Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se

Neutrophils form extracellular traps in response to Opisthorchis viverrini crude antigens and these traps are elevated in neutrophils from opisthorchiasis patients with hepatobiliary abnormalities.

Opisthorchis viverrini (Ov) infection can cause several disease conditions of the bile duct including hepatobiliary abnormalities (HBAs) and the most severe, cholangiocarcinoma (CCA). Fibrosis occurs when tissues are damaged and normal wound-healing responses are dysregulated. Neutrophils are the first cells to migrate to an infection site to protect the host from intruding extracellular pathogens through a wide range of effector mechanisms such as phagocytosis, production of reactive oxygen species, proteases, or release of neutrophil extracellular traps (NETs). In this work, we used confocal microscopy to assess whether Ov crude antigens can cause release of NETs from neutrophils from Ov-free individuals. We demonstrated for the first time that these antigens could induce release of NETs ex-vivo in a dose-dependent manner from neutrophils isolated from Ov-free individuals. Intriguingly, when we measured NETs from neutrophils isolated from Ov-infected patients, we found increased spontaneous production of NETs in patients with HBAs. Interestingly, exposure to Ov crude antigens lowered the level of NETs released by neutrophils from patients with active Ov infection regardless of HBA status. We propose that in the case of acute Ov infection, even when concentration of Ov antigens is relatively low, neutrophils can form NETs. However, when this infection becomes chronic, manifesting as a definite HBA, the levels of NET production are reduced when treated with Ov crude antigens. Excessive production of proinflammatory mediators from these NETs might have effects on the parasites, but may also lead to excessive injury of surrounding tissues resulting in HBAs and may lead eventually to the most severe complications such as CCA

DNase I and chitosan enhance efficacy of ceftazidime to eradicate Burkholderia pseudomallei biofilm cells

Abstract Biofilm-associated Burkholderia pseudomallei infection contributes to antibiotic resistance and relapse of melioidosis. Burkholderia pseudomallei biofilm matrix contains extracellular DNA (eDNA) that is crucial for biofilm establishment. However, the contribution of eDNA to antibiotic resistance by B. pseudomallei remains unclear. In this study, we first demonstrated in vitro that DNase I with the administration of ceftazidime (CAZ) at 24 h considerably inhibited the 2-day biofilm formation and reduced the number of viable biofilm cells of clinical B. pseudomallei isolates compared to biofilm treated with CAZ alone. A 3–4 log reduction in numbers of viable cells embedded in the 2-day biofilm was observed when CAZ was combined with DNase I. Confocal laser-scanning microscope visualization emphasized the competence of DNase I followed by CAZ supplementation to significantly limit B. pseudomallei biofilm development and to eradicate viable embedded B. pseudomallei biofilm cells. Furthermore, DNase I supplemented with chitosan (CS) linked with CAZ (CS/CAZ) significantly eradicated shedding planktonic and biofilm cells. These findings indicated that DNase I effectively degraded eDNA leading to biofilm inhibition and dispersion, subsequently allowing CAZ and CS/CAZ to eradicate both shedding planktonic and embedded biofilm cells. These findings provide efficient strategies to interrupt biofilm formation and improve antibiotic susceptibility of biofilm-associated infections

Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei.

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0-6 h using the plate count technique on Ashdown's agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist

Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm

Burkholderia pseudomallei biofilm resists Acanthamoeba sp. grazing and produces 8-O-4′-diferulic acid, a superoxide scavenging metabolite after passage through the amoeba

Abstract Burkholderia pseudomallei, an etiological agent of melioidosis is an environmental bacterium that can survive as an intracellular pathogen. The biofilm produced by B. pseudomallei is crucial for cellular pathogenesis of melioidosis. The purpose of this investigation is to explore the role of biofilm in survival of B. pseudomallei during encounters with Acanthamoeba sp. using B. pseudomallei H777 (a biofilm wild type), M10 (a biofilm defect mutant) and C17 (a biofilm-complemented strain). The results demonstrated similar adhesion to amoebae by both the biofilm wild type and biofilm mutant strains. There was higher initial internalisation, but the difference diminished after longer encounter with the amoeba. Interestingly, confocal laser scanning microscopy demonstrated that pre-formed biofilm of B. pseudomallei H777 and C17 were markedly more persistent in the face of Acanthamoeba sp. grazing than that of M10. Metabolomic analysis revealed a significant increased level of 8-O-4′-diferulic acid, a superoxide scavenger metabolite, in B. pseudomallei H777 serially passaged in Acanthamoeba sp. The interaction between B. pseudomallei with a free-living amoeba may indicate the evolutionary pathway that enables the bacterium to withstand superoxide radicals in intracellular environments. This study supports the hypothesis that B. pseudomallei biofilm persists under grazing by amoebae and suggests a strategy of metabolite production that turns this bacterium from saprophyte to intracellular pathogen

Intracellular survival through time of <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A), <i>Acanthamoeba</i> sp. (B) and isolate A-ST39-E1 (C).

<p>Time zero represents 3 hours after <i>B</i>. <i>pseudomallei</i> feeding. Bars represent the standard errors of the means of duplicate, three times independent experiments, * <i>p</i> < 0. 0001 using ANOVA.</p

<i>B</i>. <i>pseudomallei</i> is internalized into amoebae but could not resist digestion.

<p>CLSM micrographs show the internalized <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A-C), <i>Acanthamoeba</i> sp. (D-F) and isolate A-ST39-E1 (G-I) at 0, 3 and 6 h after kanamycin treatment. Orange fluorescence represents CellTracker^™ Orange-<i>B</i>. <i>pseudomallei</i> and green fluorescence indicates the amoebae stained with FITC-ConA for visualization.</p

Numbers of <i>Acanthamoeba</i> sp. and isolate A-ST39-E1 over time (A-B and C-D respectively) after feeding with <i>B</i>. <i>pseudomallei</i> (▲) or <i>E</i>. <i>coli</i> (positive control) (■) or deprived of bacteria as a negative control (●).

<p>Graphs and figures show no significant differences between amoebae fed on <i>B</i>. <i>pseudomallei</i> and <i>E</i>. <i>coli</i>. However, numbers of amoebae in the negative control group were significantly lower than in the pother groups (<i>p</i> ≤ 0.0001). Data are mean ± SD from duplicates of the three independent experiments.</p

Specific primers for the 18s rRNA gene of amoebae.

<p>Specific primers for the 18s rRNA gene of amoebae.</p