Disruption of Spore Coat Integrity in Bacillus subtilis Enhances Macrophage Immune Activation

Probiotics play a pivotal role in animal production by promoting growth, enhancing gut health, and modulating immune responses. Bacillus subtilis, a widely utilized probiotic, forms robust spores that exhibit exceptional resistance, making it ideal for feed applications. While B. subtilis spores have been shown to stimulate innate immune signaling, the specific contributions of spore coat proteins to immune modulation remain poorly characterized. In this study, we investigated the immunostimulatory effects of spores deficient in six key coat proteins: SpoIVA, SafA, CotE, CotX, CotZ, and CgeA. These proteins are essential for the assembly and structural integrity of the spore's multi-layered coat, and are involved in recruiting other coat components. Deletion of these genes result in defects in spore coat architecture, potentially altering spore-host interactions. Using porcine alveolar macrophages (MΦ3D4/2), we assessed cytokine responses to each mutant strain. Our findings demonstrate that the absence of specific structural proteins significantly impacts immune activation, particularly through Toll-like receptor pathways. This work provides novel insights into the immunomodulatory functions of spore coat proteins and lays the foundation for the rational design of next-generation B. subtilis-based probiotics with enhanced immunological properties for agricultural applications.