Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ

Abstract

1Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. 2The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject 3 of research interest among persistent clinical pathogens of global health importance. Our current 4 understanding of the underlying biochemical pathways responsible for biosynthesis of these 5 exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, 6 anaerobic and spore-forming bacteria, and include the important human pathogens Clostridium 7 perfringens, Clostridium botulinum, and Clostridioides difficile, among numerous others. Clostridia have 8 been reported to form biofilms composed of cellulose, although the specific loci which encode the 9 cellulose synthase have not been identified. Here, we report the discovery of a gene cluster, which we 10 named ccsABZCD, among selected bacteria within class Clostridia that appears to encode a synthase 11 complex responsible for polymerization, modification, and export of an O-acetylated cellulose 12 exopolysaccharide. To test this hypothesis, we subcloned the putative glycoside hydrolase CcsZ and 13 solved the X-ray crystal structure of both apo-and product-bound CcsZ. Our results demonstrate that 14 CcsZ is in fact an endo-acting cellulase belonging to glycoside hydrolase family 5 . This is in 15 contrast to the Gram-negative cellulose synthase, which instead encodes BcsZ, a structurally distinct GH-16 8. We further show CcsZ is capable of hydrolysis of the soluble mock substrate carboxymethylcellulose 17 (CMC) with a pH optimum of 4.5. The data we present here serves as an entry point to an understanding 18 of biofilm formation among class Clostridia and allowed us to predict a model for Clostridial cellulose 19 synthesis.20 Author summary 21 Biofilms are communities of microorganisms that enmesh themselves in a protective matrix of elf-22 produced polysaccharide materials. Biofilms have demonstrated roles in both virulence and persistence 23 among bacterial pathogens of global health importance. The class Clostridia are a polyphyletic grouping 24 of primarily Gram-positive, anaerobic and spore-forming bacteria which contain the important and well-25 studied human pathogens Clostridioides difficile, Clostridium botulinum, and Clostridium perfringens, 26among others. Bacteria belonging to class Clostridia have been anecdotally reported to form biofilms described. In this work, we identify a gene cluster, which we name ccsABZHI, for the Clostridial cellulose 29 synthase, which bears remarkable similarity to molecular machinery required for the production of 30 cellulose biofilms in other Gram-negative bacteria. We further biochemically characterize one of these 31 enzymes, CcsZ, a predicted endoglucanase which we predicted from our model should cleave cellulose 32 exopolysaccharides. We show that CcsZ is in fact capable of this activity and belongs to a broader family 33 of glycoside hydrolases with unexpected...

Extracellular succinate induces spatially organized biofilm formation inClostridioides difficile

“…We also noted that at 48h of incubation, proteins form clusters in all conditions, although this should be interpreted cautiously as PFA is known to form such protein aggregates. Interestingly, we observed that EPS are uniformly localized on top of the cells for all conditions tested, which is consistent with the expression of the ccsA gene (CD630_25450) encoding C. difficile's cellulose synthase (56), not significantly differentially regulated (Table S2) (10). Such distribution of EPS is probably due to their production only by cells localized at the top of the biofilm, by a division of labor similar to what was observed in B. subtilis (57).…”

Enzymatic dispersion of biofilms: An emerging biocatalytic avenue to combat biofilm-mediated microbial infections

“…Therefore, selectively targeting biofilms using GH enzymes might be feasible. A list of these enzymes used for biofilm dispersion is shown in Table 2 ( 135 , 139 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 ).…”