Identification and classification of novel B. pseudomallei type III secretion system effector proteins

Burkholderia pseudomallei is a Gram-negative rod-shaped bacterium and the causative agent of Melioidosis. Among an arsenal of virulence determinants, the type III secretion system (T3SS) is of particular interest because infection is attenuated in the absence of a functional T3SS. The T3SS is a needle-like apparatus that translocates virulence proteins, called effectors, directly from the bacterial cytoplasm into the host cell. These effectors subvert hose cell pathways to benefit the invading bacteria by interfering with processes like the cell cycle, apoptosis and inflammation. The type III secretion system 3 (T3SS-3) of B. pseudomallei is homologous to the Inv/Mxi-spa secretion systems of Salmonella and Shigella respectively, but under characterised in comparison, with only 8 known effectors to date. We hypothesise that many novel B. pseudomallei T3SS-3 effectors can be discovered with bioinformatic prediction. Using the prediction program EffectiveCCBD, we identified 12 putative effector genes with diverse effector-like functions and cloned these into the model organism B. thailandensis. Here we demonstrate a method of quantifying effector translocation using a β-lactamase reporter system adapted to B. pseudomallei. This assay was used to successfully quantify the translocation of a known B. pseudomallei T3SS-3 effector BopE, as well as test the bioinformatically predicted putative effectors. A novel effector, BPSS0357, with a predicted role as a copper-binding iron transport protein was successfully demonstrated to be translocated in a T3SS-3 dependent manner.<br>