Prolonged Heat Stress of GCRL163 Improves Binding to Human Colorectal Adenocarcinoma HT-29 Cells and Modulates the Relative Abundance of Secreted and Cell Surface-Located Proteins.

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 (LS) extraction, and extracellular culture fluid precipitation] were used to analyze cell surface proteins of GCRL163 by label-free quantitative proteomics after culture to the mid-exponential phase in bioreactors at pH 6.5 and temperatures of 30-45 °C. 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 extracytoplasmic proteins were exclusively obtained by LS treatment. Together with the 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 upregulated in surface or secreted protein fractions, suggesting that cell adhesion may be altered. Prolonged heat stress (PHS) increased binding of GCRL163 to human colorectal adenocarcinoma HT-29 cells, relative to acid-stressed cells. This study demonstrates that PHS influences cell adhesion and relative abundance of proteins located on the surface, which may impact probiotic functionality, and the detected novel surface proteins likely linked to the cell cycle and envelope stress.