Two major secreted proteins as probiotic effectors of Lactobacillus rhamnosus GG

The well-documented probiotic bacterium Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, named Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been previously reported to promote the survival and growth of intestinal epithelial cells. We could demonstrate that both proteins are peptidoglycan hydrolases active in septum formation and daughter cell separation of LGG by a combination of mutational analysis, zymogram and immunofluorescence microscopy. The Msp1 protein was subsequently shown to have D-glutaminyl-L-lysyl endopeptidase activity, while Msp2 appears to be an endopeptidase cleaving the peptide crosslink. Therefore, we tested the in vivo persisistence of an msp1 knock-out mutant in healthy mice. After co-administration of an equal number of CFUs of LGG wild-type and msp1 mutant to mice, the msp1 mutant was even retrieved in higher number than wild-type from feces samples. Subsequently, we also compared the effect of administration of LGG wild-type and the msp1 mutant on experimental colitis in mice. In a mild DSS-induced colitis model, we found that administration of the msp1 mutant did not significantly affect clinical parameters, i.e. body weight and macroscopical score, compared to mice receiving LGG wild-type. However, mice receiving LGG wild-type seemed to induce more of the pro-inflammatory cytokine TNF compared to the msp1 mutant. Using ELISA, the msp1 mutant was shown to secrete ca. four times the amount of Msp2 produced by the wild-type. As Msp1 and Msp2 show complementary effects when applied to host epithelial cells, Msp2 may be able to complement the Msp1 loss in an msp1 mutant. Unfortunately, we were unsuccessful in the construction of an msp2 knock-out mutant to confirm the role of Msp2. Therefore, we are currently conducting complementary assays with purified recombinant Msp1 and Msp2 proteins in dedicated model systems to further delineate the details of their probiotic signalling effects in epithelial cell