Edwardsiella ictaluri evpP is required for colonisation of channel catfish ovary cells and necrosis in anterior kidney macrophages

Abstract

Edwardsiella ictaluri is a Gram-negative facultative anaerobe that can survive inside channel catfish phagocytes. E. ictaluri can orchestrate Type VI Secretion System (T6SS) for survival in catfish macrophages. evpP encodes one of the T6SS translocated effector proteins. However, the role of evpP in E. ictaluri is still unexplored. In this work, we constructed an E. ictaluri evpP mutant (Ei Delta evpP) and assessed its survival under complement and oxidative stress. Persistence of Ei Delta evpP in catfish as well as attachment and invasion in catfish macrophage and ovary cells were determined. Further, virulence of Ei Delta evpP in catfish and apoptosis it caused in macrophages were explored. Ei Delta evpP behaved same as wild type (EiWT) under complement and oxidative stress in complex media, whereas oxidative stress affected mutant's survival significantly in minimal media (p < .05). Persistence of Ei Delta evpP in live catfish and uptake and survival inside peritoneal macrophages were similar. The attachment and invasion capabilities of Ei Delta evpP in catfish ovary cells were significantly less than that of EiWT (p < .05). Although Ei Delta evpP showed reduced attenuation in catfish, causing decreased catfish mortality compared with EiWT (44.73% vs. 67.53%), this difference was not significant. The apoptosis assay using anterior kidney macrophages indicated that the number of live macrophages exposed to Ei Delta evpP was significantly higher compared with EiWT exposed macrophages at 24-hr post-treatment (p < .05). However, there were no significant differences in the early and late apoptosis. Remarkably, necrosis in Ei Delta evpP exposed macrophages was significantly less than that of EiWT exposed macrophages at 24 hr (p < .05). Our results demonstrated that evpP is required for colonisation of catfish ovary cells and increased apoptosis and necrosis in anterior kidney macrophages.