Effect of <i>chbP</i> mutation on <i>B. pseudomallei</i> plaque formation.

<p>A) Plaque-forming efficiency. HeLa cells were infected with <i>B. pseudomallei</i> (K96243, <i>chbP</i> mutant or <i>chbP</i>/pCHBP strain) at an MOI of 20. Plaque-forming efficiency was established following staining of the infected cells with crystal violet. Plaque-forming efficiency at 21 h was calculated by the following equation: number of plaques/CFU of bacteria added per well. Asterisks indicate significant differences (P value <0.05, <i>t</i>-test) between groups. Error bars represent standard errors of the means for experiments performed in triplicate. B) Photographs of plaques. Representative images of the infected cell monolayers after infection with <i>B. pseudomallei</i> K96243, <i>chbP</i> mutant or <i>chbP</i>/pCHBP strains for 21 and 24 h. Note the reduced number of plaques and reduced plaque size of the <i>chbP</i> mutant.</p

SDS-PAGE and Western blot analysis of CHBP in <i>B. pseudomallei</i> K96243 wild-type, <i>chbP</i> mutant and <i>trans</i>-complemented strains.

<p>A) SDS-PAGE. Bacterial lysates and secreted proteins of <i>B. pseudomallei</i> K96243, <i>chbP</i> mutant or <i>chbP</i>/pCHBP strain cultured in LB broth for 6 h were separated by 12% polyacrylamide gel electrophoresis. B) Western blot analysis. The blotted proteins from A) were separately probed with anti-CHBP and anti-BopE antibodies. Molecular mass markers are shown on the left. Lanes 1–3 are bacterial cell lysates and lanes 4–6 are secreted proteins precipitated from culture supernatants.</p

Confocal micrographs indicating <i>bsaQ</i>-dependent secretion of CHBP in U937 cells infected with <i>B. pseudomallei</i>.

<p>PMA-activated U937 cells were separately infected with <i>B. pseudomallei</i> (K96243, <i>bsaQ</i> or <i>chbP</i> mutant strain). At different time points of infection (3, 6, 9 and 12 h), infected cells were stained using purified rabbit anti-CHBP antibody detected with anti-rabbit Ig-Alexa Fluor⁴⁸⁸ (Molecular Probes). The bottom panel shows the localization of CHBP and the top panel merges this signal with DIC images showing the position of infected cells. Scale bars, 10 µm.</p

Effect of <i>chbP</i> mutation on <i>B. pseudomallei-</i>induced cytotoxicity.

<p>A) HeLa cells and B) U937 cells were infected with <i>B. pseudomallei</i> (K96243, <i>chbP</i> mutant or <i>chbP</i>/pCHBP strain) with a range of MOIs. After 6 h, cytotoxicity was assessed using the CytoTox96 lactate dehydrogenase (LDH)-release kit (Promega). Asterisks indicate significant differences (P value <0.05, <i>t</i>-test) between groups. Error bars represent standard errors of the means for experiments performed in triplicate.</p

Confocal micrographs of CHBP expression and localization in U937 cells infected with <i>B. pseudomallei</i>.

<p>PMA-activated U937 cells were separately infected with three strains of <i>B. pseudomallei</i> (K96243, <i>chbP</i> mutant or <i>chbP</i>/pCHBP strain). After 6 h, infected cells were fixed, permeabilized andstained using purified rabbit anti-CHBP antibody detected with anti-rabbit Ig-Alexa Fluor⁴⁸⁸ (Molecular Probes). The bottom panel shows the localization of CHBP and the top panel merges this signal with differential interference contrast (DIC) images showing the position of infected cells. Scale bars, 20 µm.</p

SDS-PAGE and Western blot analysis of CHBP in <i>B. pseudomallei-</i>infected U937 cells.

