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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.

SUBMITTER: Nagy TA

PROVIDER: S-EPMC7285895 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledHemophagocytes are cells of the monocyte lineage that have engulfed erythrocytes and leukocytes. Hemophagocytes frequently accumulate in patients with severe acute bacterial infections, such as those caused by Salmonella enterica, Brucella abortus, and Mycobacterium tuberculosis. The relationship between hemophagocytosis and infection is not well understood. In the murine liver, S. enterica serovar Typhimurium resides within hemophagocytic macrophages containing leukocytes. Here we show that S. Typhimurium also resides within hemophagocytes containing erythrocytes. In cell culture, S. Typhimurium benefits from residence within hemophagocytes by accessing iron, but why macrophages hemophagocytose is unknown. We show that treatment of macrophages with a cocktail of the proinflammatory cytokine interferon gamma (IFN-?) and lipopolysaccharide (LPS) stimulates engulfment of nonsenescent erythrocytes. Exposure of resting or IFN-?-treated macrophages to live, but not to heat-killed, S. Typhimurium cells also stimulates erythrocyte engulfment. Single-cell analyses show that S. Typhimurium-infected macrophages are more likely to erythrophagocytose and that infected macrophages engulf more erythrocytes than uninfected macrophages within the same culture well. In addition, macrophages containing erythrocytes harbor more bacteria. However, S. Typhimurium does not promote macrophage engulfment of polystyrene beads, suggesting a role for a ligand on the target cell. Finally, neither of the two S. Typhimurium type 3 secretion systems, T3SS1 or T3SS2, is fully required for hemophagocytosis. These results indicate that infection of macrophages with live S. Typhimurium cells stimulates hemophagocytosis.ImportanceMacrophages are white blood cells (leukocytes) that engulf and destroy pathogens. Hemophagocytes, a subset of macrophages, are characteristic of severe acute infection in patients with, for instance, typhoid fever, brucellosis, tuberculosis, and leishmaniasis. Each of these diseases has the potential to become chronic. Hemophagocytes (blood-eating cells) engulf and degrade red and white blood cells for unknown reasons. The bacterial pathogen Salmonella acquires the essential nutrient iron from murine hemophagocytes. We report that Salmonella stimulates macrophages to engulf blood cells, indicating that cells of this bacterium actively promote the formation of a specialized cellular niche in which they can acquire nutrients, evade killing by the host immune system, and potentially transition to chronic infection.

Project description:Salmonella enterica are natural bacterial pathogens of humans and animals that cause systemic infection or gastroenteritis. During systemic infection, Salmonella generally reside within professional phagocytes, typically macrophages, whereas gastroenteritis is caused by infection of epithelial cells. We are only beginning to understand which host pathways contribute to Salmonella survival in particular cell types. We therefore sought to identify compounds that perturb Salmonella-host interactions using a chemical genetics approach. We found one small molecule, D61, that reduces Salmonella load in cell-line and primary macrophages but has no effect on Salmonella growth in epithelial cells or rich medium. We determined that in macrophages D61 induces LC3II, a marker of the autophagy pathway, and promotes aggregation of LC3II near Salmonella We found that D61 antibacterial activity depends on the VPS34 complex and on ATG5. D61 also reduced Salmonella load in the spleens and livers of infected mice. Lastly, we demonstrate that D61 antibacterial activity in macrophages is synergistic with the antibiotic chloramphenicol, but that this synergy is largely independent of the known autophagy-stimulating activity of chloramphenicol. Thus, a small molecule has anti-bacterial activity specifically in macrophages and mice based on the promotion of bacterial degradation by autophagy.Importance Autophagy is a conserved cellular response to metabolic stress and to invading pathogens. For many pathogens, including Salmonella, autophagy can play a detrimental or beneficial role during infection depending on the cellular context. We combined chemical genetics with single cell analyses and murine infection to dissect host-pathogen interactions. We identified a small molecule that reduces bacterial load in macrophages by increasing autophagic flux. This compound also reduces bacterial colonization of tissues in infected mice. These observations demonstrate the potential therapeutic utility of stimulating autophagy in cells and animals to curb infection.

Project description:The human enteric bacterial pathogen Salmonella enterica causes approximately 1.35 million cases of food borne illnesses annually in the United States. Of these salmonellosis cases, almost half are derived from the consumption of fresh, raw produce. Although epiphytic S. enterica populations naturally decline in the phyllosphere, a subset of phytophagous insects have recently been identified as biological multipliers, consequently facilitating the growth of bacterial populations. We investigated whether tomato leaves with macroscopic feeding damage, caused by infestation of adult Western flower thrips (Frankliniella occidentalis), support higher S. enterica populations. To explore this hypothesis, we assessed S. enterica populations in response to thrips feeding by varying insect density, plant age, and the gender of the insect. As a reference control, direct leaf damage analogous to thrips feeding was also evaluated using directed, hydraulic pressure. In a supplementary set series of experiments, groups of F. occidentalis infested tomato plants were later inoculated with S. enterica to determine how prior insect infestation might influence bacterial survival and persistence. Following an infestation period, leaves visibly damaged by adult F. occidentalis supported significantly higher S. enterica populations and resulted in greater amounts of electrolyte leakage (measured as electrical conductivity) than leaves lacking visible feeding damage. Plant age did not significantly influence S. enterica populations or estimates of electrolyte leakage, independent of initial infestation. Additionally, the gender of the insect did not uniquely influence S. enterica population dynamics. Finally, applications of aggressive water bombardment resulted in more electrolyte leakage than leaves damaged by F. occidentalis, yet supported comparable S. enterica populations. Together, this study indicates that F. occidentalis feeding is one of the many potential biological mechanisms creating a more habitable environment for S. enterica.

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Clofazimine Reduces the Survival of Salmonella enterica in Macrophages and Mice.

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