Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.
Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA).
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, 1432 Ås, Norway.|Nofima - Norwegian Institute of Food, Fisheries, and Aquaculture Research, PO Box 210, 1431 Ås, Norway.
Hanne
H
Jensen
Department of Microbiology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Box 7025, 750 07 Uppsala, Sweden.
Stefan
S
Roos
Department of Microbiology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Box 7025, 750 07 Uppsala, Sweden.
Hans
H
Jonsson
Nofima - Norwegian Institute of Food, Fisheries, and Aquaculture Research, PO Box 210, 1431 Ås, Norway.
Ida
I
Rud
Nofima - Norwegian Institute of Food, Fisheries, and Aquaculture Research, PO Box 210, 1431 Ås, Norway.
Stine
S
Grimmer
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Jan-Peter
JP
van Pijkeren
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Robert A
RA
Britton
Nofima - Norwegian Institute of Food, Fisheries, and Aquaculture Research, PO Box 210, 1431 Ås, Norway.
Lars
L
Axelsson
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000382:microbiology*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D052200:Limosilactobacillus reuteri / Q000502:physiology*; D009093:Mucus / Q000378:metabolism* / Q000382:microbiology*
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases