Mini-Tn7-bar-rfp, single-copy tagging vector based on phosphinothricin resistance, harboring rfp driven by the P_S12 promoter. After insertion aided by pTNS3-asd_Ec (29), the non-antibiotic resistance marker, which is flanked by identical FRTs, can be removed by Flp-mediated excision. Abbreviations: bar, gene encoding bialaphos/phosphinothricin resistance; FRT, Flp recombination target sequences; oriT, RP4 conjugal origin of transfer; PC_S12, promoter of the Burkholderia cenocepacia rpsL gene; P_S12, promoter of the B. pseudomallei rpsL gene; R6Kγori, π protein-dependent R6K origin of replication; Tn7L/Tn7R, left and right transposase recognition sequences; T_oT₁, transcriptional terminator.

Growth curve experiments performed with B. pseudomallei strains. (A) B. pseudomallei strains were grown in the absence of DAP. The wild-type strain and the Δasd strain complemented with a single copy of the asd gene on a site-specific transposon exhibited the same growth rates and final optical densities, while the Δasd mutant exhibited a typical DAP-dependent phenotype. (B) The B. pseudomallei Δasd mutant was tested in different concentrations of DAP, ranging from 0 μg/ml to 500 μg/ml. Compared to wild type, the Δasd mutant exhibited absence of growth without DAP. All other concentrations of DAP afforded a partial growth rate recovery and final optical density, albeit only after a lag in growth.

Infection of HeLa and RAW264.7 cells by B. pseudomallei and the Δasd strain. HeLa (A) and RAW264.7 (B) cell monolayers were infected at an MOI of 10:1. The complemented Δasd strain showed no decrease in its ability to invade and replicate within either cell line. However, the Δasd mutant in the absence of DAP could not sustain an infection in either cell line, denoted by an overall drop in bacterial numbers.

(A) Cytotoxicity of B. pseudomallei strains to the RAW264.7 murine macrophage cell line. RAW264.7 cells were infected with the Δasd mutant (in the presence or absence of DAP), the complemented mutant strain, and wild-type strain. Between 2 h and 6 h postinfection there was a slight increase in cytotoxicity associated with infection by the complemented and wild-type strains compared to the Δasd mutant-infected monolayers. By 12 h postinfection, cytotoxicities of the complement- and wild-type-infected monolayers were more obvious, while cytotoxicity caused by the Δasd mutant remained similar to the noninfected control. At 24 h postinfection, the complement- and wild-type-infected monolayers exhibited maximal cytotoxicity. (B) Microscopy and time course of the cytopathic effects of B. pseudomallei Δasd infection. Monolayers were infected at an MOI of 10:1 and then analyzed for red fluorescence at 2 h and 24 h postinfection. Differential interference contrast (DIC) images were overlaid with the red fluorescent channel. Red fluorescence indicates the presence of B. pseudomallei. Note the high bacterial levels in the complement- and wild-type-infected monolayers at 24 h and the confluent MNGC formation. This coincides with high levels of cytotoxicity at 24 h postinfection. Abbreviations: CT, noninfected control; Δasd, B. pseudomallei Δasd/rfp; Δasd + DAP, B. pseudomallei Δasd/rfp in the presence of 200 μg/ml of DAP; Δasd + complement, B. pseudomallei Δasd/complement/rfp; WT, B. pseudomallei wt/rfp. Error bars represent the SEM of three experiments. Statistical significance was determined by the two-tailed unpaired t test (***, P < 0.0005).

Intracellular replication of B. pseudomallei. RAW264.7 murine macrophage monolayers were visualized using a combination of differential interference contrast and red fluorescence microscopy 24 h postinfection with the B. pseudomallei Δasd/complement/rfp strain (A and B) and with B. pseudomallei wt/rfp strain (C and D). All macrophages in the field of view are interacting with MNGCs and are filled with intracellular bacteria about to burst into the extracellular milieu. Images in panels A and C were captured at 600× magnification, while images in panels B and D are zoomed-in images of the regions denoted in panels A and C, respectively. Note the large number of bacteria projecting out of the remaining macrophages in panels C and D. There were no bacteria and there was an absence of protrusions as well as MNGCs at all time points in monolayers infected with the Δasd mutant (data not shown).

(A) The B. pseudomallei 1026b Δasd mutant is avirulent in mice. Mice (n = 5 animals per group) were challenged i.n. with either 4,500 CFU B. pseudomallei 1026b (wild type) or 1 × 10⁷ CFU B. pseudomallei 1026b Δasd mutant, and survival was monitored. Statistical differences in survival times were determined by Kaplan-Meier curves followed by log-rank test (**, P < 0.01 for B. pseudomallei 1026b wt versus B. pseudomallei 1026b Δasd mutant). (B) Intranasal vaccination with the B. pseudomallei 1026b Δasd mutant protects mice from lethal B. pseudomallei challenge. Mice (n = 10 animals per group) were primed i.n. with 1 × 10⁷ CFU B. pseudomallei 1026b Δasd and boosted in the same manner 3 weeks later. Two weeks postboost, mice were challenged i.n. with 4 × 10³ CFU wild-type B. pseudomallei 1026b. Survival was monitored, and statistical differences in survival times were determined by Kaplan-Meier curves followed by a log-rank test (***, P < 0.0001 for vaccinated versus nonvaccinated mice). Data represen two individual pooled experiments.