Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen

Abstract

It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB(380-501) was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB(1-379) peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.

A New Addition to the Cell Plan of Anammox Bacteria: "Candidatus Kuenenia stuttgartiensis" Has a Protein Surface Layer as the Outermost Layer of the Cell

“…Many Gram-positive S-layer proteins contain a so-called Slayer homology (SLH) motif at their N terminus that is involved in anchoring the S-layer to the secondary cell wall polymers (29,65). In Gram-positive S-layer proteins without SLH domains, an Nterminal motif (having, for instance, a net positive charge) or, in some cases, a C-terminal motif seems to play a role in attachment (63,64,66). When a BLAST search with Kustd1514 was performed against the S-layer SLH consensus motif proposed previously by Engelhardt and Peters (32), no significant hits were found.…”

Role of S-layer proteins in bacteria

“…Several L. crispatus strains have been studied and the SLP domains responsible for adherence to collagen, characterized (Toba et al 1995;Sillanpaa et al 2000;Sun et al 2013). Horie et al (2002) demonstrated the ability of the L. crispatus CbsA SLP to inhibit adherence of an enterotoxic E. coli strain to laminin.…”

Probiotic Mechanisms of Action

“…A wide variety of molecules mediating the adhesion of pathogenic bacteria has been characterized. However, the understanding of the factors that mediate adhesion for Lactobacillus is extremely limited [85,86,87]. Further studies are needed for the identification and analysis of the functional significance of various components of mucous layers as well as the complex interactions of mucous layers, microbiota (including probiotics) and epithelial cells with underlying innate and adaptive immune systems [68].…”