CHARACTERIZATION OF A NOVEL ATTACHMENT VIRULENCE FACTOR FROM THE INTRACELLULAR BACTERIAL PATHOGEN Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei (Bp) is an intracellular bacterial pathogen causing melioidosis, a life-threatening disease affecting humans with a high mortality rate. Bp causes disease in estimated 165,000 people each year with approximately 89,000 fatalities. The innovative single-cell transcriptomic approach has made it possible to scrutinize differential gene expression of Bp at each stage during the infection of eukaryotic cells. Bp intracellular “TRANSITome” has led to the identification of a novel surface attachment protein BPSS0097 (Sap1) that was specifically turned on at the vacuole entry stage. A Sap1 mutant showed a significant decrease in attachment to host cells and was completely attenuated in the BALB/c mouse intranasal model of acute Bp infection. This is the first description of an attachment deficient mutant that is completely attenuated in a lethal challenge mouse model. We hypothesized that Sap1 recognizes specific host cell surface receptor molecules that facilitate the binding of Bp to the surface of host cells to initiate its intracellular infection. To test this central hypothesis, I first optimized Sap1 protein production and determined Sap1 crystal structure via collaboration. The structure of Sap1 was successfully determined at 1.30Å resolution revealing a significant similarity to Src- Homology 3 (SH3) protein family, a very abundant protein-protein interactions module in eukaryotes. The knowledge gained in the functional analysis based on Sap1 structural information laid a solid foundation for me to further identify the receptor protein from host cells to be the glycoprotein non-metastatic melanoma protein b (GPNMB). The interaction between GPNMB and Sap1 was validated to be specific and strong. Overall, I identified a new ligand/receptor interaction pair of Sap1/GPNMB and my study is the first to show that GPNMB is used by a bacterial pathogen as a gateway to attach and invade host cells. iv Furthermore, a transcriptional activator of Sap1 was identified, which also acts as a global regulator of other membrane-associated virulence factors in Bp. In summary, I accomplished a comprehensive characterization of a novel Bp attachment factor Sap1 that leads to a further understanding of Bp attachment mechanisms at the molecular level. The findings of my dissertation project will aid in future vaccine development and therapeutics for melioidosis treatment.