Disruption of the Francisella noatunensis subsp. orientalis pdpA Gene Results in Virulence Attenuation and Protection in Zebrafish

Several Francisella spp., including Franciselia noatunensis, are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella pathogenicity island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. pdpA is a member of the pathogenicity-determining protein genes carried by the FPI that are implicated in the ability of the mammalian pathogen Francisella tularensis to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis. Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (the F. noatunensis subsp. orientalis Delta pdpA1 and Delta pdpA2 strains). Both mutants were attenuated (P < 0.0001) in zebrafish challenges and displayed impaired intracellular replication (P < 0.05) and cytotoxicity (P < 0.05), all of which could be restored to wild-type (WT) levels by complementation for the Delta pdpA1 mutant. Importantly, differences were found for bacterial burden and induction of acute-phase and proinflammatory genes for the F. noatunensis subsp. orientalis Delta pdpA1 and Delta pdpA2 mutants compared to the WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with the F. noatunensis subsp. orientalis Delta pdpA1 mutant led to protection (P < 0.012) against an acute 40% lethal dose (LD40) challenge with WT F. noatunensis in the zebrafish model of infection. Taken together, the results from this study further demonstrate physiological similarities within the genus Francisella relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.