Burkholderia pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages

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

FIG. 1.

Survival characteristics of B. pseudomallei in RAW 264.7 cells. Monolayers were infected with B. pseudomallei DD503 (•) or ZP1220 (○) at an MOI of 10, and intracellular loads of bacteria were quantified at 3, 6, 12, and 18 h postinfection. The values are the means ± standard deviations of three independent experiments.

FIG. 2.

Light micrographs of RAW 264.7 cells infected with B. pseudomallei. The monolayers were visualized at 18 h postinfection and were infected as follows: (A) mock infected; (B) DD503 infected (MOI, 10); and (C) ZP1220 infected (MOI, 10). Bars = 50 μm. The micrographs are representative of at least three independent experiments.

FIG. 3.

Cellular integrity of RAW 264.7 cells infected with B. pseudomallei. Monolayers were infected with B. pseudomallei DD503 (•) or ZP1220 (○) at an MOI of 10. The percent cytotoxicity was determined by assaying for LDH release in culture supernatants at 6, 12, and 18 h postinfection. The values are the means ± standard deviations of three independent experiments performed in duplicate.

FIG. 4.

Cytokine and chemokine production by B. pseudomallei-infected RAW 264.7 cells. Culture supernatants from monolayers infected with B. pseudomallei DD503 (•) or ZP1220 (○) at an MOI of 10 or mock infected (▪) were harvested at 6, 12, and 18 h postinfection and assayed for the production of (A) TNF-α, (B) IL-1α, (C) IL-1β, (D) IL-6, (E) RANTES, and (F) GM-CSF. The values are the means ± standard deviations of three independent experiments performed in duplicate.

FIG. 5.

TEM micrographs of RAW 264.7 cells infected with B. pseudomallei. Fixed monolayers infected with B. pseudomallei DD503 (A and B) or ZP1220 (C and D) at an MOI of 10 were examined at 1 h (A), 3 h (B), 6 h (C), or 12 h (D) postinfection. The arrows indicate vacuolar membranes. Bars = 500 nm.

FIG. 6.

Confocal micrographs of RAW 264.7 cells infected with B. pseudomallei. Fixed monolayers infected with B. pseudomallei DD503 (A to C) or ZP1220 (D to F) at an MOI of 40 were examined at 6 h (A and D), 12 h (B and E), and 18 h (C and F) postinfection. Bacteria were stained red with rabbit anti-B. thailandensis polyclonal sera and anti-rabbit IgG Alexa Fluor 568, actin was stained green with Alexa Fluor 488 phalloidin, and nuclei were stained blue with DRAQ5. The micrographs are representative of at least three independent experiments.

FIG. 7.

Interactions of B. pseudomallei ΔsctU_Bp3, ΔsctU_Bp1,2,₃, and DD503 with RAW 264.7 cells. Monolayers were infected at an MOI of 10 (A to D). (A) Bacterial uptake (open bars) and survival (filled bars) in RAW 264.7 cells were determined at 3 and 18 h postinfection, respectively. (B) Culture supernatants were assayed for IL-1β production at 18 h. (C) Percent cytotoxicity was determined by assaying for LDH release in culture supernatants at 18 h. The values are the means and standard deviations of three independent experiments performed in duplicate. (D) Light micrograph of ΔsctU_Bp1,2,₃-infected RAW 264.7 cells at 18 h. Bar = 50 μm. (E and F) Confocal micrographs of RAW 264.7 cells infected with ΔsctU_Bp1,2,₃ at an MOI of 40 at 18 h postinfection. Bacteria were stained red with rabbit anti-B. thailandensis polyclonal sera and anti-rabbit IgG Alexa Fluor 568, actin was stained green with Alexa Fluor 488 phalloidin, and nuclei were stained blue with DRAQ5. The micrographs are representative of at least two independent experiments.