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

Abstract

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.

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum

“…The genome assembly confirmed the presence of a prophage located within yabG (Figure 1). yabG encodes a sporulation-specific protease shown to regulate the assembly of the spore coat in both B. subtilis and C. difficile [23][24][25]27].…”

“…Since YabG is involved in the assembly of the spore surface in B. subtilis [24,25] and spore germination in C. difficile [26,27], we hypothesized that the presence/absence of the yin element could control the function of YabG and therefore alter the spore properties. Thermal destruction assays showed that the V73 spores were slightly less heat resistant than spores of ST7B (most heat-sensitive spore subpopulations, depicted in Figure 6 in the first 20 min of heating); however, the difference was not statistically significant for the most heat-resistant spore subpopulations (depicted in Figure 6 after the first 20 min of heating).…”

“…The yabG is regulated by SigK [23] and codes for a cysteine protease that regulates the assembly of the spore coat by processing several coat proteins in B. subtilis [24,25]. YabG also processes two proteins required for spore germination in C. difficile, CspBA and Pre-pro-SleC, as well as a coat protein required for efficient host colonization [26,27]. Interestingly, yabG is the only member of the SigK regulon that is conserved across spore formers and part of a genomic signature for sporulation [28].…”