L-Alanine promotes anti-infectious properties of Bacillus subtilis S-2 spores via the germination receptor gerAA.

Bacillus species, which have two cell-type forms (vegetative cells and spores), demonstrate a variety of probiotic functions in animal feed additives and human nutrition. We previously found that the probiotic effect of Bacillus subtilis S-2 spores with high germination response to L-alanine was specifically enhanced by the L-alanine pretreatment. The germination response of Bacillus is highly associated with the germination receptors of spores. However, how L-alanine-induced germination of spores exerts anti-infectious effect in epithelial cells remains unclear. In this study, we constructed the mutant strain of B. subtilis S-2 with germination receptor gerAA knockout to further explore the role of spore germination in resisting pathogen infection to cells. The differential probiotic effects of B. subtilis S-2 and S-2 spores pretreated with L-alanine were evaluated in intestinal porcine epithelial cells (IPEC-J2) or Caco2 cells infected with enterotoxigenic Escherichia coli (ETEC) or following IL-1β stimulation. The results showed that the germination response of the S-2 spores to L-alanine was significantly reduced. Compared with the S-2 spores, the L-alanine-induced germination of B. subtilis S-2 spores significantly increased the activity of anti-adhesion of ETEC to IPEC-J2 cells and reduced the expression of inflammatory factors and cell receptors. L-alanine induction also significantly promoted the expression of autophagy-related proteins in the B. subtilis S-2 spores. These findings demonstrate that the gerAA germination receptor is essential for the probiotic function of Bacillus spores and that L-alanine treatment promotes the anti-infectious properties of the germinated spores in porcine intestinal epithelial IPEC-J2 cells. The result suggests the importance of germination receptor gerAA in helping spore germination and enhancing anti-infectious activity. The findings in the study benefit to screening of potential Bacillus probiotics and increasing probiotic efficacy induced by L-alanine as an adjuvant.