Clofazimine Reduces the Survival of Salmonella enterica in Macrophages and Mice

Abstract

Drug resistant pathogens are on the rise, and new treatments are needed for bacterial infections. Efforts toward antimicrobial discovery typically identify compounds that prevent bacterial growth in microbiological media. However, the microenvironments to which pathogens are exposed during infection differ from rich media and alter the biology of the pathogen. We and others have therefore developed screening platforms that identify compounds that disrupt pathogen growth within cultured mammalian cells. Our platform focuses on Gram-negative bacterial pathogens, which are of particular clinical concern. We screened a panel of 707 drugs to identify those with efficacy against Salmonella enterica Typhimurium growth within macrophages. One of the drugs identified, clofazimine (CFZ), is an antibiotic used to treat mycobacterial infections that is not recognized for potency against Gram-negative bacteria. We demonstrated that in macrophages CFZ enabled the killing of S. Typhimurium at single digit micromolar concentrations, and in mice, CFZ reduced tissue colonization. We confirmed that CFZ does not inhibit the growth of S. Typhimurium and E. coli in standard microbiological media. However, CFZ prevents bacterial replication under conditions consistent with the microenvironment of macrophage phagosomes, in which S. Typhimurium resides during infection: low pH, low magnesium and phosphate, and the presence of certain cationic antimicrobial peptides. These observations suggest that in macrophages and mice the efficacy of CFZ against S. Typhimurium is facilitated by multiple aspects of soluble innate immunity. Thus, systematic screens of existing drugs for infection-based potency are likely to identify unexpected opportunities for repurposing drugs to treat difficult pathogens.

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

“…We therefore established whether media that mimic the macrophage phagosome, nutrient limitation and low magnesium [ 17 , 18 ], sensitize bacteria to JD1. Bacteria were grown overnight in nutrient poor M9 medium with 1 μM magnesium (M9-lowMg), which de-stabilizes the LPS polysaccharide layer [ 25 ]. Bacteria were diluted into the same medium.…”

“…Polymyxin B (PMB) is a cAMP that at 0.5 μg/mL permeabilizes the S . Typhimurium outer, but not the inner membrane [ 25 ]. Polymyxin B nonapeptide (PMBN) is an attenuated derivative of PMB lacking the fatty acid tail.…”

“…We used a fluorescence-based cell culture assay to identify compounds with non-traditional antimicrobial capabilities from chemical and drug libraries. This approach uncovered inhibitors of bacterial efflux pumps, a stimulator of macrophage autophagy and an antibiotic used against Mycobacterium species that was unexpectedly potent against Gram-negative bacteria during infection [21,22,25]. Within the current work, we examined the mechanism of action of a compound identified by SAFIRE, JD1, that strongly reduces S. Typhimurium replication in macrophages and disrupts the bacterial cell membrane upon breaching the outer membrane barrier.…”

“…During infection of macrophages, bacterial outer membrane permeability is likely compromised by cationic antimicrobial peptides (cAMPs), which are ubiquitous in body fluids and are also present in phagosomes [17,18,35]. Polymyxin B (PMB) is a cAMP that at 0.5 μg/mL permeabilizes the S. Typhimurium outer, but not the inner membrane [25]. Polymyxin B nonapeptide (PMBN) is an attenuated derivative of PMB lacking the fatty acid tail.…”

“…Clofazimine is a lipophilic (LogP 7.7) clinical antibiotic that damages membranes [95][96][97][98] and is used to treat infections with Mycobacterium leprae and tuberculosis [99][100][101][102]. Clofazimine is not active against Gram-negative bacteria in standard microbiological media [103][104][105] but is has efficacy in macrophages and mice against S. Typhimurium [25]. While JD1 is likely too lipophilic (LogP 12.5) to become a lead compound for antibiotic discovery, our observations demonstrate the feasibility of developing lipophilic antibiotics that target Gram-negative bacterial cell membranes and may ultimately have therapeutic value.…”