Disruption of the Francisella noatunensis orientalis pdpA gene results in virulence attenuation and protection in zebrafish

Authors: HANSEN, JOHN - Western Fisheries Research Center; RAY, KARINA - Western Fisheries Research Center; CHEN, PATRICK - Western Fisheries Research Center; YUN, SUSAN - University Of California, Davis; ELLIOTT, DIANE - Western Fisheries Research Center; CONWAY, CARLA - Western Fisheries Research Center; CALCUTT, MICHAEL - University Of Missouri; PURCELL, MAUREEN - Western Fisheries Research Center; Welch, Timothy - Tim; SOTO, ESTEBAN - University Of California, Davis

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/10/2021
Publication Date: 10/15/2021
Citation: Hansen, J.D., Ray, K., Chen, P., Yun, S., Elliott, D., Conway, C., Calcutt, M., Purcell, M., Welch, T.J., Soto, E. 2021. Disruption of the Francisella noatunensis orientalis pdpA gene results in virulence attenuation and protection in zebrafish. Infection and Immunity. 89(11):220-21. https://doi.org/10.1128/.
DOI: https://doi.org/10.1128/IAI.00220-21

Interpretive Summary: Infectious diseases are the most significant impediment to efficient aquatic animal production causing both economic loss and animal welfare concerns. To develop improved strategies for disease control, a better understanding of the host-pathogen interactions leading to the disease state are needed. Several Francisella species are important emerging pathogens of farmed tilapia and cod yet little is known regarding the factors that allow these bacteria to be pathogenic in fish. In this paper we show that the PdpA protein of Francisella noatunensis (Fn) is required for this pathogen’s virulence against zebrafish. We demonstrated that a defined mutant lacking the PdpA protein was unable to cause disease, lacked the ability to grow inside cells, and lacked cytotoxic activity. The mutant also provided protection against infection when applied as a live vaccine. This finding broadens our general understanding of the factors bacterial pathogens of fish need to cause disease and may be useful in the design of safe vaccines.

Technical Abstract: Several Francisella spp. including F. 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 encoded by the FPI that are implicated in the ability of the mammalian pathogen, F. 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. Since polarity can be an issue in gene dense regions, we generated two different marker-based mutants in opposing polarity (Fno'pdpA1 and 'pdpA2). 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. Importantly, differences were found for bacterial burden and induction of acute phase and pro-inflammatory genes for Fno-'pdpA1 and 'pdpA2 compared to WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with Fno-'pdpA1 led to protection (p<0.012) against an acute lethal-dose 40 challenge with WT Fno in the zebrafish model of infection. The results from this study further demonstrate physiological similarities within the genus relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.