A Multi-armed Approach for Identifying Circulating Bacterial Proteins in Melioidosis

The detection of pathogen-associated macromolecules in clinical samples is a powerful alternative to more traditional gold standards of diagnosing infectious diseases. Burkholderia pseudomallei, the causative agents of melioidosis, is a facultative, intracellular bacteria categorized as a Tier 1 Select Agent by the United States Federal Government for its capacity for large-scale dissemination, elevated rates of mortality and morbidity, and minimal medical countermeasures in place. B. pseudomallei is prevalent in tropical regions and is intrinsically resistant to many first-line antibiotics. The current gold standard for diagnosing melioidosis is blood culture, however this method is inadequate and timely. The multifaceted display of clinical presentations of melioidosis further emphasize the need for specific, yet rapid diagnostics. To further characterize viable bacterial targets present during an infection, a multi-armed approach was used to analyze clinical melioidosis samples through direct and indirect platforms. First, the In vivo Microbial Antigen Discovery (InMAD) platform utilized syngeneic CD1 mice to indirectly detected bacterial proteins in clinical samples in conjunction with a high-density nucleic acid protein array (HD-NAPPA). Secondly, patient serology was evaluated on the HD-NAPPA to determine generated antibody response in the host as these proteins may be shed targets from these intracellular pathogens. Third, protein profiling by liquid chromatography with tandem mass spectrometry (LC-MS/MS) would depict proteins in an unbiased proteomic methodology. Converging data from each approach resulted in multiple targets of interest to be evaluated as biomarkers of melioidosis.