| Vibrio parahaemolyticus(V.parahaemolyticus)is a Gram-negative halophilic bacterium in a wide variety of environments,including oceans,coasts,and estuaries.As an important zoonosis pathogen,V.parahaemolytic infection can not only cause acute hepatopancreas necrosis in shrimp,but also cause acute gastroenteritis in human,and in severe cases,it can also lead to sepsis,necrotizing fasciitis and wound infection,which brings great distress and serious threat to the global aquaculture industry and public health.Transition metal is an essential nutrient element for almost all organisms.Zinc(Zn)is the second most abundant trace element in bacteria,which can play a catalytic and regulatory role as a cofactor for a variety of proteins and enzymes.However,excessive zinc has toxic effects.The host can use nutritional immunity to capture zinc surrounding the pathogen so that it cannot be used by the pathogen or to combat infection by imposing zinc toxicity.Bacteria have also evolved mechanisms to maintain zinc homeostasis against host cell defenses.In this study,the transcriptome change of V.parahaemolyticus to Zn-deficient and Znreplete conditions were explored by RNA sequencing analysis.Three putative Zn uptake systems regulated by the Zur regulator were screened,namely the ZnuABC,ZnuABC Ⅱ,and ZrgABCDE systems.The expression of three genes encoding zinc binding proteins(zrgA,znuA,znuA Ⅱ)under zinc-deficient conditions was verified by quantitative Real-Time PCR,and the results were consistent with RNA sequencing,which showed that the expression of all three genes was significantly upregulated.The corresponding gene deletion strains were constructed,including single-gene deletion strains ΔzrgA,ΔznuA,ΔznuA II,double-gene deletion strainsΔzrgAΔznuA,ΔzrgAΔznuA Ⅱ,ΔznuAΔznuAⅡ,and three-gene deletion strainsΔzrgAΔznuAΔznuAⅡ,and the gene complementary strain CΔzrgA.The growth of each strain under different conditions was measured by growth curve.The results showed that ΔzrgA,ΔzrgAΔznuA,ΔzrgAΔznuA Ⅱ,ΔzrgAΔznuA ΔznuA Ⅱ showed obvious growth defects under the condition of Zn deficiency.In addition,the growth defects of ΔzrgAΔznuA,ΔzrgAΔznuA Ⅱ,ΔzrgAΔznuAΔznuA Ⅱ strains were more obvious than those of ΔzrgA,while the growth ofΔznuAΔznuAⅡ double-gene deletion strains showed no difference compared with WT strains.The results indicated that zrgA was the main Zn uptake gene in V.parahaemolyticus,while znuA and znuA Ⅱ played a secondary role.The effects of zrgA on the motor capacity of V.parahaemolyticus were analyzed by motility test and quantitative Real-Time PCR.The results showed that the swimming capacity of ΔzrgA was significantly decreased under zinc deficiency conditions,but the expression of polar flagella genes associated with swimming capacity was not down-regulated in the mutant.Thus,the decreased mobility observed inΔzrgA may be the result of growth defects.The effect of zrgA on the pathogenicity of V.parahaemolyticus was investigated using a zebrafish infection model,and the results showed that zrgA had no effect on the pathogenicity of V.parahaemolyticus.The regulatory effect of Zur on zrgA was analyzed by quantitative Real-Time PCR and β-galactosidase activity.The results showed that the expression of zrgA gene was significantly up-regulated in Δzur.The βgalactosidase activity of Δzur carrying pDM8-PzrgD-zrgE plasmid was significantly higher than that of WT strain carrying the same plasmid.The results showed that Zur inhibited the expression of zrgA and released the restriction in the absence of Zn.In conclusion,V.parahaemolyticus upregulates the expression of Zur regulon in response to Zn limitation and is more dependent on ZrgABCDE system for Zn acquisition.According to the results of RNA sequencing,VP_RS04700(zntA)gene was most significantly down-regulated under the Zn-restricted condition,which was speculated to be related to Zn efflux in V.parahemolyticus.The expression of zntA under different metal stress conditions was analyzed by quantitative Real-Time PCR.The results showed that zntA was up-regulated to varying degrees under the conditions of excessive Zn,Cu,Co and Ni.The deletion strain ΔzntA and the complementary strain CΔzntA were constructed,and the growth of each strain under different conditions was analyzed by growth curves.The results showed that ΔzntA strains were sensitive to excessive Zn and Ni.Due to the presence of toxic neutralization between metals,it was tested to see if the toxicity of Zn could be neutralized by other metals,and the results showed that adding ferrous ions to the medium in the presence of excessive Zn could restore its growth.The results of spot dilution assays showed that the number of colonies formed by ΔzntA strains did not decrease after 3 hour treatment with excessive Zn or Ni,indicating that Zn and Ni had antibacterial effects on the growth of ΔzntA strains.The metal content of these strains was analyzed by inductively coupled plasma mass spectrometry(ICP-MS)under different growth conditions.The results showed that the accumulation of Zn in ΔzntA strains was significantly higher than that in WT and CΔzntA strains,indicating that zntA was the efflux pump of Zn in V.parahaemolyticus.Under the condition of excess Ni,Ni accumulation in ΔzntA strains was not different from that in WT,while Zn accumulation was significantly increased.Moreover,there was no difference in the growth of the three strains under the condition of excess Ni when TPEN was added to the medium to remove Zn.The results indicated that the growth defects of ΔzntA strain under the condition of excessive Ni were caused by excessive accumulation of Zn.Meanwhile,ΔzntA strain was more sensitive to oxidative stress induced by H2O2 in the presence of Zinc.The competition infection experiment showed that the competition ability of ΔzntA strain was significantly reduced during V.parahaemolyticus.The regulatory effect of ZntR on zntA was analyzed by quantitative Real-Time PCR and β-galactosidase activity.The results showed that the expression level of zntA in ΔzntR strains was significantly decreased.The β-galactosidase activity of WT strain carrying pDM8-PzntA plasmid was significantly higher than that of ΔzntR strain carrying the same plasmid,and the β-galactosidase activity of WT strain carrying pDM8-PzntA plasmid after being incubated under Zn,Cu,Co and Ni conditions was significantly higher than that under normal conditions.The results indicate that ZntR positively regulates the expression of zntA and responds to the induction of Zn,Cu,Co and Ni.These results indicate that ZntR activates zntA expression in V.parahaemolyticus for Zn efflux,and ZntA-mediated Zn efflux is associated with oxidative stress tolerance and pathogenicity in V.parahaemolyticus.In conclusion,in this study,the genes related to Zn uptake and efflux in V.parahaemolyticus were screened and identified by RNA sequencing analysis,and their biological functions were explored,which revealed the relevant mechanism of maintaining Zn homeostasis in V.parahaemolyticus,and provided a theoretical basis for the subsequent development of metal-based antibacterial programs to manage infectious diseases. |
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