Deficiency in the msbB Gene Reduced the Salmonella Typhimurium Virulence Through Mechanisms Beyond LPS Modification

The <i>Salmonella enterica</i> serovar Typhimurium (ST) mutant lacking the <i>msbB</i> gene (Δ<i>msbB</i>) has been widely studied as a candidate for attenuated bacterial vectors in therapeutic applications. Deletion of <i>msbB</i> results in LPS with under-acylated lipid A, which lowers endotoxicity while maintaining structural integrity. This attenuation has traditionally been attributed to reduced TLR4 activation due to weaker interaction between the modified lipid A and TLR4. In our study, we confirmed that Δ<i>msbB</i> ST was less lethal than wild-type (WT) ST in a mouse sepsis model. However, this difference persisted even in TLR4- and caspase-11-deficient mice, suggesting that LPS signaling is not the primary determinant of virulence. In vitro, bone marrow-derived macrophages (BMDMs) from TLR4- or caspase-11-deficient mice showed only modest reductions in ST-induced cell death and cytokine production. Importantly, Δ<i>msbB</i> ST behaved similarly to WT ST in these assays, further indicating that LPS-mediated signaling is not central to the observed attenuation. Our previous studies showed that ST-induced mortality in mice is primarily mediated through NLRC4 activation. Using qPCR and immunoblotting, we found that expression of NLRC4 activators was diminished in the Δ<i>msbB</i> strain. Additionally, the mutant exhibited increased outer membrane permeability-likely contributing to its heightened antibiotic sensitivity-and reduced motility due to lower flagellin protein levels. In summary, the attenuation of virulence observed in the Δ<i>msbB</i> strain is not directly due to altered LPS-TLR4 interactions, but rather an indirect effect of diminished expression of virulence factors that activate the NLRC4 inflammasome.