TnaA And Eha Regulates TnaA Affects The Survival In Macrophages And Virulence Of Edwardsiella Tarda

Edwardsiella tarda?E.tarda?is a gram-negative rod bacterium of a member of the Enterobacteriaceae.The bacterium has a wide range of hosts and can cause diseases of fish,amphibians,reptiles and mammals.The pathogen can bring out great hazard in aquaculture industry.E.tarda is a facultative intracellular parasite and its hemolysis-related gene?abbreviated as eha?is an important transcriptional regulatory gene.Tryptophanase?tnaA?can catalyze tryptophan to generate indole and the indole has some characteristic of bacterial quorum-sensing signal,which can participate in the regulation of various physiological activities of bacteria.The purpose of this study is investigating to research the mechanism the tnaA and the transcriptional regulator Eha to regulate tnaA to affect the bacterial survival in the macrophages and virulence.Firstly,the differential expression of tnaA gene in ET13 wild strains and eha deletion strains were detected by qRT-PCR.The results showed that Eha affects the transcriptive expression of tnaA.The promoter region of tnaA gene was inserted into plasmid pBAD-lacZ to generate recombinant plasmid pBAD-lacZ-P_tnaA.Electric shock method was used to introduce the recombinant plasmid into ET13 and?eha strains respectively,and then pBAD-lacZ-P_{tna A}-ET13 strain and pBAD-lacZ-P_tnaA-?eha strain were obtained.After the difference of galactosidase activity between the two strains at pH6.3 and 0.10%H₂O₂ was detected,the results showed that eha had a direct regulatory effect on tnaA.Secondly,a suicide plasmid pHM5 was used to construct tnaA deletion strain??tnaA?and tnaA complementary strain?ComptnaA?of ET13.The tnaA was over-expressed in?eha strain to produce?eha-tnaA strain.The concentrations of indole produced by wild type ET13,ComptnaA,?eha-tnaA and?tnaA strains during their growth process were determined by dimethylaminobenzaldehyde spectrophotometry.The results showed that tnaA deletion affected ET13 to product of the indole when the bacteria grew during 3-12h.When the above four strains were respectively infected with RAW264.7 macrophagess,we compared their survival ability in macrophages.The results showed that tnaA could promote the survival and reproduction of ET13 within macrophages,and?eha-tnaA strain could partially restore the survival ability of?eha strain in the cells.In order to further explore tnaA and Eha regulate tnaA to affect intracellular survival mechanism of E.tarda,the intracellular survival environments of macrophages were simulated in vitro to detect the survival rate of these bacteria within acidic conditions and their sensitivity to hydrogen peroxide?H₂O₂?.The results showed that tnaA deletion could affect the survival rate of E.tarda in acidic environment,and increase the sensitivity of the bacteria to H₂O₂.Furthermore,the results of Bafilomycin A1showed that the acidification of macrophages could inhibit the proliferation of the above strains within macrophages,and tnaA gene could regulate the bacterial resistance against macrophage acidification.After the differences of reactive oxygen species?ROS?produced by macrophages strains infected by above bacterial strains were detected,the results showed that tnaA deletion could affect the resistance of E.tarda to the ROS production by macrophages.Western blot was used to detect the macrophage autophagy infected by E.tarda,and it was found that these strains could induce autophagy of macrophages,while tnaA had no effect the bacteria to resist autophagy of macrophages.Lastly,when mice were infected by E.tarda with intraperitoneal injection,the results of the survival rates of ET13 wild type and?tnaA strains in mice showed that?tnaA can invade the liver,spleen,kidney and lungs of mice,but the numbers of?tnaA in these organs were significantly lower than ET13 wild type.After the median lethal dose(LD₅₀)of ET13 wild type in mice and zebrafish were compared with the one of?tnaA,the results showed that the LD₅₀of?tnaA in mice was 3.62 times lower than the one of the wild strains,and the LD₅₀ of?tnaA in the zebrafish was 14.36 times lower than the one of the wild type.At the same time,LD₅₀ from zebrafish infected by the wild type was about 1.6×10³ times lower than the one from mice.It is indicated that only a lower dose of bacteria was required to produce LD₅₀ for the zebrafish.Consequently,the zebrafish was more sensitive as host infected by E.tarda.The above results showed that tnaA or tnaA regulated by Eha was in favour of E.tarda to resist the environment within macrophages that killed bacteria such as ROS and high acidification,and in favour of the virulence of bacteria.This study provided an important clue for the pathogenic mechanism and network regulatory system of E.tarda.