Diaconescu-Grabari, S. | bioRxiv Science

bioRxiv2025

Pantetheinamides that inhibit the growth of intracellular Salmonella Typhimurium

The metabolic adaptability of intracellular pathogens, such as the Gram-negative Salmonella enterica serovar Typhimurium (STm), enables their survival in nutrient-restricted host environments, while also presenting an opportunity for selective antimicrobial targeting. Herein, we report the synthesis and screening of a small pantetheinamide library aimed at inhibiting the proliferation of STm within macrophages. Two lead compounds exhibited selective activity against intracellular STm and reduced bacterial burden in a murine colitis model. Mechanistic studies suggest their primary mode of action to be the inhibition of coenzyme A (CoA) biosynthesis via the PanD-PanZ regulatory complex, which is absent in host cells. Importantly, resistance pressure to our compound was shown to be significantly stronger in nutrient-limited media compared to nutrient-rich media, supporting the targeting of nutrient stress as a strategy to delay resistance. This work highlights the value of inhibiting pathogen-specific metabolic vulnerabilities in combination with host defence mechanisms to develop novel antimicrobials.

microbiology↗

bioRxiv2025

Metabolic and genomic adaptations of Salmonella Typhimurium grown on itaconate

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.

microbiology↗