title: Burkholderia pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages.
abstract: Burkholderia pseudomallei is a facultative intracellular pathogen capable of surviving and replicating within eukaryotic cells. Recent studies have shown that B. pseudomallei Bsa type III secretion system 3 (T3SS-3) mutants exhibit vacuolar escape and replication defects in J774.2 murine macrophages. In the present study, we characterized the interactions of a B. pseudomallei bsaZ mutant with RAW 264.7 murine macrophages. Following uptake, the mutant was found to survive and replicate within infected RAW 264.7 cells over an 18-h period. In addition, high levels of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1alpha and IL-1beta, were detected in culture supernatants harvested from infected monolayers. The subcellular location of B. pseudomallei within infected RAW 264.7 cells was determined, and as expected, the bsaZ mutant demonstrated early-vacuolar-escape defects. Interestingly, however, experiments also indicated that this mutant was capable of delayed vacuolar escape. Consistent with this finding, evidence of actin-based motility and multinucleated giant cell formation were observed between 12 and 18 h postinfection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs exhibited the same phenotype as the bsaZ mutant, indicating that functional T3SS-1 and T3SS-2 did not appear to be responsible for the delayed escape phenotype in RAW 264.7 cells. Based upon these findings, it appears that B. pseudomallei may not require T3SS-1, -2, and -3 to facilitate survival, delayed vacuolar escape, and actin-based motility in activated RAW 264.7 macrophages.
authors.affiliation: Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, 903 South 4th, Hamilton, MT 59840, USA.
authors.forename: Mary N
authors.initials: MN
authors.lastname: Burtnick
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brett
authors.forename: Vinod
authors.initials: V
authors.lastname: Nair
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Warawa
authors.forename: Donald E
authors.initials: DE
authors.lastname: Woods
authors.forename: Frank C
authors.initials: FC
authors.lastname: Gherardini
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000145:classification / Q000235:genetics / Q000502:physiology*; D016207:Cytokines / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000382:microbiology* / Q000648:ultrastructure; D051379:Mice; D009154:Mutation; D010641:Phenotype; D014617:Vacuoles / Q000382:microbiology*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D016207:Cytokines