title: Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga.
abstract: We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Lamothe
authors.forename: Sandra
authors.initials: S
authors.lastname: Thyssen
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000048:Acanthamoeba / Q000382:microbiology*; D000818:Animals; D042602:Burkholderia cepacia complex / Q000166:cytology / Q000254:growth & development* / Q000472:pathogenicity*; D002455:Cell Division; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D014617:Vacuoles / Q000382:microbiology*
chemical_list: D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D018942:Macrolides; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases