Effect of
            <i>tcdR</i>
            Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

Girinathan, Brintha P.; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N.; Sorg, Joseph A.; Dupuy, Bruno; Govind, Revathi

doi:10.1128/msphere.00383-16

Cited by 36 publications

(53 citation statements)

References 63 publications

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“…Mutation of tcdR has been reported to significantly reduce levels of both toxin gene expression and toxin protein production in vitro 33,34 . Nearly five-fold fewer tcdR vegetative cells were recovered in the feces relative to wild type at day 1 (p=0.0314, Kruskal-Wallis with Dunn's correction for multiple comparisons); however, there was no difference by day 3, nor were significant differences detected in fecal spores between the two strains at either day ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Clostridioides difficileexploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

Fletcher

Pike

Parsons

et al. 2020

Preprint

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Introductory paragraphClostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Using tcdR mutants in two strains of C. difficile, we defined how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression, and the gut microbiota to determine how inflammation by the host may be beneficial to C. difficile. Here we show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to compete against C. difficile for the same essential nutrients released from collagen degradation.

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Section: Resultsmentioning

confidence: 99%

Clostridioides difficileexploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

Fletcher

Pike

Parsons

et al. 2020

Preprint

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“…To quantify this, we determined the EC 50 values for L-alanine in C. difficile UK1 spores, C. difficile RS07 spores, and C. difficile RS07 palr2 spores. Although spore germination is a multienzyme process, these kinetic measurements allow for the quantitative interaction of the spores with activators and inhibitors of germination (27)(28)(29)(30)(31)(32)(33)(34). When C. difficile UK1 spores were suspended in buffer supplemented with TA and increasing concentrations of L-alanine, we observed an increase in the germination rate.…”

Section: Difficile Rs07 Spores More Readily Germinate In Response mentioning

confidence: 88%

A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate

Shrestha

Lockless

Sorg

2017

Journal of Biological Chemistry

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has become one of the most common bacterial pathogens in hospital-acquired infections in the United States. Although is strictly anaerobic, it survives in aerobic environments and transmits between hosts via spores. spore germination is triggered in response to certain bile acids and glycine. Although glycine is the most effective co-germinant, other amino acids can substitute with varying efficiencies. Of these, l-alanine is an effective co-germinant and is also a germinant for most bacterial spores. Many endospore-forming bacteria embed alanine racemases into their spore coats, and these enzymes are thought to convert the l-alanine germinant into d-alanine, a spore germination inhibitor. Although the Alr2 racemase is the sixth most highly expressed gene during spore formation, a previous study reported that Alr2 has little to no role in germination of spores in rich medium. Here, we hypothesized that Alr2 could affect l-alanine-induced spore germination in a defined medium. We found that mutant spores more readily germinate in response to l-alanine as a co-germinant. Surprisingly, d-alanine also functioned as a co-germinant. Moreover, we found that Alr2 could interconvert l- and d-serine and that Alr2 bound to l- and d-serine with ∼2-fold weaker affinity to that of l- and d-alanine. Finally, we demonstrate that l- and d-serine are also co-germinants for spores. These results suggest that spores can respond to a diverse set of amino acid co-germinants and reveal that Alr2 can accommodate serine as a substrate.

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“…C. difficile strains (Table S1) were grown in TY (Tryptose and Yeast extract) agar or broth culture in an anaerobic chamber which is maintained at 10% H 2 , 10% CO 2 and 80% N 2 as described previously (27–30). Lincomycin (Linc 20ug/ml) and thiamphenicol (Thio; 15 ug/ml) were added to the culture medium when required.…”

Section: Methodsmentioning

confidence: 99%

“…The control plasmid pBA040 with promoter less gusA was created by digesting with KpnI and SacI to remove tet promoter and then self-ligated after creating blunt ends. Plasmids were introduced into R20291 and R20291:: spo0A strains through conjugation as described previously (15, 27). The transconjugants were grown in TY medium in the presence of thiamphenicol (15 ug/ml) overnight.…”

Section: Methodsmentioning

confidence: 99%

“…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). We have also demonstrated that CodY can directly bind to sin locus upstream DNA to transcriptionally repress its expression (15).…”

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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Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

Cited by 36 publications

References 63 publications

Clostridioides difficileexploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

Clostridioides difficileexploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate

Spo0A suppressessinlocus expression inClostridioides difficile

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