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Tigecycline suppresses toxin A and B production and sporulation in Clostridium difficile.

ABSTRACT: Clostridium difficile infection (CDI) is mediated by potent extracellular toxins and is spread largely via bacterial spores. We and others have shown that some antibiotics stimulate C. difficile toxin production in a strain-specific manner; however, the effects of newer anti-C. difficile antibiotics on this process remain to be investigated.The effects of the protein synthesis inhibitor tigecycline on sporulation and toxin A and toxin B production were compared in historical (strain 9689) and hypervirulent BI/NAP1/027 (strain 5325) isolates of C. difficile in vitro.Tigecycline at 1/4× MIC stimulated an increased and earlier toxin A and/or B gene expression in both the historical and the hypervirulent strains, although a commensurate increase in toxin protein production was observed only in the 9689 strain. In fact, in the hypervirulent 5325 strain, toxin production was dramatically suppressed. By comparison, subinhibitory concentrations of vancomycin and metronidazole also stimulated increased protein toxin production by the historical, but not the hypervirulent, strain. In addition, tigecycline dose-dependently reduced viable spore production by both the 9689 and 5325 strains. Vancomycin treatment also suppressed spore formation in both C. difficile strains; however, metronidazole, while reducing spore formation in the 9689 strain, stimulated a near 2 log increase in spore production by the 5325 isolate.In summary, these findings suggest that the treatment of CDI patients with tigecycline could effectively both control disease progression and limit its spread by disrupting sporulation.

SUBMITTER: Aldape MJ

PROVIDER: S-EPMC4267498 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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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:UnlabelledClostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficileImportanceThis study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation in C. difficile and that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains.

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Tigecycline suppresses toxin A and B production and sporulation in Clostridium difficile.

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