The Identification Of Novel Virulence Related Factors In Erysipelothrix Rhuiopathiae And Moledular Characterization Of Quinolones Resistance In Erysipelothrix Rhuiopathiae

Swine erysipelas caused by E.rhusiopathiae is the most prevalent and economically important of these diseases.E.rhusiopathiae could cause sepsis,endocarditis,polyarthritis.In China,swine erysipelas has emerged at an alarming rate,developing from scattered occurrences on a small number of farms to systemic outbreaks in many provinces since 2012 and inflicting huge financial losses.Although E.rhusiopathiae acts as a pathogen severely harmful to the pig industry,the studies on pathogenesis and immunogenic mechanism were relatively inadequate.This knowledge gap has become one of the most important hindrances to controlling infection.It was especially important for study of E.rhusiopathiae pathogenesis to identify novel virulence-associated factor and clarify its role in the infection.The present study tried to systematically identify virulence factors.Since the surface proteins of Gram-positive bacteria play a vital role in adhering to and intruding into host cell,the cell-wall-associated proteins(CWPs)were chosen as object of this study.CWPs that are differentially expressed between a high-virulence strain and a low-virulence strain were investigated by iTRAQ combined LC-MS/MS analysis.the identified potential virulence factors were verified.In the early 1990,swine erysipelas in china was controlled by vaccination and some other measures.Nowdays,swine erysipelas burst afresh.Some questions call for solution.Is the traditional vaccine unuseful? Does the bacterial resistance to drug changed a lot? In order to provide scientific instruction for prevention of E.rhusiopathiae infection,the drug-resistant test of E.rhusiopathiae clinical isolates was performed.The resistance rates to quinolones was high.Then we study the molecular mechanism of quinolones resistance in E.rhusiopathiae.The principal results were described as following: 1.Comparative Proteomics Analysis of Cell wall Proteins of Erysipelothrix rhusiopathiae StrainsA total of 100 CWPs showed significant differences in expression.Among the 100 differentially expressed proteins,57 proteins were up-regulated and 43 proteins were down-regulated in the high-virulence strain.The proteins that were up-regulated in the high-virulence strain were mostly ABC transporter proteins and adhesion proteins,and the proteins that were down-regulated in the high-virulence strain were mainly stress-response proteins.Selected proteins that were up-regulated in the high-virulence strain were verified by Western blot to support the iTRAQ data.The iTRAQ results showed that a sugar-binding protein Sbp was up-regulated by 1.73-fold in the high-virulence strain.The Sbp protein was reported to be involved in the bacterial twin-arginine-translocation pathway and is important to the virulence of several bacterial pathogens.To verify the role of sbp in E.rhusiopathiae infection,a sbp-deletion mutant was constructed.Disruption of sbp led to decreased adhesive and invasive ability to PIEC,as well as the survival rate in blood of healthy pig.Experiments in pig model showed that the sbp-deletion mutant caused reduced mortality,which suggested that Sbp is an important virulence related factor of E.Rhusiopathiae.The virulence related proteins of bacteria also act as important protective antigen.The protective efficacy of the virulence related factor Sbp remains to be exaluated.Experiments in pig model found that the purified recombinant Sbp could elicit a significant humoral antibody response,and to confer 66.7% protection against a lethal dose of E.rhusiopathiae infection.Antibody test found the humoral antibody response last at least 6 months.The results indicate that Sbp may act as an effective component of a new vaccine.2.molecular characterization of quinolones resistance in Erysipelothrix rhusiopathiaeIn this study,the E-test were used to detect the minimum inhibitory concentration(MIC)of E.rhusiopathiae isolates to ciprofloxacin(CIP)and levofloxacin(LEV)respectively.According to the guidelines of the Clinical and Laboratory Standards Institute(CLSI),the resistance and susceptibility of all isolates were determined: The resistance rate of ciprofloxacin was 48.8%.Then,via sequence and analyse of DNA gyrase(GyrA,Gyr B)and DNA topoisomerase IV(ParC,ParE),2 quinolone resistance related mutations were found.In order to prove the role of mutations of DNA gyrase and DNA topoisomerase IV for QNs resistance,in study,the site-directed mutagenesis was performed respectively: Gyr A(90D?N)and ParC(81S?I).The results showed that GyrA site mutations could increase the MICs of Erysipelothrix rhusiopathiae by 3 times to CIP.