Viruslence Protein Regulated Peroxidase TagD Protects Vibrio Cholerae From Oxidative Stress And Its Physiological Function

Cholera is an old and prevalent sweepingly acute intestinal infection deadly infectious disease caused by Vibrio cholerae. Its clinical disease is characterized by the passage of voluminous stools of rice water character and vomiting. Cholera leads to the patients rapid dehydration and lack of body fluid electrolyte, serious time to cause the patient to produce muscle weakness and even shock and death. Vibrio cholerae is a Gram-negative bacteria, its size is short and adhered single flagella and fimbriae. Extensive studies have shown that the ability of V. cholerae strains to cause severe enteric infection in humans dependents on the expression of cholera toxin and a pilus colonization factor known as the toxin-coregulated pilus.When pathogenic microbial invades and infects the host, the host cell will start the defense mechanisms to produce ROS, it can kill microoganism by destroying the membrane lipids, proteins, DNA and other cellular components. But pathogenic microorganisms produce a variety of enzymes to resist ROS produced by the host cell in order to ensure the infection efficiency. One of them is thiol peroxidase (Tpx). Tpx (20kDa) usually has two cysteine residues determine protein function. The research about Tpx is more widely and deeply in Escherichia coli、Salmonella and Helicobacter pylori, while the study in Vibrio cholerae has not been reported. And the gene tagD expresses the thiol peroxidase locates in the epidemic pathogenicity island, as the regulon of ToxR. So in our experiment, we choose Vibrio cholerae thiol peroxidase TagD as the research object.First, we constructed tagD (vc0824) mutant through in-frame deletion. Under normal condition, the mutant and widle-type strain have no difference in sensitivity to H2O2. However, under the condition simulated host intestin environment, the mutant strain is more sensitive to H2O2than widle-type strain.We known that the cysteine is important to the function of proxidase, so we constructed TagD site-mutagenesis complementary strain. Interestingly, when the cysteine residuse at positions Cys-59and Cys-93was evaluated through single-site-directed mutagenesis with serine residuses, the complementary and mutant strain have no difference in sensitivity to H2O2. The result show the Cys-59and Cys-93is functional amino acid residuses of TagD.tagD mutant is more sensitive to oxidative stress in virulence induced medium. And in Vibrio cholerae, the tagD is a regulon of ToxR, so well as ToxT. So we constructed Luminescence reporter strain, then we find the ToxT can also regualte the expression of tagD. And there are two possible ToxT binding sites in the promoter region of tagD. Furthermore, the expression of toxT also has effect on the sensitivity of Vibrio cholerae to H2O2.At the last of the experiment, we find that functional TagD is a homodimer and it can be broken under hydrogen peroxide stress. Detection of colonization ability showed that the mutant which lacking tagD gene can colonize in the intestine of infant mouse as well as wild type. However, the individual colonization ability of mutant is weaker than wild-type strain in the intestine of Zebrafish.Above all, the viruslence gene regulated proxidase TagD can protect bacteria from oxidative stress and is helpful to pathogenicity of Vibrio cholerae.