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Toxin A-Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype.

ABSTRACT: BACKGROUND:Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. METHODS:An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). RESULTS:There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). CONCLUSIONS:We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

SUBMITTER: Lin Q

PROVIDER: S-EPMC7286367 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Toxin A-Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype.

Lin Qianyun Q Pollock Nira R NR Banz Alice A Lantz Aude A Xu Hua H Gu Limei L Gerding Dale N DN Garey Kevin W KW Gonzales-Luna Anne J AJ Zhao Mingwei M Song Linan L Duffy David C DC Kelly Ciaran P CP Chen Xinhua X

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 20200601 12

<h4>Background</h4>Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B.<h4>Methods</h4>An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and ...[more]

PMID: 31400280

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Project description:The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits.IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.

Project description:Clostridioides difficile infection (CDI) is a toxin-mediated diarrheal 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 multispecies 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.IMPORTANCE Clostridioides 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 the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate 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. 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, species-specific sensing mechanism.

Dataset Information

Toxin A-Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype.

Publications

Toxin A-Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype.

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