Abstract
Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.
Highlights
Bacterial surface (S-) layers are crystalline arrays of selfassembling, proteinaceous subunits called surface layer (S-layer) proteins (Slps), with molecular masses ranging from 40 to 200 kDa (Sara & Sleytr, 2000)
Identification of extracellular and putative S-layer associated protein (SLAP) from modified lithium chloride (LiCl) extraction Previous work characterizing the S-layer of lactobacilli used 5 M LiCl salt to extract S-layer protein (Slp) efficiently with less lethality than http://mic.sgmjournals.org
Cells were treated with 5 M LiCl, solubilizing all non-covalently bound Slp and proteins associated with the Slayer, and the proteins were extracted via dialysis
Summary
Bacterial surface (S-) layers are crystalline arrays of selfassembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa (Sara & Sleytr, 2000). Present as the outermost component of the cell wall, S-layers are found in many species of eubacteria and archaea, and in varying environments. Slayer lattices are 5–25 nm thick and form oblique (p1, p2), square (p4) or hexagonal (p3, p6) symmetries, as observed by freeze-etched electron microscopy (Sleytr et al, 2001). Further structural observations have revealed that S-layers are highly porous in nature, with pores occupying up to 70 % of the cell surface (Sleytr & Beveridge, 1999). Previous work comparing amino acid sequences from different. Three supplementary figures are available with the online version of this paper
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