Type ? Secretion System Transports Zn²⁺ To Combat Oxidative Stresses In Burkholderia Thailandensis

Zinc is essential for life by serving both catalytic and structural roles within a large number of proteins.During an infection,Zn²⁺ is not readily accessible to the invadingbacterium in the vertebrate host because the host attempts to restrict the availability of essential micronutrients.To acquire sufficient zinc for survival and pathogenesis,bacteria have evolved a number of efficient zinc transporters.In this study,we revealed the role of a Burkholderia type ? secretion system in Zinc acquisition and characterized a novel zinc scavenging pathway related to T6SS-4.The results are as follows:1?A putative Zur binding site Zur-Box was found in T6SS-4 promoter region.We thus examined the interaction between Zur and T6SS-4 promoter by electrophoresis mobility shift assay?EMSA?,then the result point to regulation of T6SS-4 by the zinc-responsive regulator Zur.Deletion of zur markedly enhanced the expression of T6SS-4 genes?icmF4,clpV4 and hcp4?.Transcript levels of T6SS-4 genes?icmF4,clpV4 and hcp4?and tse Z gene,determined by qRT-PCR,showed that Zur represses T6SS-4 genes and tseZ expression.But oxidative stress relieved the repression of Zur to T6SS-4 transcription.2 ? We constructed T6SS-4 genes?icmF4,clpV4 and hcp4?and tseZ gene deletion mutants,compared the survival rate in various mutant strains exposed to H₂O₂ or CHP,and found that the survival rate of T6SS-4 genes and tseZ gene deletion mutants declined.We also found the content of intracellular hydroxyl radicals in each mutant strain was higher than in the wild type strain.So T6SS-4 genes and tseZ gene is crucial for protection against oxidative damage3?Deletion of clpV4 diminished the effect of exogenous Zn²⁺onincreasing the survival rate of the bacteria under oxidative stress conditions.The intracellular Zn²⁺contents in bacteria challenged with CHP and H₂O₂ using?ICP-MS?,deletion of clpV4 significantly reduced intracellular Zn²⁺contents and such defects were almost fully recovered by complementation the clpV4 gene.We concluded that T6SS-4 participate in Zinc acquisition under oxidative stress.4?By protein secretion assay,we found that TseZ is a substrate secreted by T6SS-4.TseZ can specifically bind Zn²⁺ and also participate in Zinc acquisition under oxidative stress.Exogenous Tse Z protein also protected the ?clpV4 mutant from oxidative stress.This suggests that,after T6SS-4-mediated translocation,Zn²⁺ uptake by TseZ occurred independently of the secretion system.In conclusion,in this research,we discovered that under oxidative stresses,Burkholderia thailandensis T6SS-4 secrets the zinc-binding effector TseZ for Zn²⁺ acquisition to protect bacteria from oxidative damage.