Abstract
Spores of Clostridium difficile play a key role in the dissemination of this important human pathogen and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly with a clear miss-assembly of the outermost layers of the spore coat. The CotA protein is most probably subject to post-translational modification and could play a key role in stabilising the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore although for the cotD, cotE and sodA mutants enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed as enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. cotD encoding a manganese catalase, sodA a superoxide dismutase (SOD) and cotE a bifunctional enzyme with peroxiredoxin and chitinase activity. These enzymes being exposed on the spore surface would play a role in coat polymerisation and detoxicification of H(2)O(2). Two additional proteins, CotF (a tyrosine rich protein and potential substrate for SodA) and CotG (a putative managanese catalase) were shown to be located to the spore surface.