Functional Analysis Of Two-component Systems EnvZ/OmpR In Aeromonas Hydrophila

Aeromonas hydrophila is a Gram-negative,rod-shaped,opportunistic pathogenic bacterium,which causes motile Aeromonas septicaemia（MAS）in aquaculture industry,leading to great economic losses.A.hydrophila can also cause diarrhea,sepsis and necrotizing fasciitis in humans.Therefore,it is very important to study the pathogenic mechanism of A.hydrophila.Two-component System（TCS）is a major strategy of microbes in controlling their expression profiles in response to changes in the environment.EnvZ/OmpR is one of the classical TCSs.In this study,envZ/ompR double-gene mutant was constructed.Further biological characteristics and pathogenicity ofΔenvZ/ompR were evaluated.The results were as follows:1.Construction ofΔenvZ/ompRAfter homology alignment and protein function prediction,BFW97＿22565/BFW97＿22560 gene of ZYAH72 were named as envZ/ompR.To investigate the function of EnvZ/OmpR in A.hydrophila,ΔenvZ/ompR was successfully constructed via recombinant strategy,using pRE112 as a suicide vector,after positive chloramphenicol screening combined with negative sucrose screening.2.Biological characteristics ofΔenvZ/ompRThe capacity ofΔenvZ/ompR biofilm formation decreased by 19.9%compared with wild-type strain ZYAH72,indicating that TCS EnvZ/OmpR promotes the formation of bacterial biofilm.ΔenvZ/ompR had no defect in swimming assay,and the flagella ofΔenvZ/ompR was found to be intact after transmission electron microscope observation,which indicated that the EnvZ/OmpR did not participate in the regulation of the synthesis and assembly of the flagella of A.hydrophila.The cytotoxicity of theΔenvZ/ompR was evaluated by detecting the release of LDH in the supernatant after bacterial infection CIK cells.The results showed that there was no significant difference between the cytotoxicity ofΔenvZ/ompR and wild-type strain.The growth of A.hydrophila in different salt concentration medium showed no dependence on the EnvZ/OmpR.However,qRT-PCR results showed that EnvZ/OmpR up regulate the transcription of omp F in hypotonic solution and up regulated omp C in hypertonic solution,indicating that the EnvZ/OmpR involved in osmotic stress response in A.hydrophila.The specific mechanism needs further investigation.3.Pathogenicity ofΔenvZ/ompRIn grass carp blood killing assay,after one hour incubation,wild-type strain ZYAH72increased 3.82 times compared with the initial amount,andΔenvZ/ompR was only 90.1%of the original bacteria,indicating that the resistance ofΔenvZ/ompR to blood killing was significantly reduced compared with wild-type strain.Zebra intraperitoneal injection and scratching infection models were used to evaluate the LD₅₀.ΔenvZ/ompR and wild-type strain exhibited no significant difference via i.p.infection route.However,the LD₅₀ ofΔenvZ/ompR increased by 4.80 times via scratching infection,suggesting that EnvZ/OmpR could promote the dissemination of A.hydrophila from skin wound to whole body.Organ bacteria load ofΔenvZ/ompR infection group was significantly lower than that of wild-type strain infection group,supporting that EnvZ/OmpR promoted the systemic invasion ability of bacteria.qRT-PCR results showed that host expression of the proinflammatory cytokines TNF-αand IL-6 caused byΔenvZ/ompR infection were significantly reduced,and the clinical symptoms ofΔenvZ/ompR infected fish were mild,indicating that EnvZ/OmpR was involved in the pathogenic process of A.hydrophila.In summary,this study preliminarily explored the function of TCS EnvZ/OmpR in A.hydrophila,and found that it was involved in the regulation of biofilm formation,in response to osmotic stress and involved in pathogenicity especially in dissemination in host.This study provides a scientific basis for the study of the pathogenic regulatory mechanism of A.hydrophila.