Metabolic and genomic adaptations of Salmonella Typhimurium grown on itaconate
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Salmonella enterica ser. Typhimurium (STm) is an enteric pathogen that causes almost 100 million cases of salmonellosis a year. A hallmark of STm is their ability to survive and replicate in the macrophages that phagocytose them. Inside the Salmonella -containing vesicles (SCV) STm is exposed to nutrient limitation and antimicrobial molecules, including itaconate, a dicarboxylate produced at millimolar concentrations by activated macrophages. STm has an itaconate degradation pathway that allows it to detoxify itaconate and convert it into pyruvate and acetyl-CoA. In the SCV, STm undergoes extensive metabolic reprogramming in response to the nutrient limitation and high stress environment, and strict catabolite control is employed at various stages of infection, depending on carbon source availability. The extent to which itaconate is used as a carbon source by STm is not currently well understood. Here, we explore the ability of STm to grow in media containing itaconate as the sole carbon source, identify the genes of the itaconate response operon necessary for itaconate degradation, and confirm their importance during infection of macrophages. Growth on itaconate was substantially slower than in carbon-rich or acetate media, with multiple subcultures allowing for increased growth rates. Genomic analysis suggests that mutations in the RpoS-stress response and increased expression of the DctA transporter improve growth on itaconate. Using metabolomics, it was revealed that growth on itaconate resulted in substantially reduced levels of gluconeogenesis compared to growth on acetate and increased glutathione disulfide levels, indicating a high level of oxidative stress. Overall, our results point towards the STm itaconate degradation pathway having a more important role in detoxifying itaconate, rather than using it as a carbon source inside the macrophage. Additionally, this study establishes media conditions that would enable the high throughput discovery of potential inhibitors of itaconate degradation.
Author Summary
Salmonella enterica serovar Typhimurium (STm) is commonly linked to foodborne illness, but it is also known to cause severe systemic infections. Due to this and the prevalence of antibiotic resistance, the World Health Organization has identified STm as a high-priority pathogen. STm resilience during infection is due in part to its ability to thrive in a wide range of environments, including the hostile conditions inside macrophages—immune cells designed to kill invading bacteria. STm survives in part by degrading itaconate, an antimicrobial molecule produced by macrophages. While the importance of this degradation pathway for STm intracellular survival is known, the broader benefits of itaconate metabolism remain unclear. To explore this, we cultured STm in a medium using itaconate as the only carbon source. Although initial growth was slow, STm adapted over time through mutations that enhanced itaconate import and catabolism. We identified the genes required for growth on itaconate and used metabolomics to reveal that itaconate causes widespread metabolic perturbations and high oxidative stress. Our findings support itaconate degradation as a promising antimicrobial target and define a medium suitable for screening potential inhibitors.
