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

Abstract

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.

Inhibition of Multiple Staphylococcal Growth States by a Small Molecule that Disrupts Membrane Fluidity and Voltage

“…All strains tested were unable to grow in the presence of high concentrations of the compound, with values of half minimum inhibitory concentrations (MIC 50 ) ranging from 43 - 93 µM ( Fig 1 A, B ). In contrast, S. Typhimurium with an intact outer membrane grew normally even at 150 µM of D66, a concentration approaching the limit of solubility (11). Thus, bacteria lacking an outer membrane barrier have increased susceptibility to D66 upon out-growth from overnight cultures.…”

“…D66 inhibits S. Typhimurium survival in macrophages and curbs systemic infection in mice. However, D66 has been challenging to study in broth because it has difficulty passing through the Gram-negative bacterial outer membrane barrier and is expelled by efflux pumps (11). We found that in Gram-positive bacteria, the compound prevented regrowth from stationary phase, suggesting that it can reach a bacterial target(s).…”

“…These works have identified compounds that lack antibacterial activity in standard microbiological media but inhibit bacterial growth under broth conditions that mimic the macrophage phagosome environment. For instance, such compounds prevent S. Typhimurium replication in broth in growth media or altered genetic backgrounds that increase outer membrane permeability and/or inactivate efflux pumps (7,9,(11)(12)(13)(14). Thus, the relative impermeability of the Gram-negative cell envelope in standard media complicates study of the mechanism of action for these compounds.…”

“…The compound is minimally toxic to mammalian cells and mice, prevents the replication of S. Typhimurium in macrophages, and curbs infection in mice. Under broth conditions that permeabilize the outer membrane or reduce efflux pump activity, D66 inhibits the growth of Gram-negative bacteria and disrupts voltage across the inner membrane (11). In Gram-positive bacteria, we found that exposure to the compound inhibited growth, rapidly increased membrane fluidity, and disrupted membrane voltage without permeabilizing cells.…”

Salmonella enterica Infections Are Disrupted by Two Small Molecules That Accumulate within Phagosomes and Differentially Damage Bacterial Inner Membranes

“…This approach identified molecules that are not antibacterials but that work with host innate immunity to kill bacteria and are therefore anti-infectives. These compounds include efflux pump inhibitors, a stimulator of macrophage autophagy, and two compounds that damage bacterial inner membranes and reduce bacterial tissue load in mice ( 13 – 17 ). Here, we describe two additional membrane-active, anti-infective compounds, JAV1 and JAV2, which notably have amine groups that are protonatable near pH 6, suggesting compound sequestration within low-pH vesicles, such as the SCV ( 18 , 19 ).…”