Regulation of the Gene for Alanine Racemase Modulates Amino Acid Metabolism with Consequent Alterations in Cell Wall Properties and Adhesive Capability in spp.

, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, mutants with gene deletions (Δ) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of knockouts on pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the gene. Furthermore, deletion in S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δ strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δ strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the gene that regulates amino acid metabolism in influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of spp.