, and contains several genes of unknown function. We report on the characterization of a signature gene, csiA, which, as we show, is involved in the control of sporulation initiation in C. difficile. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of csiA results in increased sporulation, and increased expression of spo0A per cell. Spo0A also drives transcription of the spoIIA and spoIIG operons, coding for the first forespore-(σF) and mother cell-specific (σE) RNA polymerase sigma factors. Strikingly, deletion of csiA increases expression of the spoIIG operon, but not that of the spoIIA operon. Increased expression of spoIIG results in increased production and proteolytic activation of pro-σE, suggesting that normally, the levels of active σE are limiting for sporulation. While other regulatory proteins affect both sporulation and several processes during the transition phase of growth, including toxin production or motility, deletion of the csiA gene does not alter the expression of the genes coding for the TcdA and TcdB cytotoxins or the genes involved in motility. Thus, our results establish that CsiA acts to modulate sporulation by reducing expression of the spo0A gene.

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“…Recently, we reported the identification and characterization of master regulator SinR in C. difficile that was found to regulate sporulation, toxin production, and motility (15). SinR in the Gram-positive model organism B. subtilis is well characterized and is known to regulate multiple pathways, including sporulation, competence, motility, and biofilm formation (16–18).…”

Section: Introductionmentioning

confidence: 99%

“…In C. difficile also the sin locus is a two-gene operon and encodes for SinR and SinR’. In our initial characterization of the C. difficile sin locus, we have shown that disruption of sin locus (absence of both SinR and SinR’) resulted in asporogenic, less toxic, and less motile phenotype (15). Another study which reports that…”

Section: Introductionmentioning

confidence: 99%

“…This led to the assumption that unlike B. subtilis , the C. difficile sin locus is transcribed from a single upstream promoter. Real-time RT PCR analysis of the cells grown in vitro , showed sin locus expression at 10-12 hour time point, indicating its tight regulation (15). From various gene expression data, we can observe that mutations in sigH and spo0A positively influence the expression of sinRR’ (23–26) and mutations in tcdR to downregulate their expression (27).…”

Section: Introductionmentioning

confidence: 99%

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Spo0A suppressessinlocus expression inClostridioides difficile

Dhungel

Govind

2020

Preprint

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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...

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Pleiotropic roles of Clostridium difficile sin locus

Cited by 51 publications

References 73 publications

In vivocommensal control ofClostridioides difficilevirulence

In vivocommensal control ofClostridioides difficilevirulence

A regulatory protein that represses sporulation inClostridioides difficile

Spo0A suppressessinlocus expression inClostridioides difficile

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