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

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Abstract

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-2ΔgerAA 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-2ΔgerAA spores to L-alanine was significantly reduced. Compared with the S-2ΔgerAA 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.

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Funding

This research was funded by the National Natural Science Foundation of China (No. 32072767 and 31672455), the Fundamental Research Funds for the Central Universities, South-Central Minzu University (Grant Number: CZY21001, CZP22003), and University-Enterprise R&D Cooperation Projects (HZY21089 and HZY23010).

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Authors and Affiliations

  1. College of Life Science, South-Central Minzu University, No. 182, Minyuan Road, Wuhan City, 430074, China

    Shuang Lu, Xianying Liao, Wei Lu, Li Zhang, Kai Na & Xiaohua Guo

  2. CABIO Bioengineering (Wuhan) Co., Ltd, Wuhan City, 430074, China

    Xiangyu Li

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  1. Shuang Lu
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  2. Xianying Liao
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  3. Wei Lu
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  4. Li Zhang
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Contributions

XG designed the experiments. SL, XL, WL, and KN performed the experiments. SL, LZ, and XL analyzed the experimental data. SL and XG wrote this paper. All authors read and approved the final manuscript.

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Correspondence to Xiaohua Guo.

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Lu, S., Liao, X., Lu, W. et al. L-Alanine promotes anti-infectious properties of Bacillus subtilis S-2 spores via the germination receptor gerAA. Probiotics & Antimicro. Prot. (2023). https://doi.org/10.1007/s12602-023-10121-2

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