Structural and functional characterization of the surface layer protein of Lactobacillus brevis ATCC 8287
Year: 2009
Type: dissertation
Abstract: Bacterial surface (S) layers are proteinaceous arrays found on the surface of hundreds of bacterial species, including several species of lactobacilli. They are composed of numerous identical, non-covalently bound subunits, which completely cover the cell surface forming a symmetric, porous, lattice-like structure. Several functions for S-layers have been found, but no common one probably exists. S-layer proteins have a wide application potential in nanobiotechnology as well as in health-related applications such as vaccine design. In this work, the structure and function of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 and the expression of the slpA gene were studied. SlpA was identified as a two-domain protein, in which the N-terminal domain is responsible for binding to the cell wall and the C-terminal domain for forming the regular polymer. The domain organization is thus reversed compared with other hitherto characterized Lactobacillus S-layer proteins. Conserved carbohydrate binding motifs were identified in the N-terminal, positively charged amino acid sequences of SlpA and five other Lactobacillus brevis S-layer proteins. The component in the cell wall interacting with SlpA was shown to be something other than teichoic or lipoteichoic acid, in contrast to the cell wall receptors of S-layer proteins previously characterized in lactobacilli. The structure of the C-terminal self-assembly domain was studied in more detail using cysteine scanning mutagenesis and targeted chemical modification. Importantly for the potential future applications of SlpA as a display vehicle of foreign peptides, four amino acid segments with high surface accessibility in the assembled form of SlpA were detected. The 46 mutated residues could be grouped according to their location in the lattice: in the protein interior, on the inner surface of the lattice, on the outer surface of the lattice and on the subunit interface or the pore region of the lattice. L. brevis ATCC 8287 very efficiently adheres to cultured human epithelial cells representing the human gut, bladder and blood vessels, while the removal of the S-layer abolishes the binding. This binding was shown to be mediated by SlpA by using flagellum display. Hybrid flagella carrying fragments from the N-terminal part of SlpA bound to epithelial cells and to fibronectin, while flagella carrying the C-terminal part were unable to bind. The smallest fragment conferring binding to Int 407 cells comprised amino acids 66-215 in mature SlpA. The gene encoding SlpA is preceded by two promoters. By separating them on reporter plasmids, both of the promoters were shown to be used in L. brevis in all growth phases. More upstream region was needed for the full activity of the upstream promoter than for the downstream promoter. The promoter activities seen at the reporter enzyme level were also seen at the mRNA level, suggesting transcriptional rather than translational regulation of slpA. Three potential regulatory motifs were identified in the upstream region of slpA. Both promoters retained their activities under selected conditions mimicking the intestinal environment in vitro.
Source: Työväentutkimus Vuosikirja
Author: Ulla Hynönen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1811393792