Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

Abstract

The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called "hypervirulent" epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCEHow Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile. TFP are also required for surface motility, which has not previously been demonstrated for C. difficile. Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis

“…S9). We also examined transcription of the major type IV pilin, pilA1 , and the flagellar subunit, fliC , which are involved in swarming and swimming motility, respectively (Purcell et al ., ). C. difficile flagella and pilin are detected by the innate immune system and can elicit a response that may impact pathogen clearance or virulence (Pechine et al ., ; Yoshino et al ., ; Maldarelli et al ., ; Batah et al ., ; Ghose et al ., ; Pechine and Collignon, ).…”

Clostridium difficile colitis: pathogenesis and host defence

“…High levels of intracellular c-di-GMP repress flagellum expression and thereby motility 91 , and also repress the synthesis of TcdA and TcdB 92 . Concurrently, c-di-GMP activates another riboswitch that induces the expression of that interact with the intestinal epithelium and contribute to C. difficile aggregation and biofilm formation 93,94 . In this manner, c-di-GMP acts as a key signal that can switch C. difficile between a highly motile, toxin-producing state and a strongly adherent biofilm-producing state.…”

Phase variation of a signal transduction system controlsClostridioides difficilecolony morphology, motility, and virulence

“…undifferentiated by colony morphology) R20291 is characterized by initial (∼ 24 hours) growth restricted to the site of inoculation of an agar surface, then spreading tendrils of growth between 24 and 96 hours (36). Type IV pili contribute to surface motility, so the non-piliated pilB1 mutant was included as a control (35, 36). In this assay, bacteria isolated from rough colonies exhibited greater surface motility after 48 hours compared to undifferentiated (WT) R20291 (Figures 2D,E).…”