title: Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.
abstract: Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SS(Bsa)) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SS(Bsa) activity. Although intracellular movement was essential for cell-cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell-cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SS(Bsa)-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.
authors: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors: Christopher T
authors: CT
authors: French
authors: Isabelle J
authors: IJ
authors: Toesca
authors: Ting-Hsiang
authors: TH
authors: Wu
authors: Tara
authors: T
authors: Teslaa
authors: Shannon M
authors: SM
authors: Beaty
authors: Wayne
authors: W
authors: Wong
authors: Minghsun
authors: M
authors: Liu
authors: Imke
authors: I
authors: Schröder
authors: Pei-Yu
authors: PY
authors: Chiou
authors: Michael A
authors: MA
authors: Teitell
authors: Jeff F
authors: JF
authors: Miller
mesh_terms: D058947:Bacterial Secretion Systems / Q000502:physiology*; D016957:Burkholderia pseudomallei / Q000472:pathogenicity* / Q000502:physiology*; D002459:Cell Fusion; D002460:Cell Line; D003584:Cytological Techniques / Q000379:methods; D003600:Cytosol / Q000382:microbiology*; D006801:Humans; D007834:Lasers; D008554:Melioidosis / Q000635:transmission*; D008856:Microscopy, Fluorescence; D037521:Virulence Factors
chemical_list: D058947:Bacterial Secretion Systems; D037521:Virulence Factors