Structural and functional elucidation of the trimeric autotransporter adhesin BpaC from Burkholderia pseudomallei

The trimeric autotransporter adhesin (TAA) BpaC plays a central role in the initial infection stages of the Gram-negative Burkholderia pseudomallei. The actual function of this protein on a biochemical level was undetermined at the start of this project. I endeavoured to identify binding partners of BpaC and associate the individual domains of the solvent-accessible part of the protein with specific binding events. To this end, I analysed the protein sequence using a combination of bioinformatic tools and TAA specific sequence rules to accurately determine secondary structure prevalences, domain borders, and likely binding partners. I designed, expressed, and purified multiple constructs of various lengths and composition, that all together cover the full solvent-accessible domain of BpaC, making every part of the protein accessible for functional or structural experiments. On a structural level, I solved parts of the C-terminal head domain of BpaC which, by extension, can be used to deduce the structural model of the full head domain. This model provided the basis for the introduction of a new subcategory of left-handed parallel beta-rolls named BpaC-like head domains; based on the unique surface charge properties of the C-terminal located head domain of BpaC. On a functional level, the identification of likely homology models for the individual domains of BpaC resulted in the prediction of binding partners of BpaC. Some of the candidates, namely extracellular matrix proteins from humans like fibronectin and collagen, were used in binding assays. These experiments revealed a preference of BpaC to bind to fibronectin, collagen I and collagen II. However, a clear domain-ligand association could not be determined. Lastly, a new hypothetical infection model is provided in which BpaC acts together with type I pili in the initial steps of adhesion of Burkholderia pseudomallei in a three stage process.