Deciphering novel enzymatic and non-enzymatic lysine lactylation in <i>Salmonella</i>

Lysine lactylation, a novel post-translational modification, is involved in multiple cellular processes. The role of lactylation remains largely unknown, especially in bacteria. Here, we identified 1090 lactylation sites on 469 proteins by mass spectrometry in <i>Salmonella</i> Typhimurium. Many proteins involved in metabolic processes, protein translation, and other biological functions are lactylated, with lactylation levels varying according to the growth phase or lactate supplementation. Lactylation is regulated by glycolysis, and inhibition of L-lactate utilization can enhance lactylation levels. In addition to the known lactylase in <i>E. coli</i>, the acetyltransferase YfiQ can also catalyse lactylation. More importantly, L-lactyl coenzyme A (L-La-CoA) and <i>S,D</i>-lactoylglutathione (LGSH) can directly donate lactyl groups to target proteins for chemical lactylation. Lactylation is involved in <i>Salmonella</i> invasion of eukaryotic cells, suggesting that lactylation is crucial for bacterial virulence. Collectively, we have comprehensively investigated protein lactylome and the regulatory mechanisms of lactylation in <i>Salmonella</i>, providing valuable insights into studying lactylation function across diverse bacterial species.