Yersinopine is a primary zinc acquisition system for Yersinia pseudotuberculosis

Yersinia pestis is a vector-borne pathogen that evolved from the enteric pathogen Yersinia pseudotuberculosis. Despite different routes of infection, both pathogens need to overcome host-mediated zinc limitation. Y. pestis relies on the transporter ZnuABC and the metallophore yersiniabactin, but surprisingly, disruption of these systems in Y. pseudotuberculosis did not limit growth during zinc limitation, suggesting the presence of additional mechanisms. Here, we identify the opine-type metallophore yersinopine, encoded by the cnt operon, as a primary zinc uptake system in Y. pseudotuberculosis. While a znuA ybtX mutant was not attenuated for growth in metal-limited medium, cnt znuA and cnt ybtX mutants both had significant growth defects. Moreover, a znuA ybtX cnt mutant was completely attenuated for growth, which was rescued by zinc but not iron supplementation. While the cnt operon is present in Y. pestis, growth of analogous cnt mutants was not altered. Comparison of the cnt operons indicated the presence of two frameshift mutations in the predicted yersinopine importer cntQ in Y. pestis, suggesting that it is a pseudogene. Indeed, introduction of the same mutations into cntQ in Y. pseudotuberculosis znuA ybtX phenocopied a znuA ybtX cnt mutant. Finally, yersinopine also contributed to Y. pseudotuberculosis fitness in the presence of calprotectin and during infection in the murine model of yersiniosis. These data establish yersinopine as a primary zinc acquisition system in Y. pseudotuberculosis that is not required for Y. pestis to overcome zinc-mediated nutritional immunity and show divergent evolution in zinc acquisition strategies in these closely related species.IMPORTANCEZinc sequestration by the host is a major component of nutritional immunity that restricts bacterial growth during infection. Here, we identify the opine metallophore yersinopine as a primary zinc acquisition system in Yersinia pseudotuberculosis. Yersinopine promotes fitness during calprotectin-mediated zinc starvation and contributes to virulence during infection. In contrast, yersinopine does not support zinc uptake in Yersinia pestis due to inactivating mutations in the protein required for uptake of zinc-bound yersinopine. These findings establish yersinopine-dependent zinc acquisition as a critical virulence factor in Y. pseudotuberculosis and reveal a previously unrecognized evolutionary divergence in metal acquisition strategies among closely related Yersinia species.