The probiotic[i] Propionibacterium freudenreichii[/i] surface proteome

Surface proteins are key actors of the complex interactions between bacteria (pathogens, commensals or symbionts) and their host. In beneficial (probiotic) bacteria, they participate in competition with pathogens, adhesion to the host cells, and immunomodulation. We investigated such proteins in the beneficial bacterium Propionibacterium freudenreichii, consumed both in Swiss-type cheeses and probiotic preparations. P. freudenreichii genome was sequenced and annotated, the localization of the encoded proteins was predicted using SurfG+. A combination of 3 biochemical methods confirmed surface exposure of P. freudenreichii proteins: shedding, shaving and labelling. Shedding consisted in the extraction of cell-wall associated proteins using guanidine, followed by trypsinolysis of the extracted proteins. Shaving consisted in enzymatic hydrolysis of surface protruding proteins which were accessible to trypsin in situ on live bacteria. For labeling, an NHS-ester-cyanine was added to live bacteria in order to label surface proteins, prior to 2-D electrophoresis and detection of fluorescent protein spots. For the 3 methods, the resulting tryptic peptides were identified by NanoLC-MS/MS on a Q-TOF mass spectrometer This combination of methods allowed identification of surface layer type-proteins, lipoproteins, proteins associated to the cell wall, to the membrane, or predicted to be secreted, as well as moonlighting proteins predicted to be cytoplasmic. Some of these proteins are known to participate in adhesion and in the modulation of the immune response by probiotics. This work constitutes a decisive step in the elucidation of P. freudenreichii ability to interact with host cells and in the understanding of protein sorting in this bacterium.