Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

Abstract

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. 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 expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. 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 truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules 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 failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsAmediated collagen binding represents a true tissue adherence property of L. crispatus.

Effect of Environmental Stresses on S-Layer Production in Lactobacillus acidophilus ATCC 4356

“…It has been proposed that S-layer plays a role as a protective sheath in Lactobacillus acidophilus ATCC 4356. In addition, some studies identified that the S-layer proteins of Lactobacilli were important for hydrophobicity, autoaggregation and adherence of this bacteria to different host surfaces (Frece et al, 2005;Greene & Klaenhammer, 1994;Hynonen et al, 2002;Kos et al, 2003;Pelletier et al, 1997;Sillanpaa et al, 2000;Toba et al, 1995;Vadillo-Rodriguez et al, 2004Van der Mei et al, 2003).…”

The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K

“…It demonstrates 37% similarity to Biofilm Associated Protein (BAP) found in bovine Staphylococcus aureus strains 33 . We further identified four paralogous proteins predicted to mediate autoaggregation and adherence to collagen 34 . They have a central region rich in Ser/Thr/Gln, demonstrate physical properties quite similar to cell surface-layer proteins, and can be further divided into two sets.…”

Development of New Products: Probiotics and Probiotic Foods

“…Moreover, the hydrophobicity of the cell surface of the strain Lactobacillus acidophilus ATCC 4356 can be varied in accordance with the change of the ionic strength of the medium [43]. In a sequencing study of the S-layer proteins in Lactobacillus acidophilus, Lactobacillus crispatus and Lactobacillus helveticus a high degree of homology in one third of their Cterminus is demonstrated [36].…”

“…The molecular weight of the S-layer proteins in lactobacilli ranges from 40 kDa to 60 kDa and they are one of the smallest known S-layer proteins [31,36,39,40]. Compared with many other S-layer proteins, which are of acidic nature, those in lactobacilli are characterized by high values of their isoelectrical points [41].…”

“…The gene for the S-layer protein has been sequenced and cloned in Lactobacillus brevis [33], Lactobacillus acidophilus [34], Lactobacillus helveticus [35] and Lactobacillus crispatus [36].…”