Hemolysin Co-regulated Family Proteins Hcp1 and Hcp2 Contribute to Pathogenesis.

is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. strain 93-146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double mutants (ΔΔ, and ΔΔ) were generated and characterized. Catfish peritoneal macrophages were able to kill Δ better than ΔΔΔ, and wild-type (WT). The attachment of Δ and ΔΔ to ovary cells significantly decreased compared to WT whereas the cell invasion rates of these mutants were the same as that of WT. Mutants exposed to normal catfish serum showed serum resistance. The fish challenges demonstrated that Δ and ΔΔ were attenuated completely and provided excellent protection against WT infection in catfish fingerlings. Interestingly, Δ caused higher mortality than that of WT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with Δ and ΔΔ, their attenuation and protection were significantly higher compared to WT and sham groups, respectively. Taken together, our data indicated that () is involved in virulence in catfish while is involved in adhesion to epithelial cells and survival inside catfish macrophages.