<p>A) Protein from lysates of U937 cells infected at an MOI of 2 with <i>B. pseudomallei</i> K96243, <i>chbP</i> mutant, <i>chbP</i>/pCHBP strain or <i>bsaQ</i> mutant for 3 or 6 h were separated by 12% polyacrylamide gel electrophoresis and blotted with anti-CHBP and anti-BopE antibodies. Molecular mass markers are shown on the left. Panel B shows data from an identical experiment, except using an MOI of 100.</p

Plaque forming, invasion efficiencies and net intracellular survival and replication of <i>B</i>. <i>pseudomallei</i> wild-type and its derivative strains.

<p>(A) Plaque forming and (B) invasion efficiencies of <i>B</i>. <i>pseudomallei</i> K96243 wild-type, 6H4 mutant or 6H4/pME1039 complemented strains in infect HeLa cells. Plaque-forming efficiency was calculated as: number of plaques/bacterial CFU added per well. Percent invasion was determined as: (number of intracellular bacteria post infection/number of CFU added) × 100. <i>B</i>. <i>pseudomallei</i> K96243 wild-type (black bar), 6H4 mutant (white bar) and the 6H4/pME1039 complemented (checked bar) strains were used to infect (C) HeLa and (D) J774A.1 macrophage cells. Intracellular loads of bacteria were enumerated at 4, 6, 8, and 24 h p.i. Asterisks indicate significant differences (<i>P</i> < 0.05, <i>t</i>-test) between wild-type and its derivative strains. Results are presented as standard errors of the means for experiments done in triplicate, with three independent experiments.</p

<i>B</i>. <i>pseudomallei bpsl1039</i> 6H4 mutant is attenuated in mice.

<p>Survival of BALB/c mice inoculated <i>via</i> the intranasal route with <i>B</i>. <i>pseudomallei</i> K96243 wild-type (●), 6H4 (▼) and 6H4/pME1039 complemented (◆) strains were determined (n = 5 per group). The survival curves of mice infected with wild-type and 6H4 were significantly different (<i>P</i> < 0.01). Data were analysed using the Log-rank (Mantel-Cox) test with a Bonferroni correction.</p

Effect of nitrate and anaerobic culture conditions on <i>B</i>. <i>pseudomallei</i> growth.

<p><i>B</i>. <i>pseudomallei</i> was inoculated in M9 minimal medium supplemented with (+NaNO₃) or without (-NaNO₃) sodium nitrate, and incubated under aerobic or anaerobic culture conditions. (A) anaerobic and (B) aerobic kinetic growth curves of <i>B</i>. <i>pseudomallei</i> cultured in the presence of nitrate. <i>B</i>. <i>pseudomallei</i> K96243 wild-type (black circle), 6H4 mutant (white circle), 6H4/pME1039 complementation (white square) strains were grown for 72 h. Every 24 h, the numbers of viable bacteria were determined by plating on LB agar for colony count. (C) Growths of <i>B</i>. <i>pseudomallei</i> K96243 wild-type (black bar), 6H4 mutant (white bar) and 6H4/pME1039 (checked bar) strains under aerobic (+O₂) and anaerobic (-O₂) culture conditions at 48 h after bacterial inoculation. Results are presented from at least three replicates with three independent experiments. Asterisks indicate statistically significant differences (<i>P</i> < 0.05, <i>t</i>-test).</p

Nitrate reductase activity of <i>B</i>. <i>pseudomallei</i> wild-type and its derivative strains.

<p><i>B</i>. <i>pseudomallei</i> K96243 wild-type, 6H4 mutant and 6H4/pME1039 complemented strains were cultured in LB medium supplemented with 40 mM sodium nitrate. The nitrate reductase activitiy of each <i>B</i>. <i>pseudomallei</i> strain was determined under permeabilised (dot bar) or unpermeabilised conditions (black bar). The level of nitrate reductase activity was measured at absorbance 420 nm and 540 nm. Results are presented as standard errors of the means for experiments done in quadruplicate with two independent experiments. Asterisks indicate significant differences (<i>P</i> < 0.05, <i>t</i>-test).</p