List of strains.

Host-derived short-chain fatty acids (SCFAs) are essential for Salmonella Typhimurium (STM) virulence. Formate, an SCFA found in the ileum, enhances STM invasion, but the role of the intracellular formate pool in STM pathogenesis remains poorly understood. Deletion of the pflB gene, which encodes pyruvate-formate lyase, depletes this intracellular pool, leading to reduced flagellation and increased expression of pathogenicity island-1 genes (hilA and prgH). This response is driven by elevated intracellular pH and membrane damage, triggering a shift from adhesion to invasion. This transition is regulated by the membrane-bound extra cytoplasmic sigma factor RpoE via the CsrA/csrB pathway. Replenishing the intracellular formate pool enabled STM ΔpflB to use formate as a signalling molecule to modulate virulence. Our findings underscore the critical role of intracellular formate in maintaining pH balance and coordinating the regulation of flagellar and SPI-1 genes, emphasizing the need to fine-tune pflB expression across intestinal regions for optimal STM invasion.