Pattern analysis of conditional essentiality (PACE)-based heuristic identification of an in vivo colonization determinant as a novel target for the construction of a live attenuated vaccine against Edwardsiella piscicida.

Edwardsiella piscicida is the aetiological agent of fish edwardsiellosis, causing huge economic losses in aquaculture industries. The use of a live attenuated vaccine (LAV) will be an effective strategy to control the disease in farmed fish. Thus, methods facilitating exploration of targets used for construction of an LAV will be of great significance. Previously, we devised an algorithm termed pattern analysis of conditional essentiality (PACE) to perform genome-wide analysis of the temporal dynamic behaviour of E. piscicida mutants colonizing turbot. Here, we correlated the conditional essentiality patterns of the PACE-derived colonization determinants with that of the aroC gene encoding chorismate synthase, the established target for LAV construction in E. piscicida, and identified ETAE_0023 as a novel valuable LAV target. ETAE_0023 encodes an uncharacterized DcrB family protein. Deletion of ETAE_0023 dramatically impaired E. piscicida invasion capability in ZF4 cells as well as colonization in fish and resulted in in vivo clearance at ∼30 days post-infection. ΔETAE_0023 showed an ∼2500-fold higher 50% lethal dose (LD) than that of the wild type strain. Vaccination with ΔETAE_0023 by intraperitoneal (i.p.) injection upregulated expression of immune factors, i.e., IL-1β, IgM, MHC-I and MHC-II, and produced significantly high levels of E. piscicida-specific IgM as well as serum bactericidal capacities in turbot. Moreover, a single i.p. inoculation with ΔETAE_0023 generated significant protection comparable to the established WED LAV strain in turbot against challenge with the wild type strain after 5 weeks of vaccination. Taken together, we demonstrated a PACE-based method for heuristic identification of targets for LAV construction and presented ΔETAE_0023 as a new LAV candidate against edwardsiellosis.