Updates to Clostridium difficile Spore Germination

Abstract

Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringensC. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.

Keywords: Clostridium difficile; germination; spores.

FIG 1

Anatomy of bacterial spores. Bacterial spores are composed of several layers, including a dehydrated spore core (A), an inner membrane (B), a germ cell wall (C), a spore cortex (D), a spore coat/outer membrane (E), and an exosporium (F). The spore cortex is a thick layer of modified peptidoglycan where ∼50% of N-acetylmuramic acid side chains are removed to produce muramic acid delta-lactam rings, which are depicted in red.

FIG 2

Bacillus anthracis l-alanine germination model. Nutrients enter the spore through GerP_ABCDEF complex (A), which facilitates movement of l-alanine through the spore coat and outer membrane to the spore inner membrane, where it binds to GerK and/or GerL germination receptors (B). These germinant-receptor interactions lead to slight core rehydration (C) and release of Ca-DPA from the spore core (D). Ca-DPA travels through the cortex (or can be added exogenously) and binds to CwlJ (E). This binding activates CwlJ, initiating hydrolysis of the cortex peptidoglycan (F). This leads to full core rehydration and spore outgrowth.

FIG 3

The cspBAC operon: structural map of the conserved catalytic triad. (A and B) Schematic of Csp proteases in C. perfringens (A) and C. difficile (B). Intact catalytic residues are in black, and mutated residues are in red. The asterisk indicates the YabG cleavage site. (C) Superposition of the C. difficile Csp mutated catalytic residues (model from I-TASSER structure prediction) with the C. perfringens CspB (PDB 4i0W) (70). (D) Map of the mutations reported by Francis et al. on CspC (I-TASSER structure prediction), showing both internal mutations in ribbon diagrams (top) and mutations on the protein surface (bottom) (44). Catalytic triad residues are highlighted in green, mutated residues that result in a loss of germination are highlighted in red, and the G457R mutation is highlighted in yellow.

FIG 4

Comparison of proposed models of Clostridium difficile germination. (A) “Germinosome” model based on the findings from Francis et al., Adams et al., and Fimlaid et al., where proposed Csp protein-protein interactions lead to activation of SleC (44, 70, 94). (B) “Lock-and-key” model modified from that of Kochan et al., where CspC-taurocholate interactions facilitate cogerminant entry into the spore (66).