Structure of core assembly of the Clostridioides difficile germinosome.

Abstract

The germinosome is the machinery of Clostridioides difficile that sets in motion the process of spore germination to vegetative bacteria. Three highly-regulated proteins-CspA, CspB and CspC-serve as key instigators of germination. We report that CspA and CspB exist independently as homodimers in solution. In the presence of CspC, a picomolar complex of CspA:CspC forms. Furthermore, we document that CspA binds to the germinant, taurocholate, and that the complex CspA:CspC:taurocholate serves as the receptor for glycine, the co-germinant. We report high-resolution X-ray and cryo-EM structures for the three proteins, and for the CspA:CspC:taurocholate complex. These structures show how the CspA:CspC heterodimer recognizes taurocholate and reveal that specific structural features in the three Csp proteins avoid the recognition of the germinant by homodimers. Remarkably, the homodimer of CspB organizes itself in a supramolecular fibril assembly comprised of a three-stranded right-handed superhelix. These proteins are targets for interference with the transition from spore to the vegetative form of C. difficile.