A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host. Author summaryAs bacterial resistance to existing antibiotics increases and expands, scientists are exploring new approaches to combatting bacterial infections. There is a special need for antibiotics against Gram-negative bacteria, which are difficult to treat and can cause devastating infections. One underexplored possible antimicrobial target for Gram-negative bacteria is the bacterial cell membrane, a structure essential for viability. Here we describe a small molecule that inhibits a Gram-negative bacterial infection in host cells and mice. This molecule disturbs, but does not permeabilize, bacterial cell membranes under growth conditions that mimic infection. These data indicate that subtle bacterial membrane damage caused by a small molecule augments host innate immune defenses and enables bacterial killing, suggesting a new approach to antibacterial therapy.