A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

Abstract

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

An Oral Fluorouracil Prodrug, Capecitabine, Mitigates a Gram-Positive Systemic Infection in Mice

“…Typhimurium in minimal than in a standard rich medium, indicating that screening in nutrient-poor media facilitated the identification of capecitabine. These observations highlight that screens for new antibacterials performed under conditions that mimic infection identify different compounds than screens performed in traditional media ( 15 , 56 – 58 ). They also underscore the value of working with students in a CURE format on screens of biological interest ( 59 , 60 ).…”

Staphylococcal Bacterial Persister Cells, Biofilms, and Intracellular Infection Are Disrupted by JD1, a Membrane-Damaging Small Molecule

“…1A ). In S. Typhimurium, MIC 95 s ranged from 14 to 90 μM, depending on the conditions used to compromise the outer membrane ( 30 ). These data indicate that JD1 is similarly effective against Gram-positive bacteria and against Gram-negative bacteria that have sustained outer membrane damage.…”

“…JD1 immediately disrupted the inner membrane and membrane potential of S . Typhimurium but much more slowly reduced respiration and ATP accumulation ( 30 ). To establish whether the compound acts similarly in S. aureus , cells were loaded with the fluorescent probe 3,3′-dipropylthiadicarbocyanine iodide [DiSC 3 (5)], which accumulates within and is quenched by polarized cell membranes.…”

“…8C ), suggesting that nutrient limitation potentiates JD1. Moreover, at concentrations that are not toxic to host cells ( 30 ), JD1 reduced the survival of intracellular S. aureus USA300 better than vancomycin.…”

“…JD1 (406 g/mol) is a small aromatic molecule that facilitates the killing of the Gram-negative pathogen Salmonella enterica serovar Typhimurium in mammalian macrophages and epithelial cells and reduces bacterial load in mice. In broth culture, JD1 damages the inner membrane if conditions permeabilize the outer membrane and/or the RND family AcrAB-TolC efflux pump is inactivated ( 30 ). We therefore examined the effect of JD1 on Gram-positive bacteria, which lack an outer membrane and may facilitate JD1 access to the cell membrane in persister cells and biofilms.…”