Sequence and phylogenetic analysis of the gene for surface layer protein, slpA, from 14 PCR ribotypes of Clostridium difficile

Abstract

Clostridium difficile is the commonest cause of antibiotic-associated diarrhoea, with the hospitalized elderly being at particular risk. The organism makes a crystalline surface protein layer (S-layer), encoded by the slpA gene, the product of which is cleaved to give two mature peptides which associate to form the layer. The larger peptide (high molecular weight; HMW), derived from the C-terminal portion of the precursor, is relatively conserved, whereas the smaller peptide (low molecular weight; LMW), derived from the N-terminal portion of the precursor, is a dominant antigen which substantially forms the basis for serotyping of isolates. PCR ribotyping is a more discriminatory typing method, based on the intergenic rRNA. We obtained the sequence for slpA and some flanking DNA from a collection of C. difficile strains of 14 ribotypes isolated from elderly patients. Sequences from different ribotypes were compared with one another and with published sequences. Sequences from C. difficile ribotypes 046 and 092 were identical. Sequences from ribotype pairs 005 and 054, 012 and 046/092, 014 and 066 and 031 and 094 differed by 1-3 nt in the slpA gene. There were ultimately nine ribotypes or groups of ribotypes with very different slpA sequences, particularly in the region encoding the LMW peptide. The sequence from ribotype 002 was very different from previously published sequences. The DNA segment sequenced included the 59 315 bp of a secA homologue, encoding a putative transport protein required for peptide secretion across the plasma membrane. The amino acid sequences of the predicted HMW peptides were aligned and a neighbour-joining tree was produced using 10 000 bootstrap replicates. The predicted SecA N-terminal region was similarly analysed. For both SlpA and SecA, a strong association was found between ribotypes 012, 046/092, 017, 031 and 094. Ribotypes 001 and 078 formed part of this clade for SlpA but not SecA, indicating independent evolution for slpA and secA, presumably because they come under different selection pressures.

Localization of the Clostridium difficile Cysteine Protease Cwp84 and Insights into Its Maturation Process

“…The cleavage at the N-terminal domain is heterogeneous, with the major cleavage site identi- fied at Lys-91, in accordance with the recent results from de la Riva et al (11). It is noteworthy that the consensus cleavage site of SlpA into the mature S-layer proteins is also located after a serine residue (13,14), but no other consensus motif has been recovered in Cwp84. The C-terminal cleavage site has been presumably identified at Lys-518.…”

The LMW surface-layer proteins of Clostridium difficile PCR ribotypes 027 and 001 share common immunogenic properties

“…Both SLPs are encoded by a single gene, slpA, and are produced from the post-translational cleavage of a common precursor: the HMW SLP is derived from the Cterminal portion of the precursor, whereas the LMW SLP is derived from the N-terminal portion (Calabi et al, 2001). Sequence variability is particularly evident in the LMW SLP but is also present in the HMW SLP, giving rise to a range of protein sizes Eidhin et al, 2006). Studies on S-layer adhesive properties have demonstrated that chemical removal of the SLPs or treatment with anti-SLP antibodies can abolish adherence of C. difficile to human HeLa or mouse 929 cells .…”

Clostridium difficileinfection

“…43 We and others have shown that SlpA is a major contributor to C. difficile adherence, and that inhibition of adherence can be exploited as a strategy to prevent C. difficile binding to biotic surfaces. 44 SLPs have also been implicated in immune modulation associated with CDI; 45 thus, these proteins are critical non-toxin virulence factors.…”