A Conserved Switch Controls Virulence, Sporulation, and Motility inC. difficile

Abstract

SUMMARYSpore formation is required for environmental survival and transmission of the human enteropathogenicClostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulateC. difficileSpo0A activity are not defined. In the well-studiedBacillusspecies, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function inC. difficile, we created a null mutation of thespo0Eortholog and assessed sporulation and physiology. Thespo0Emutant produced significantly more spores, demonstrating Spo0E repressesC. difficilesporulation. Unexpectedly, thespo0Emutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function inB. subtilisrevealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, we found that Spo0E orthologs are widespread among prokaryotes, suggesting that Spo0E performs conserved regulatory functions in diverse bacteria.