Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66

Anti-infectives include molecules that target microbes in the context of infection but lack antimicrobial activity under conventional growth conditions. We previously described D66, a small molecule that kills the Gram-negative pathogen <i>Salmonella enterica</i> serovar Typhimurium (<i>S.</i> Typhimurium) within cultured macrophages and murine tissues, with low host toxicity. While D66 fails to inhibit bacterial growth in standard media, the compound is bacteriostatic and disrupts the cell membrane voltage gradient without lysis under growth conditions that permeabilize the outer membrane or reduce efflux pump activity. To gain insights into specific bacterial targets of D66, we pursued two genetic approaches. Selection for resistance to D66 revealed spontaneous point mutations that mapped within the <i>gmhB</i> gene, which encodes a protein involved in the biosynthesis of the lipopolysaccharide core molecule. <i>E. coli</i> and <i>S.</i> Typhimurium <i>gmhB</i> mutants exhibited increased resistance to antibiotics, indicating a more robust barrier to entry. Conversely, <i>S.</i> Typhimurium transposon insertions in genes involved in outer membrane permeability or efflux pump activity reduced fitness in the presence of D66. Together, these observations underscore the significance of the bacterial cell envelope in safeguarding Gram-negative bacteria from small molecules.