A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections

We recently described a protein <i>O</i>-glycosylation pathway conserved in all species of the <i>Burkholderia</i> genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. <i>Burkholderia mallei</i>, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of <i>B. mallei</i> infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated <i>Burkholderia</i> glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the <i>B. cenocepacia</i> K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of <i>B. mallei</i>. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with <i>B. pseudomallei, B. mallei, B. multivorans,</i> and <i>B. cenocepacia</i>. Our results indicate that protein <i>O</i>-glycosylation in <i>Burkholderia</i> can be exploited as a biomarker for diagnosis of <i>Burkholderia</i>-associated infections.