Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins ofClostridium difficile

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Abstract
Recent investigations of theClostridium difficilegenome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence:slpAencodes the precursor of the two proteins of the S-layer, P36 and P47, whereascwp66encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused onslpA,cwp66, andcwp84,the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein inEscherichia coli. Polymorphism ofcwp66andcwp84genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. TheslpAgene is strongly transcribed during the entire growth phase as a bicistronic transcript;cwp66is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. Thecwp84gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern ofcwp84, suggests a significant role in the physiopathology ofC. difficilefor the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.