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

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in 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. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that mainly infects horses, mules, donkeys and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia 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 previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. 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 B. pseudomallei, B. mallei, B. multivorans and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.IMPORTANCEGlanders is a severe zoonotic disease caused by the Gram-negative bacterium Burkholderia mallei, which affects horses, mules and donkeys, as well as humans. B. mallei is also considered a category B biothreat agent. Due to insufficient pathognomonic symptoms in the early stages of glanders, diagnosis can be difficult. Complement fixation is the most accurate and reliable serological test prescribed by the World Organization for Animal Health; however, this test has a considerable number of false-positive results. We have recently described a conserved protein O-glycosylation pathway present in all species of the Burkholderia genus; we also demonstrated that Burkholderia-infected humans develop anti-glycan antibodies. Here, we exploited this system to construct and evaluate a synthetic glycoengineered protein antigen as a diagnostic tool for glanders. Our results show 100 % specificity in the detection of antibodies from infected horses, indicating that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.