Screening And Functional Identification Of Brucella Secretory Proteins

Brucellosis is a worldwide zoonosis caused by Brucella,a facultative intracellular bacteria,and seriously threats human health and animal husbandry.Upon entry into host cells,Brucella resides within a membrane-bound compartment known as the Brucella-containing vacuole(BCV).The secretory proteins translocated by Brucella are thought to control BCV’s trafficking along the endocytic pathways and disturb the immune response from host cell and consequently,aid the bacterial intracellular replication and diffusion.Recently,25 secretory proteins have been identified,but more secretory proteins need to be characterized,compared with other pathogenic bacteria.In this study,in order to screen the secretory proteins and reveal their functions,the periplasmic proteins,outer membrane proteins and extracelluar proteins were collected to perform TEM-1 test and three secretory proteins that they were BMEII0580,BMEII0581 and BMEII0607,were identified.According to the functional annotation,BMEII0580 is probable blue-copper protein YacK precursor,BMEII0581 is superoxide dismutase(Cu-Zn),and BMEII0607 is ferric anguibaction-binding protein.Then,the gene knockout strains and its conplemented strains were constructed to perform the infection assay of cells and mice.The results indicate that the secretory proteins are not essential to the bacterial virulence.By bioinformatics analysis,BMEII0580 exhibits a high similarity(42%identical)to the multicopper oxidase from E.coli and has a typical Tat signal motif and T1,T2,T3 copper sites.First,in order to determine its nature like the multicopper oxidase,the in vitro expression pattern of BMEII0580 by copper was detected,and the results showed that the expression of BMEII0580 was up-regulated.And,like other multicopper oxidases,the procaryotic expressed and purified BMEII0580 is able to oxidase 2,2 =-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),dimethoxyphenol and para-phenylenediamine.Additionlly,BMEII0580 has ferroxidase activity.By copper tolerance test,compared with the wild type strain 16M,the gene deletion strain showed significantly growth defect.In conclusion,our results confirm that BMEII0580 is a multicopper oxidase and contributes to the copper tolerance of B.melitensis 16M.Theoretical analysis shows that the secretory proteins translocated by the intracellular bacteria Burcella druing the infection are able to constantly elicit a specific immune response.Accordingly,in this study,the identified secretory proteins were used to develop Bovine Brucella-Ab indirect enzyme-linked immunosorbent assay(iELISA)after procaryotic expressed and purified.The results showed that BMEII0581 was effectively able to distinguish the positive and negative serum and used to establish iELISA.The specificity and sencitivity of iELISA were 91.67%and 95.83%,respectively and showed this protein’s good prospect in Brucellosis diagnosis.