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Infection and Immunity, July 2008, p. 2991-3000, Vol. 76, No. 7
0019-9567/08/$08.00+0     doi:10.1128/IAI.00263-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Mary N. Burtnick,^1, Paul J. Brett,^1, Vinod Nair,² Jonathan M. Warawa,¹ Donald E. Woods,³ and Frank C. Gherardini^1*

Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840,¹ Research Technologies Section, RTB, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840,² Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada³

Received 25 February 2008/ Returned for modification 18 April 2008/ Accepted 22 April 2008

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

Published ahead of print on 28 April 2008.

Editor: A. J. Bäumler

M.N.B. and P.J.B. contributed equally to this work.