Biology subjects

Anjuwon-Foster, B. R.

Publications and source records attributed to Anjuwon-Foster, B. R..

2 recordsLinked to original sources

bioRxiv2018

RstA is a Major Regulator of Clostridioides difficile Toxin Production and Motility

Clostridioides difficile infection (CDI) is a toxin-mediated disease. Several factors have been identified that influence the production of the two major C. difficile toxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified a C. difficile regulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes and rstA transcription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds the rstA promoter via the predicted DNA-binding domain. Through mutational analysis of the rstA promoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes, tcdA and tcdB, as well as the promoters for the sigD and tcdR genes, which encode regulators of toxin gene expression. Complementation analyses with the Clostridium perfringens RstA ortholog and a multi-species chimeric RstA protein revealed that the C. difficile C-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.\n\nIMPORTANCEClostridioides difficile is an anaerobic, gastrointestinal pathogen of humans and other mammals. C. difficile produces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside of the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. In addition, the ability for RstA to bind DNA and repress toxin production requires the species-specific domain predicted to respond to small quorum-sensing peptides. This study provides a novel regulatory link between C. difficile sporulation and toxin production. Further, our data suggest that C. difficile toxin production is regulated through a direct sensing mechanism.

microbiology↗

bioRxiv2018

Characterization of flagellar and toxin phase variation in Clostridium difficile ribotype 012 isolates

Clostridium difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a \"flagellar switch\" upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting the strain is phase-locked. To determine whether a \"phase locked\" flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase OFF, non-motile, and attenuated for toxin production. We isolated low frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production, and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase ON derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains, and perhaps other ribotypes, which could impact disease progression and diagnosis.\n\nImportanceClostridium difficile causes diarrheal disease resulting in significant morbidity and mortality in many countries. C. difficile produces flagella that enhance bacterial motility, and secretes toxins that promote diarrheal disease symptoms. Previously, we found that production of flagella and toxins are co-regulated via a flippable DNA element termed the \"flagellar switch\", which mediates the phase variable production of these factors. C. difficile can exist as flagellated, motile, toxigenic bacteria (\"flg ON\") or aflagellate, non-motile, nontoxigenic bacteria (\"flg OFF\") due in part to the flagellar switch orientation. Here we evaluate multiple isolates of C. difficile ribotype 012 strains and find them to be primarily flg OFF. Some, but not all, of these isolates showed the ability to switch between flg ON and OFF states. These findings suggest heterogeneity in the ability of C. difficile ribotype 012 strains to phase vary flagellum and toxin production, which may broadly apply to pathogenic C. difficile.

microbiology↗