The Dual Role of RsiP in Regulating Virulence and Host Adaptation in Bacillus anthracis

<i>Bacillus anthracis</i> displays susceptibility to penicillin despite harboring a β-lactamase gene, a phenotype governed by the anti-sigma factor RsiP. While RsiP represses σ^P-dependent β-lactamase expression, its broader roles in physiology and virulence remain unclear. This study aimed to define the global regulatory functions of RsiP beyond antibiotic resistance. Deletion of <i>rsiP</i> significantly upregulated the <i>nprR</i> gene, which is an important quorum-sensing (QS) system regulator and enhanced protease secretion. The Δ<i>rsiP</i> mutant caused higher mortality in cellular and <i>Galleria mellonella</i> models and triggered elevated inflammatory cytokines (IL-6, IL-1β, TNF-α, MIP-2) in macrophages models. Surprisingly, in DBA/2 mice models, Δ<i>rsiP</i> was attenuated, with increased host survival and reduced bacterial loads. Competitive indices (CI) confirmed fitness defects in mice (spleen CI = 0.39; liver CI = 0.42). These defects were not due to altered oxidative stress tolerance but were attributed to impaired macrophage internalization of Δ<i>rsiP</i> spores, reducing early colonization. Our findings indicate that RsiP not only modulates β-lactam resistance but also influences extracellular protease activity and host adaptation.