Burkholderia pseudomallei Type III Secretion System Mutants Exhibit Delayed Vacuolar Escape Phenotypes in RAW 264.7 Murine Macrophages

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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-α), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1α and IL-1β, 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.