Pathogenicity Analysis Of The TypeⅡ TA System HigBA In Edwardsiella Piscicida And Attenuated Vaccine Construction

Edwardsiella piscicida(formerly included in E.tarda)is one of the leading pathogen threatening worldwide fresh and seawater aquaculture industries and has been considered as a model organism for studying intracellular and systemic infections.Toxin-antitoxin(TA)systems are omnipresent in bacteria and archaea and participate widely,and their main role is to participate in the formation of the persister cells and thereby help the bacteria in stress responses.However,the roles of TA systems are almost completely unknown in aquatic pathogenic bacteria.To probe the function of the E.piscicida type Ⅱ TA system HigBA,by protein blast we identified and characterized HigBA,where HigB is the toxin and Hig A is the antitoxin.Co-transcriptional profiling experiments showed that higB and hig A are expressed in a bicistronic operon.To determine the biological role of HigBA,two markerless higB and higBA in-frame mutant strains,TX01ΔhigB and TX01ΔhigBA,were constructed.First,we comparatively analyzed wild strain TX01 and in-frame mutant strains TX01ΔhigB and TX01ΔhigBA differences between stress resistance.Experimental results showed that TX01ΔhigB and TX01ΔhigBA grew consistently under normal conditions compared with the wild strain TX01.Compared to the wild strain TX01,TX01ΔhigB exhibited markedly reduced resistance against oxidative stress,but TX01ΔhigBA remained unchanged.Subsequently,we analyzed and compared the physiological performance of the three strains,which showed reduced biofilm forming ability TX01ΔhigB for but increased biofilm forming ability for TX01ΔhigBA compared with wild strain TX01;the motility of TX01ΔhigB increased,but that of TX01ΔhigBA did not change significantly.Resistance against host serum killing is an important characteristic of E.piscicida.Therefore,we analytically compared the resistance of the 3strains to host serum killing.Compared to the wild strain TX01,TX01ΔhigB exhibited markedly compromised tolerance to host serum stress,but TX01ΔhigBA remained unchanged.There were no significant differences in the antioxidant stress capacity,biofilm forming ability and antiserum killing ability between the complementary strains TX01ΔhigBC and wild strains TX01.These results demonstrate that loss of higB significantly reduces bacterial resistance to stressful conditions and attenuates bacterial biofilm formation,while also resulting in a significant increase in bacterial motility;However,loss of higBA did not affect bacterial anti oxidative stress capacity,motility and antiserum killing,and only enhanced bacterial biofilm formation.The above results suggest that higB is likely to be involved in the pathogenicity of the bacteria.Therefore,we performed pathogenicity experiments.In vitro infection experiments showed that deletion of higB dramatically attenuated the adhesion ability of bacteria in FG cells(flounder parotid gland cells)as well as the infection and proliferation abilities within mouse macrophage cell RAW264.7.We performed in vivo bacterial dissemination and virulence analysis experiments by infecting tilapia,and the experimental results showed that the number of bacteria was decreased in the spleen of TX01ΔhigB infected fish compared with the wild strain TX01,while no significant changes were observed in the spleen of TX01ΔhigBA infected fish;after 25 days of monitored period,TX01ΔhigB infected tilapia exhibited a 63.3% of survival rate,but TX01-infected tilapia exhibited only 13.3% of survival rate,whereas no change was observed in tilapia infected with TX01ΔhigBA(23.3%).The backcomplemented strain TX01ΔhigBC did not differ significantly from the wild type TX01 in either in vitro or in vivo infection experiments.These findings indicated that HigB is essential for responding adverse circumstance and pathogenicity of E.piscicida.Next,we performed an analysis of the regulated mechanism of higBA.First,the predicted higba promoter P236 was cloned,linked to the p SC11 and transformed into E.coli DH5α.By X-gal plate detection,DH5α/p SZ236 had promoter activity.DH5α/p SZ236 was cotransformed with overexpression vector p T3,hig A overexpression vector p THig A and higBA overexpression vector p THigBA to obtain recombinant strains DH5α/p SZ236/p T,DH5α/p SZ236/p THig A and DH5α/p SZ236/p THigBA.β-Galactosidase activity experiments showed that the activity value of DH5α/p SZ236/p THig A was 2086 Miller units,which was lower than that of DH5α/p SZ236/p T(2488 Miller units);Whereas the activity value of DH5α/p SZ236/p THigBA was only 0.7 Miller units,a significant decrease of about 3000 fold compared to DH5α/p SZ236/p T.To further determine the regulatory function of Hig A,the Hig A protein was expressed and purified in vitro and subjected to gel shift assay(EMSA),which revealed that Hig A was able to directly bind to the HigBA promoter P236.These results suggested the expression of operon higBA was directly regulated by Hig A,which was promoted by HigB.Based on the attenuated virulence of TX01ΔhigB,we further investigated its potential as a live vaccine.For this purpose,tilapia were immunized with TX01ΔhigB by intramuscular injection.The tilapia were challenged with TX01 at four weeks post-vaccination.The results showed an immune protection rate(RPS)of 54.6% for TX01ΔhigB 25 days after challenge.Moreover,specific antibody production in sera of vaccinated tilapia was determined.ELISA analysis showed that specific serum antibodies were detected in TX01ΔhigB-vaccinated fish at 2–4 weeks,and the highest antibody titer was observed at 4 weeks.Vaccination experiment showed that TX01ΔhigB induced effective protection against lethal E.piscicida infection.In summary,HigBA both plays an important role in stress resistance and pathogenicity of E.piscicida and can regulate higBA.This study provides new insights into the biological activity of type Ⅱ TA system HigBA in aquatic pathogenic bacteria and contributes to understand the pathogenesis of E.piscicida.