Burkholderia malleiCluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

ABSTRACTBurkholderia malleiis 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 tssEmutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the tssEmutants 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 tssEmutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssEmutants 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. malleiby RAW 264.7 cells.