Contribution Of The Spx Regulators And NADH Oxidase To The Pathogenicity Of Streptococcus Suis Serotype 2

Streptococcus suis(S.suis)is an important zoonotic pathogen responsible for significant economic losses to the swine industry worldwide and severe threats to public health.It causes a variety of diseases in pigs,including meningitis,septicemia,arthritis,pneumonia,and even acute death.S.suis infections in humans lead to meningitis and streptococcal toxic shock-like syndrome(STSLS).Based on differences in capsular polysaccharides,33 serotypes(types 1 to 31,33,and 1/2)have been described for S.suis.Among them,serotype 2 is considered the most virulent and the most frequently isolated serotype in association with diseases worldwide.By 2012,the total number of S.suis infections in humans was close to 1600 cases,doubling the number published in 2009.Remarkably,2 large outbreaks of S.suis epidemics occurred in China in 1998(Jiangsu Province)and in 2005(Sichuan Province),which resulted in not only deaths of a large number of pigs,but also many human cases and deaths.In addition,S.suis was identified as the leading cause of adult meningitis in Vietnam,the second most common cause in Thailand and the third most common cause of community-acquired bacterial meningitis in Hong Kong.During infection of the host,S.suis encounters various stress conditions and requires a regulatory network to sense and respond to environmental signals.Currently,some two-component regulatory systems(TCSs)and common regulators have been described and shown to modulate environmental adaptation and pathogenesis in S.suis.Spx proteins are a group of global transcriptional regulators that are highly conserved among low-GC-content Gram-positive bacteria.Spx has been reported to regulate stress response and virulence in many bacterial species.However,its role in S.suis remains unclear.In this study,we explored the function of Spx in S.suis 2,and found that the two Spx regulators modulate stress tolerance and pathogenesis in S.suis 2.On the basis,four Spx A1-regulated genes(0350 ? nox ? tpx and cop A)were selected,and their roles in oxidative stress tolerance and virulence of S.suis 2 were studied.The results indicated that NADH oxidase contributes to oxidative stress tolerance and virulence of S.suis 2.The main contents are as following:1.Functional study of the Spx regulators in S.suis 2Two Spx proteins were identified in S.suis 2,and designated Spx A1 and Spx A2,respectively.A multiple alignment was performed of S.suis Spx proteins with the Spx regulators from other bacteria.The high level of homology exhibited by these proteins suggests that Spx may paly an important role in S.suis.Two mutant strains(?spx A1 and?spx A2)lacking the spx genes were constructed through homologous recombination.To investigate the role of Spx in stress response of S.suis 2,growth curves of the mutants under various stress conditions were compared with those of the wild type(WT)strain.The results showed that Spx A1 affects oxidative stress tolerance,while Spx A2 affects tolerance to Na Cl and SDS.Both Spx regulators affect nutrition metabolism of S.suis 2.A murine infection model was used to assess the pathogenicity of the mutant and WT strains.The results demonstrated that the two Spx regulators modulate the ability of S.suis 2 to colonize the tissues and trigger the inflammatory response of mice.Global gene transcription profiles showed that a large number of genes which can be classified into several functional categories were differentially expressed in each mutant strain,revealing the global regulatory role of Spx.2.NADH oxidase contributes to oxidative stress tolerance and pathogenicity of S.suis 2Based on the microarray data and literature consultion,four Spx A1-regulated genes were selected to explore their roles in oxidative stress tolerance and virulence of S.suis 2.Mutants of the four genes were constructed through homologous recombination.For preliminary research on the role of the four genes in oxidative stress tolerance,growth curves of the mutants under normal and oxidative stress conditions were compared with those of the WT strain.A murine infection model was used to assess the virulence of the mutant and WT strains.The results showed that NADH oxidase is involved in tolerance to oxidative stress and virulence of S.suis 2.Analysis of bacterial loads in the tissues of mice and induction of inflammatory cytokines,as well as experimental infection of piglets further confirmed that NADH oxidase is required for the pathogenicity of S.suis 2.The inactive forms of NADH oxidase were generated with point mutations.Our results revealed that the enzymatic activity of NADH oxidase plays a key role in oxidative stresstolerance and a secondary role in virulence.Overexpression of NADH oxidase in ?spx A1 led to increased ability to resist to oxidative stress and enhanced virulence,suggesting that NADH oxidase plays an important part in the regulatory effects of Spx A1 on oxidative stress tolerance and virulence.