A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile.

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance. Author summary: Clostridioides difficile, an anaerobic spore-forming bacterium, colonizes the gastro-intestinal tract when, as during antibiotic treatment, the protective effect of the microbiota is disrupted. A leading agent of nosocomial infections, causing a range of symptoms from mild diarrhea to life-threatening conditions, the organism is recognized as a global and urgent threat. Infection begins with the ingestion of spores, which will germinate in response to bile salts. Two proteinaceous spore surface layers, the coat and the exosporium, play a crucial role in infection and colonization, as they contribute to spore resistance, binding to host cells and the interaction with and the response to germinants. The yabG gene, part of a genomic signature for sporulation, codes for a cysteine protease, with residues required for catalysis embedded in a CheY-like response regulator receiver domain. YabG is required for proper morphogenesis of the spore surface layers, germination and host colonization. YabG also regulates the mother cell line of gene expression by allowing the expression of genes required for assembly of the coat and exosporium. While this latter function has not been described for other organisms, the general role of yabG in the assembly of the spore surface layers is likely to be conserved among spore-formers. [ABSTRACT FROM AUTHOR]