title: Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages.
abstract: Burkholderia mallei is a facultative intracellular pathogen that causes severe disease in animals and humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant growth defects were observed in comparison to controls. In addition, analysis of infected monolayers by differential interference contrast and fluorescence microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from lysosome-associated membrane protein 1-positive vacuoles. Curiously, however, following entry into the cytosol, the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in growth and actin-based motility following uptake of B. mallei by RAW 264.7 cells.
authors.affiliation: Department of Microbiology and Immunology, University of South Alabama, Mobile, AL 36688, USA.
authors.forename: Mary N
authors.initials: MN
authors.lastname: Burtnick
authors.forename: David
authors.initials: D
authors.lastname: DeShazer
authors.forename: Vinod
authors.initials: V
authors.lastname: Nair
authors.forename: Frank C
authors.initials: FC
authors.lastname: Gherardini
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brett
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D042726:Burkholderia mallei / Q000166:cytology / Q000235:genetics* / Q000254:growth & development*; D002460:Cell Line; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D009154:Mutation; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D051907:Lysosomal Membrane Proteins; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1