Burkholderia pseudomallei BimC Is Required for Actin-Based Motility, Intracellular Survival, and Virulence

<p>The intracellular pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans and various animals, is capable of survival and movement within the cytoplasm of host cells by a process known as actin-based motility. The bacterial factor BimA is required for actin-based motility through its direct interaction with actin, and by mediating actin polymerization at a single pole of the bacterium to promote movement both within and between cells. However, little is known about the other bacterial proteins required for this process. Here, we have investigated the role of the bimC gene (bpss1491) which lies immediately upstream of the bimA gene (bpss1492) on the B. pseudomallei chromosome 2. Conserved amongst all B. pseudomallei, B. mallei and B. thailandensis strains sequenced to date, this gene encodes an iron-binding protein with homology to a group of proteins known as the bacterial autotransporter heptosyltransferase (BAHT) family. We have constructed a B. pseudomallei bimC deletion mutant and demonstrate that it is defective in intracellular survival in HeLa cells, but not in J774.1 macrophage-like cells. The bimC mutant is defective in cell to cell spread as demonstrated by ablation of plaque formation in HeLa cells, and by the inability to form multi-nucleated giant cells in J774.1 cells. These phenotypes in intracellular survival and cell to cell spread are not due to the loss of expression and polar localization of the BimA protein on the surface of intracellular bacteria, however they do correlate with an inability of the bacteria to recruit and polymerize actin. Furthermore, we also establish a role for bimC in virulence of B. pseudomallei using a Galleria mellonella larvae model of infection. Taken together, our findings indicate that B. pseudomallei BimC plays an important role in intracellular behavior and virulence of this emerging pathogen.</p

Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence

This is the final version. Available on open access from Nature Research via the DOI in this recordPhospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.Mahidol UniversityThailand Research Fun

Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil

Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection.

Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei

<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

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

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

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