Purification and Characterization of a Surface Protein from Lactobacillus fermentum 104R That Binds to Porcine Small Intestinal Mucus and Gastric Mucin

Abstract

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidaselabeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesionpromoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesionpromoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.Proteinaceous surface appendages or coverings, e.g., fimbriae, flagella, or surface (S)-layers with affinity for mammalian extracellular matrix (55) or mucosa (17, 28), have been extensively characterized for many pathogenic bacteria. It is thought that adhesion to mammalian extracellular matrix components is a prerequisite for bacterial colonization and invasion to subepithelial tissues (55) Cell surface-adhesive proteins from nonpathogenic bacteria and their role in colonization has not been as thoroughly characterized.Lactobacillus, one of the common indigenous organisms of the gastrointestinal tracts of mammals (46,48,49) and a potential probiotic microbe that contributes to the health of the host (11,16), has the capacity to adhere to epithelial cells (5,6,9,10,16,21,23,24,33,34,44,49) and mucus gel (25,32,35,38,41,51,52) from the intestinal tracts of different species. Lactobacillus surface proteins have been proposed to be involved in colonization of gastrointestinal epithelial cells and mucosa of mammals (9,12,21,41,44,53). Collagen binding by lactobacilli (1, 47) is known, and purification of collagen binding proteins from L...

In vitro evaluation of adhesion and aggregation abilities of four potential probiotic strains isolated from guppy (Poecilia reticulata)

“…It has been reported that lipoteichoic acids are one of the factors responsible for adhesion of Lactobacillus johnsonii LaI (Granato et al 1999). In addition, adhesion-promoting proteins, which are present on the cell surface of L. fermentum and L. brevis have been isolated and characterized (Rojas et al 2002). Several strains of Enterobacter spp.…”

A Mucus Adhesion Promoting Protein, MapA, Mediates the Adhesion ofLactobacillus reuterito Caco-2 Human Intestinal Epithelial Cells

“…Strains 104R 12) and LEM83 22) were isolated from pig, ML1 from mouse, 17) and LB54 from chicken (E. Satoh, personal communication). All of these strains were cultured in MRS broth (Difco, Detroit, MI) at 37 C under anaerobic conditions.…”

“…Another piece of the puzzle is demonstration of the mucus-binding ability of Lactobacillus fermentum, which was mediated by a mucus adhesion promoting protein (MapA) from L. fermentum 104R. [8][9][10]12) This strain was reidentified as L. reuteri on the basis of DNA relatedness (Satoh, unpublished data). MapA is known as a cell-surface protein with a molecular weight of 26 kDa and a theoretical pI of 9.7.…”

“…Therefore, the adhesion of LAB to the GI tract or colonization to the GI tract by these organisms has been investigated using mucus, tissue samples, cell lines, and components of the extracellular matrix. [5][6][7][8][9][10][11][12][13][14] Lactobacillus reuteri is one of LAB found in the GI tracts of many animals, and it is believed to benefit the host in various ways. 15,16) Several studies have shown that oral administration of this species causes immunomodulation in host animals.…”

Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG

“…Thus far, the precise molecular mechanisms that differentiate the colonization ability between autochthonous and allochthonous intestinal lactobacilli remain undefined (42), although they are likely to be partly dependent on a diverse range of cell surface adhesion molecule-mediated interactions with the host intestinal mucosa. With that being said, there are a growing number of reports in the literature that indicate that lactobacillar adherence to the intestinal mucosal layer is mediated by surface proteins with a mucus-binding capacity (15,21,22,25,30,32,33,41). Moreover, homology-driven genome mining in several Lactobacillus spp.…”