Virulence from the rhizosphere: membrane fusion and the burkholderia intercellular life cycle

Burkholderia pseudomallei is a Tier 1 Select Agent that causes the often fatal human disease melioidosis. B. pseudomallei and related species invade a variety of cell types, replicate in the cytoplasm, and efficiently spread to adjacent cells. Using genetic dissection and photothermal nanoblade delivery, which allows the efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells, we have investigated temporal and spatial requirements for virulence determinants in the Burkholderia intracellular life cycle. The Bsa type III secretion system is essential for escape from primary endosomes, and by nanoblade delivery we demonstrate that cell-cell spread is dependent on the activity of a type VI secretion system that functions downstream from T3SS-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleated cells (MNCs) in diverse cell types. By infection and nanoblade delivery, we observe a remarkable correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies show that plaque formation represents MNC death. Our data show that the primary means for intercellular spread by B. pseudomallei and related species involves T6SS-mediated membrane fusion, and we propose a model in which a unique activity at the C terminus of VgrG5, which is positioned at the tip of the T6SS apparatus, is responsible for fusion following injection across the membranes of adjacent cells