Unveiling the dance of evolution: Pla-mediated cleavage of Ymt modulates the virulence dynamics of .

Unlabelled: has recently evolved into a highly lethal flea-borne pathogen through the pseudogenization of extensive genes and the acquisition of exogenous plasmids. Particularly noteworthy are the newly acquired pPCP1 and pMT1 plasmids, which encode the virulence determinants Pla and murine toxin (Ymt), crucial for subcutaneous infection and survival within flea vector of , respectively. This study reveals that Pla can cleave Ymt at K299 both and expressing Ymt displays enhanced biofilm formation and increased blood survival, indicating significant roles of Pla-mediated Ymt cleavage in these phenotypes. Intriguingly, although both the ancestral form of Pla and the prevalent Pla-I259T variant in modern strains are capable of cleaving Ymt at K299, the cleavage efficiency of Pla-I259T is only half that of the ancestral variant. In subcutaneous infection, mice infected with ΔK299A show significantly prolonged survival compared to those infected with Δ. Similarly, infection with ΔI259T also results in extended survival compared to Δ infection. These data demonstrate that the I259T substitution of Pla mitigates the enhanced virulence of in mice caused by Pla-mediated Ymt cleavage, thereby prolonging the survival period of infected animals and potentially conferring advantages on the transmission of to the next host. These findings deepen our understanding of the intricate interplay between two newly acquired plasmids and shed light on the positive selection of the Pla-I259T mutation, providing new insights into the virulence dynamics and transmission mechanisms of .

Importance: The emergence of as a highly lethal pathogen is driven by extensive gene pseudogenization and acquisition of exogenous plasmids pPCP1 and pMT1. However, the interplay between these two plasmids during evolution remains largely unexplored. Our study reveals intricate interactions between Ymt and Pla, two crucial virulence determinants encoded on these plasmids. Pla-mediated cleavage of Ymt significantly decreases survival in mouse blood and enhances its virulence in mice. The prevalent Pla-I259T variant in modern strains displays reduced Ymt cleavage, thereby extending the survival of infected animals and potentially increasing strain transmissibility. Our findings shed light on the nuanced evolution of , wherein reduced cleavage efficiency is a positive selection force, shaping the pathogen's natural trajectory.