Characterization of three predicted zinc exporters inidentifies ZntR-ZntA as a powerful zinc and cadmium efflux system not required for virulence and unveils pathogenic Brucellae heterogeneity in zinc homeostasis.

Abstract

causes non-zoonotic ovine brucellosis of worldwide distribution and is responsible for important economic losses mainly derived from male genital lesions and reproductive fails. Studies about the virulence mechanisms of this rough species (lacking lipopolysaccharide O-chains) are underrepresented when compared to the main zoonoticspecies that are smooth (with O-chains). Zinc intoxication constitutes a defense mechanism of the host against bacterial pathogens, which have developed efflux systems to counterbalance toxicity. In this study, we have characterized three potentialzinc exporters, including the ZntA ortholog previously studied in. Despite an in-frame deletion removing 100 amino acids fromZntA, the protein retained strong zinc efflux properties. Only indirect evidence suggested a higher exporter activity forZntA, which, together with differences in ZntR-mediated regulation ofexpression betweenand, could contribute to explaining why the Δmutant ofis attenuated while that ofis virulent. Additionally,ZntA was revealed as a powerful cadmium exporter contributing to cobalt, copper, and nickel detoxification, properties not previously described for theortholog. Deletion mutants for BOV_0501 and BOV_A1100, also identified as potential zinc exporters and pseudogenes in, behaved as theparental strain in all tests performed. However, their overexpression in the Δmutant allowed the detection of discrete zinc and cobalt efflux activity for BOV_0501 and BOV_A1100, respectively. Nevertheless, considering their low expression levels and the stronger activity of ZntA as a zinc and cobalt exporter, the biological role of BOV_0501 and BOV_A1100 is questionable. Results presented in this study evidence heterogeneity among pathogenic Brucellae regarding zinc export and, considering the virulence ofΔ, suggest that host-mediated zinc intoxication is not a relevant mechanism to controlinfection.