Investigation of Key Mechanisms of Contact Dependent Growth Inhibition Systems in \u3ci\u3eBurkholderia cepacia\u3c/i\u3e Complex Species

Burkholderia cepacia complex is a group of closely related environmental bacteria that can exacerbate disease in immunocompromised individuals. B. cepacia bacteria are equipped with an arsenal of mechanisms to cooperate and compete for nutrients in various polymicrobial environments. Contact-dependent growth inhibition (CDI) systems are antagonistic mechanisms widespread among proteobacteria. CDI systems consist of Two Partner Secretion pathway proteins that function to deliver the toxic C-terminal portion of the large effector exoprotein (termed ‘BcpA’ in Burkholderia-type CDI systems) to the cytoplasm of a compatible recipient bacterial cell upon direct cell to cell contact. The translocation of BcpA out of the producing cell is mediated by the outer membrane transporter, BcpB. While the production of a cognate immunity protein, BcpI, prevents autotoxicity and mediates the discrimination between kin and non-kin cells. Many Burkholderia CDI systems contain an accessory protein, BcpO, that can enhance specific CDI mediated killing, however the precise role remains unknown. This body of work identifies key factors and mechanisms that influence CDI activity in Burkholderia species. Studies contained in this dissertation are outlined into two major sections: (i) the examination of the role the accessory lipoprotein, BcpO, plays in B. dolosa AU0158 CDI activity; (ii) the identification of the interaction between CDI systems produced within the same B. cepacia complex bacterium. Previous work identified BcpO as an accessory protein required for maximum CDI activity for two CDI systems; however, the precise function of BcpO remains to be understood. Work done to understand the role of BcpO demonstrates that the accessory protein likely functions in a system specific manner. Immunoprecipitation and mass spectrometry analysis was used to identify potential binding partners of BcpO. Though unsuccessful in elucidating the role of BcpO, inquiry into BcpO led to the discovery of relaxed specificity among the BcpB transporters. This work shows that various B. cepacia complex BcpB proteins can secrete cognate and non-cognate BcpA substrates. The promiscuity among BcpB transporters influences the interplay between CDI systems produced within the same B. cepacia complex bacterium. This work demonstrates that genes that encode the CDI systems of B. dolosa AU0158 may not function independently. Instead, the presence of multiple CDI system proteins is required to induce specific BcpA mediated CDI killing. The examination of BcpB flexibility also led to the identification of a fourth B. dolosa CDI system capable of mediating interbacterial competition. All together this dissertation adds valuable information that drives the field of CDI forward. Understanding the interactions that occur between distinct CDI systems provides further insight into the complexity of bacterial antagonism