Roles of EvpP in Edwardsiella piscicida-Macrophage Interactions.

Edwardsiella piscicida is found to be an important facultative intracellular pathogen with a broad host range. These organisms can replicate and survive within host macrophages to escape from the subversion of the immune defense. E. piscicida-macrophage interaction is very important in determining the outcome of edwardsiellasis. As an effector protein of E. piscicida T6SS, EvpP has been determined to be a very important virulence factor for E. piscicida, although its precise role in E. piscicida-macrophage interactions is not yet clear. In this study, the roles of EvpP in E. piscicida-macrophage interactions were characterized. Here, we constructed the deletion mutants of evpP (ΔevpP) and complementation (ΔevpP-C) by the allelic exchange method. Compared to wild type strain (WT), ΔevpP was found to be attenuated for growth within macrophages. In line with this observation, we found its survival capacity was lower than WT under oxidative and acid stress in vitro, which simulate conditions encountered in host macrophages. Attenuation of ΔevpP also correlated with enhanced activation of macrophages, as reflected by augmented NO production in ΔevpP-treated macrophages. Moreover, compared to WT, ΔevpP induced markedly increased apoptosis of macrophages, characterized by increased Annexin V binding and the activation of cleaved caspase-3. These findings provided strong evidence that EvpP is involved in the process of E. piscicida-macrophage interactions and is required for its survival and replication in macrophages. Thus, we propose that EvpP might be an important factor that controlling the fate of E. piscicida inside macrophages. To further exploring the underlying mechanism of EvpP action, the cDNA library was constructed from E. piscicida-infected macrophages and a yeast two-hybrid screen was performed to search for cellular proteins interacting with EvpP. Ribosomal protein S5 (RPS5) was identified as a target of EvpP. Furthermore, the interaction was validated with co-immunoprecipitation assay. This result implies that the observed effect of EvpP on macrophages might be related to RPS5-mediated regulation, contributing to a better understanding of the mechanisms of EvpP involved in E. piscicida-macrophage interactions.

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

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 (EiΔevpP) and assessed its survival under complement and oxidative stress. Persistence of EiΔevpP in catfish as well as attachment and invasion in catfish macrophage and ovary cells were determined. Further, virulence of EiΔevpP in catfish and apoptosis it caused in macrophages were explored. EiΔevpP 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 EiΔevpP in live catfish and uptake and survival inside peritoneal macrophages were similar. The attachment and invasion capabilities of EiΔevpP in catfish ovary cells were significantly less than that of EiWT (p < .05). Although EiΔevpP 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 EiΔevpP 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 EiΔevpP 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.

A new target for the old regulator: H-NS suppress T6SS secretory protein EvpP, the major virulence factor in the fish pathogen Edwardsiella tarda.

UNLABELLED: The evpP gene in fish pathogen Edwardsiella tarda, coding the T6SS secretory protein EvpP and carrying an evpA-evpO independent promoter region, was crucial for host cell invasion. The transcription of evpP was positively regulated by either the two-component system EsrA-EsrB or iron concentration, and its overexpression was known to enhance the invasion ability in our previous study. This work demonstrated that the H-NS protein, a pleiotropic regulator of gene expression, was a new transcriptional modulator of evpP gene. The results showed that in vivo the transcriptional level of evpP was downregulated by H-NS and in vitro this global regulator interacted directly with evpP promoter region. Moreover, DNase I footprinting experiments mapping the interaction regions of H-NS and evpP revealed that this global regulator bound to evpP promoter and neighbouring areas at multiple sites. We provided a new insight into evpP regulation network and demonstrated the repression of H-NS to the transcription of evpP gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Recently, the devastating fish disease edwardsiellosis caused by Edwardsiella tarda has been widely concerned. The xenogeneic silencing of the classic regulator H-NS to the T6SS secretory protein EvpP, which played an important role in the virulence of Edw. tarda, was firstly reported in this study. It raised a better understanding of the virulence regulation of EvpP and provided more information about the complex infection mechanism of this pathogen. Our findings would contribute to the development of live attenuated vaccines against edwardsiellosis thus reducing the economic losses caused by this bacterium.