Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus

The cbsA gene of Lactabacillus crispatus strain JCM 5810, encoding a protei n that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coil. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expresse d as a His tag fusion protein, purified by affinity chromatography, and sho wn to bind solubilized as well as immobilized type I and TV collagens. Thre e other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collag ens only weakly, and sequence comparisons of CbsA with these S-layer protei ns were used to select sites in cbsA where deletions and mutations were int roduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncate d peptides from CbsA were constructed by gene fusion. Analysis of these mol ecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that f ailed to polymerize into a periodic S-layer did not bind collagens, suggest ing that the crystal structure with a regular array is optimal for expressi on of collagen binding by CbsA. Strain JCM 5810 was found to contain anothe r S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, nas transcribed under laboratory c onditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to c ollagen-containing regions in the chicken colon, suggesting that CbsA-media ted collagen binding represents a true tissue adherence property oft. crisp atus.