Bordetella and Burkholderia species are significant human and animal pathogens. Bordetella pertussis, B. parapertussis and B. bronchiseptica colonize respiratory epithelium to cause human pertussis and pertussis-like respiratory illnesses, while Burkholderia pseudomallei and its less-virulent relative Burkholderia thailandensis are soil organisms that can behave as opportunistic pathogens and survive as facultative intracellular parasites. Bordetella and Burkholderia are well equipped with virulence determinants, including multiple protein secretion systems. In the first section of this dissertation I describe an analysis of the Bordetella type III secretion (T3SS) effector protein BteA, which is necessary and sufficient for the induction of rapid toxicity in a variety of mammalian cell lines. We identify a N-terminal domain of BteA that mediates its localization to lipid raft domains of mammalian cells following ectopic expression and T3SS-mediated translocation by B. bronchiseptica. The rest of the dissertation is focused on the secretion systems and virulence mechanisms in B. pseudomallei and B. thailandensis. Unlike Bordetella, which contains a single injection-type T3SS and one known effector protein BteA, Burkholderia pseudomallei contains three injection T3SSs that function in mediating its interactions with other organisms in the rhizosphere and in mammalian pathogenesis. In the second section of the dissertation, the roles of T3SS and other virulence mechanisms are closely examined in the intracellular lifecycle of Burkholderia. Bacteria were delivered directly into the cytoplasm of mammalian cells using a photothermal nanoblade device, bypassing invasion and the need to escape from endosomes. Activity of the Burkholderia secretion apparatus T3SS (T3SSBsa) was critical for endosome escape following infection, but was not required for subsequent actin polymerization, intercellular spread and the fusion of host cell membranes to form multinucleate giant cells (MNGCs) following cytosolic nanoblade delivery of bacteria. Motility, mediated by either the Fla2 flagellar system or actin polymerization, was required for cell-cell spread and MNGC formation by B. thailandensis and B. pseudomallei, as was the activity of a type VI secretion (T6SS) system. We conclude that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles. The third section of the dissertation describes further analysis of the Fla2 flagellar T3SS and B. pseudomallei injection T3SS effector proteins. Using confocal immunofluorescence microscopy, we discovered that fla2 encodes multiple, lateral flagellar filaments and is activated following infection of mammalian cells. Surprisingly, constitutive expression of a Fla2 response regulator protein, Frr, strongly reduced plaquing efficiency in B. pseudomallei and B. thailandensis. I also describe the identification and analysis of novel B. pseudomallei effector protein (Bep) candidates. Specific phenotypes for six Bep proteins were observed following transfections of HeLa cells; BepA exhibited perinuclear localization, BepB resulted in cytotoxicity and alterations in cell morphology, BepC bundled actin, BepD decorated microtubules, and BepE and BepF rearranged actin and altered cell morphology. However, strains carrying deletion mutations in single Bep effector candidate loci did not exhibit defects following infection of mammalian cells. A single strain containing deletions in BopA, BopC and BopE, the three previously known T3SSBsa effectors, exhibited reduced plaque formation in RAW264.7 macrophages. Of the three effectors, only BopA was seen to play a major role in B. pseudomallei intracellular pathogenesis.
