From Virulence to Vulnerability: New Insights on Disrupting the d ‐Alanine Pathway and Its Influences on Polymyxin Resistance in Riemerella anatipestifer

Alanine racemase (Alr), a pyridoxal 5'-phosphate-dependent enzyme critical for d-alanine synthesis in bacterial peptidoglycan, represents a promising antimicrobial target. Riemerella anatipestifer is a notable bacterial pathogen, mainly affecting waterfowl. Its multidrug resistance and virulence characteristics pose significant challenges for treatment and control measures. Previous joint multi-omics analyses indicated that polymyxin affects only the alanine metabolic pathway in this R. anatipestifer. To explore the physiological function of Alr in R. anatipestifer, we constructed an Alr mutant strain of R. anatipestifer and evaluated its phenotypic traits and interspecies competitiveness in comparison with the wild-type strain. We discovered that Alr deletion was lethal to R. anatipestifer. A minimal supplement of d-Ala (50 μg/mL) was necessary for the optimal growth of the Alr mutant. The depletion of d-alanine in the growth medium led to cell lysis and cell wall perforation in the Alr mutant strain. In vitro assays for invasion and survival as well as in vivo virulence assays involving the Alr mutant exhibited attenuated phenotypes. Overall, we have demonstrated that Alr functions as a unique alanine racemase in R. anatipestifer. d-cycloserine (DCS) fails to inhibit Alr significantly, reduces bacterial viability, and confirms that Alr alone was capable of rescuing d-alanine auxotrophy in R. anatipestifer. However, the exogenous addition of d-alanine was unable to restore its polymyxin resistance phenotype and virulence, suggesting that the Alr gene exhibits activity beyond just alanine racemase functions. These findings identify Alr as a new target for polymyxin antibiotics and indicate that its role in virulence may serve as a potential therapeutic approach.