Cloning and characterization of in vivo expressed genes of Burkholderia pseudomallei in bacteremic melioidosis patients

Abstract

Melioidosis is an infectious disease caused by a soil saprophyte gram negative bacterium. Burkholderia pseudomallei. The known mayor endemic areas of this disease are Southeast Asia and Northern Australia. Melioidosis cases have been reported from every parts of Thailand, with highest incidence rate in the northeastern part of the country. The pathogenesis of melioidosis remains unknown. Currently, the bacterial pathogenesis study is focused on in vivo expressed genes detection. Two approaches were performed to detect in vivo expressed genes by using the immunoscreening experiment. The first experiment, In vivo induced antigen technology ( IVIAT) approach, the pooled melioidosis sera were extensively absorbed with in vitro grown cells. whole cell lysate, whole cell lysate and denature of B. pseudomallei. The antibody titre against the whole cell lysate in the absorbed serum has been demonstrated to be decreased more than 1,000 times. The expressed genomic library in plasmid pET 30a was screened with the absorbed serum and the signal was detected using ECL chemiluminescence. Thirty one positive clones were isolated from screening of 13,000 library clones. Only 8 clones could be successfully sequenced and DNA sequence analysis of the insert DNA at the S tag primer of all 8 clones by using BLAST and Macvector programn revealed similarity with hypothetical protein. Western blot was performed with expressed protein from 19 positive clones randomly selected from the 31 positive clones, all expressed protein reacted with the melioidosis, S-tag and normal serum. The inclusion body preparation was isolated from the clone number 23 and western blot was performed with 16 normal sera and 16 melioidosis sera. The protein reacted with all of the tested sera. The second approach, the λ ZAPll expressed genornic library of B. pseudornallei was screened with pooled melioidosis serum preabsorbed with E. coli host cell. The positive clones were detected by using protein A-CDP star chemiluminescence. All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis. The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology. At least 6 in vivo genes were identified by this approach. Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (typo three secretion protein). Another two were coded for conserved hypothetical proteins. The last Iwo isolated genes were groEL (a chaperonine protein) and a transmembrane protein. All of the isolated genes are potentially useful as diagnostic for melioidosis. The gmhA gene was amplified from the B. pseudomallei genome using PCR and expressed in pRSETA vector. The expressed protein band of gmhA gene did not react with the pooled absorbed melioidosis serum in western blot experiment. The nature of antigenicity of protein encoded by gmhA may be conformational antigeniciy that lost the antigeniciy during SDS-PAGE denature process.