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

Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840; Research Technologies Section, RTB, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840; and Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada

^* To whom correspondence should be addressed. Email: fgherardini{at}niaid.nih.gov.

	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 (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 TNF-, IL-6, GM-CSF and RANTES but not IL-1 and IL-1 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 post-infection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs displayed 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 clusters 1, 2 or 3 to facilitate survival, delayed vacuolar escape and actin-based motility in activated RAW 264.7 macrophages.