LAUSR

Lactobacillus casei group bacteria improve cheese ripening and may interact with host intestinal cells as probiotics, where surface proteins play a key role. Three complementary methods (trypsin shaving [TS], LiCl-sucrose extraction [LS] and extracellular culture fluid [ECF] precipitation) were used to analyze cell-surface proteins of L. paracasei GCRL163 by label-free quantitative proteomics after culture to mid-exponential phase in bioreactors at pH 6.5 and temperatures of 30ºC to 45ºC. A total of 416 proteins, including 300 with transmembrane, cell-wall anchoring and secretory motifs, and 116 cytoplasmic proteins were quantified as surface proteins. Although LS caused significantly greater cell lysis as growth temperature increased, higher numbers of extra-cytoplasmic proteins were exclusively obtained by LS treatment. Together with increased positive surface charge of cells cultured at supra-optimal temperatures, proteins including cell wall hydrolases Msp1/p75 and Msp2/p40, α-fucosidase AlfB, SecA and a PspC-domain putative adhesin were up-regulated in surface or secreted protein fractions, suggesting that cell adhesion may be altered. Prolonged heat stress increased binding of L. paracasei GCRL163 to human colorectal adenocarcinoma HT-29 cells, relative to acid-stressed cells. This study demonstrated that prolonged heat stress influences cell adhesion and relative abundance of proteins located at the surface, which may impact probiotic functionality, and detected novel surface proteins likely linked to the cell cycle and envelope stress.