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

Additional file 1 of Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Additional file 1: Figure S1. Primers used in (A) multiplex HRM for the detection of 15 G6PD mutations and (B) G6PD gene sequencing. Table S1. Primers used in multiplex HRM assays. Table S2. Primers used for site-directed mutagenesis. Figure S2. The frequency distribution of G6PD enzyme activity in (A) males and (B) females. Figure S3. Box plot of G6PD activity for each variant among (A) malaria-positive males, (B) malaria-positive females, (C) malaria-negative males and (D) malaria-negative females. Figure S4. Secondary structure analysis of G6PD variants by circular dichroism. Table S3. Melting temperature (Tm) values of recombinant G6PD proteins by thermal shift assay. Mutations were ranked in order of stability, from most stable to least stable. Table S4. Thermal inactivation of G6PD variants as reported by T1/2. Mutations were ranked in order of stability, from most stable to least stable. Table S5. Stability of G6PD variants in the presence of Gdn-HCl as reported by C1/2. Mutations were ranked in order of stability, from most stable to least stable. Table S6. Susceptibility of G6PD variants to trypsin digestion. Mutations were ranked in order of stability, from most stable to least stable. Table S7. Structural characteristics of the dimer and tetramer interfaces (t = 100 ns). Table S8. Average values of the trajectory analyses performed on the WT and variants. Figure S5. Ligand binding pocket occupancy heatmap indicating the presence (orange) and absence (turquoise) of hydrogen bonds (t = 100 ns). Figure S6. Superimposition and structural deviations of the simulated variants against the WT (red) at the mutation site, dimer and tetramer interfaces (t = 100 ns). (A) Gaohe, (B) Valladolid, (C) Canton, (D) Viangchan, (E) Gond, (F) Gaohe + Viangchan, (G) Valladolid + Viangchan, and (H) Canton + Viangchan

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

Gene co-localization of <i>B</i>. <i>pseudomallei bpsl1039-bpsl1040</i> and RT-PCR analysis.

<p>(A) Organization of <i>B</i>. <i>pseudomallei bpsl1039-bpsl1040</i> genes and the location of primer pairs used in RT-PCR analysis. (B) RT-PCR analysis using primers 1039-F2 and 1040-R showed co-transcription of <i>B</i>. <i>pseudomallei bpsl1039-bpsl1040</i> genes (lane 3). Lanes 1 and 2 represent positive and negative controls, respectively, using wild-type genomic DNA and the extracted RNA, respectively. A negative RT-PCR control (lane 2) confirms that the band observed in the positive reaction is not DNA contamination. (C) RT-PCR analysis of <i>bpsl1040</i> expression, using primers 1040-F and 1040-R, was performed in <i>B</i>. <i>pseudomallei</i> wild-type (K96243) and 6H4 mutant. The 6H4 mutant showed the absence of <i>bpsl1040</i> expression. <i>B</i>. <i>pseudomallei</i> 16S rRNA gene was amplified as control. Lane M represents 1 Kb DNA marker ladder.</p

3D structural model of the <i>pkdhfr</i> 91P mutation and molecular docking of pyrimethamine in the active site.

<p>*Image panel attached separately. a) 3D structural model of <i>pkdhfr</i> with pyrimethamine bound at the active site. Mutations found in this study were shown as ball and stick coloured yellow. Equivalent residues of <i>pkdhfr</i> known to be related to pyrimethamine resistance in <i>pfdhfr</i> were presented as stick coloured dark pink and pyrimethamine shown as stick coloured magenta. b) Modelled <i>pkdhfr</i> binding site interaction with pyrimethamine. Pyrimethamine molecule was presented as stick and amino acid residues of <i>pkdhfr</i> were presented as line with carbon, nitrogen, oxygen and chloride colored as dark grey, blue, red and green, respectively. Hydrogen bonds are shown as green dashed line. c) 2D ligand interaction diagram of modelled pyrimethamine binding with all surrounding residues in active site of <i>pkdhfr</i> showing only contact with Ile13, Asp53, Ile 173 and Thr194. Hydrogen bonds are shown as dashed line. <i>Figure was generated by Discovery Studio Visualizer–Accelrys</i>.</p

Baseline features of patients with <i>pkdhfr</i> sequenced.

<p>Data are N (%) unless otherwise indicated. No significant differences (p<0.05) were seen between those with pkdhfr mutations and wild type.</p

Relative distribution of <i>pkdhfr</i> genotypes from a tertiary and district referral hospitals in Sabah (with district administrative borders).

<p>*Image attached separately. a) All mutations. b) Wild-type. c) T91P mutations. d) R34L mutations.</p

<i>Pkdhfr</i> genotypes among patients enrolled at QEH, Kudat District Hospital and Kota Marudu District Hospital.

<p><i>Pkdhfr</i> genotypes among patients enrolled at QEH, Kudat District Hospital and Kota Marudu District Hospital.</p