Potential Immunogenic Polypeptides of Burkholderia pseudomallei Identified by Shotgun Expression Library and Evaluation of Their Efficacy for Serodiagnosis of Melioidosis

Abstract

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

Cross-Species Comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei Quorum-Sensing Regulons

“…It is unclear where B. mallei, which is a host-restricted bacterium, might come into contact with benzoate. However, the BenB ortholog in B. pseudomallei was identified as an antigen in human sera, suggesting that this factor is produced in vivo during melioidosis (51). We note that we did not see the ben operon in our B. pseudomallei QS regulon.…”

Comparison of in-house IgM and IgG ELISAs for the serodiagnosis of melioidosis in Malaysia

“…Despite the discovery and report of numerous potential serodiagnostic markers for melioidosis, the results were variable. The identified targets such as BPSS1904, BPSL3130 [ 24 ], recombinant truncated flagellin [ 25 ], OmpA [ 26 ], TssD-5 [ 27 ], OPS [ 22 ] and HCP1 [ 32 ] showed sensitivity and specificity of 71–95 % and 88–98 %, respectively. However, details of the geographical location (endemic status), type of strain and evaluation with a large number of samples will be necessary to provide the most effective serodiagnostic markers for Malaysian local populations.…”

The Optimization of Igm In-House ELISA for the Laboratory Diagnosis of Melioidosis in Malaysia

“…The optimal time for the incubation period for the substrate is 30 minutes. As compared to other studies which used o-phenylenediamine (OPD) as a substrate, the test reaction need to be stopped by adding a small amount of sulfuric acid (H2SO4) after a set time of incubation [16,27,28]. Although OPD produces a rapid color change in ELISA, it is toxic and proper ISSN: 2469-5807 precaution need to be taken to prevent direct contact with the substrate.…”