Characterization of the Burkholderia cenocepacia TonB Mutant as a Potential Live Attenuated Vaccine.

Burkholderia cenocepacia is an opportunistic pathogen prevalent in cystic fibrosis patients, which is particularly difficult to treat, causing chronic and eventually fatal infections. The lack of effective treatment options makes evident the need to develop alternative therapeutic or prophylactic approaches. Vaccines, and live attenuated vaccines, are an unexplored avenue to treat B. cenocepacia infections. Here we constructed and characterized a B. cenocepacia tonB mutant strain, which was unable to actively transport iron, to test whether this single gene deletion mutant (strain renamed GAP001) protected against an acute respiratory B. cenocepacia lethal infection. Here we show that the mutant strain GAP001 is attenuated, and effective at protecting against B. cenocepacia challenge. Intranasal administration of GAP001 to BALB/c mice resulted in almost complete survival with high degree of bacterial clearance.

Comparing in vitro and in vivo virulence phenotypes of Burkholderia pseudomallei type G strains.

Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains.

Burkholderia cepacia Complex Vaccines: Where Do We Go from here?

Burkholderia comprises a wide variety of environmental Gram-negative bacteria. Burkholderia cepacia complex (Bcc) includes several Burkholderia species that pose a health hazard as they are able to cause respiratory infections in patients with chronic granulomatous disease and cystic fibrosis. Due to the intrinsic resistance to a wide array of antibiotics and naturally occurring immune evasion strategies, treatment of Bcc infections often proves to be unsuccessful. To date, limited work related to vaccine development has been performed for Bcc pathogens. In this review, we have gathered key aspects of Bcc research that have been reported in recent years related to vaccine efforts, virulence, immune responses, and animal models, and use this information to inform the research community of areas of opportunity toward development of a viable Bcc vaccine.

Microarray analysis of the Escherichia coli response to CdTe-GSH Quantum Dots: understanding the bacterial toxicity of semiconductor nanoparticles.

BACKGROUND: Most semiconductor nanoparticles used in biomedical applications are made of heavy metals and involve synthetic methods that require organic solvents and high temperatures. This issue makes the development of water-soluble nanoparticles with lower toxicity a major topic of interest. In a previous work our group described a biomimetic method for the aqueous synthesis of CdTe-GSH Quantum Dots (QDs) using biomolecules present in cells as reducing and stabilizing agents. This protocol produces nanoparticles with good fluorescent properties and less toxicity than those synthesized by regular chemical methods. Nevertheless, biomimetic CdTe-GSH nanoparticles still display some toxicity, so it is important to know in detail the effects of these semiconductor nanoparticles on cells, their levels of toxicity and the strategies that cells develop to overcome it. RESULTS: In this work, the response of E. coli exposed to different sized-CdTe-GSH QDs synthesized by a biomimetic protocol was evaluated through transcriptomic, biochemical, microbiological and genetic approaches. It was determined that: i) red QDs (5 nm) display higher toxicity than green (3 nm), ii) QDs mainly induce expression of genes involved with Cd+2 stress (zntA and znuA) and tellurium does not contribute significantly to QDs-mediated toxicity since cells incorporate low levels of Te, iii) red QDs also induce genes related to oxidative stress response and membrane proteins, iv) Cd2+ release is higher in red QDs, and v) QDs render the cells more sensitive to polymyxin B. CONCLUSION: Based on the results obtained in this work, a general model of CdTe-GSH QDs toxicity in E. coli is proposed. Results indicate that bacterial toxicity of QDs is mainly associated with cadmium release, oxidative stress and loss of membrane integrity. The higher toxicity of red QDs is most probably due to higher cadmium content and release from the nanoparticle as compared to green QDs. Moreover, QDs-treated cells become more sensitive to polymyxin B making these biomimetic QDs candidates for adjuvant therapies against bacterial infections.

Monounsaturated fatty acids are substrates for aldehyde generation in tellurite-exposed Escherichia coli.

Reactive oxygen species (ROS) damage macromolecules and cellular components in nearly all kinds of cells and often generate toxic intracellular byproducts. In this work, aldehyde generation derived from the Escherichia coli membrane oxidation as well as membrane fatty acid profiles, protein oxidation, and bacterial resistance to oxidative stress elicitors was evaluated. Studies included wild-type cells as well as cells exhibiting a modulated monounsaturated fatty acid (MUFA) ratio. The hydroxyaldehyde 4-hydroxy 2-nonenal was found to be most likely produced by E. coli, whose levels are dependent upon exposure to oxidative stress elicitors. Aldehyde amounts and markers of oxidative damage decreased upon exposure to E. coli containing low MUFA ratios, which was paralleled by a concomitant increase in resistance to ROS-generating compounds. MUFAs ratio, lipid peroxidation, and aldehyde generation were found to be directly related; that is, the lower the MUFAs ratio, the lower the peroxide and aldehyde generation levels. These results provide additional evidence about MUFAs being targets for membrane lipid oxidation and their relevance in aldehyde generation.

Reduction of the monounsaturated fatty acid content of Escherichia coli results in increased resistance to oxidative damage.

Reactive oxygen species (ROSs) affect several macromolecules and cellular components in eukaryotic and prokaryotic cells. In this work, the effect of various ROS-generating compounds on the Escherichia coli membrane was studied. Membrane fatty acid profiles, oxidative damage levels and bacterial resistance to these toxicants were determined. Studies included wild-type cells as well as a strain exhibiting a modified monounsaturated fatty acid (MUFA) profile (accomplished by overexpressing the ß-hydroxyacyl acyl carrier protein dehydratase-encoding gene, fabA). Levels of membrane MUFAs and oxidative damage markers decreased slightly upon toxicant exposure with a concomitant increase in cell resistance to these ROS-generating compounds. A direct relationship between MUFAs and lipid peroxidation was observed. The lower the MUFA the lower the peroxide levels, suggesting that MUFAs are targets for membrane lipid oxidation.

Production of dimethyl triselenide and dimethyl diselenenyl sulfide in the headspace of metalloid-resistant Bacillus species grown in the presence of selenium oxyanions.

A Bacillus species harvested from the environment is metalloid resistant and, when grown anaerobically in complex growth medium and amended with the selenium oxyanion selenate, selenite, or selenocyanate, produces volatile organoselenium compounds in bacterial culture headspace. Two novel compounds so far undetected in bacterial culture headspace, CH3Se2SCH3 and CH3SeSeSeCH3, are produced and can be detected using solid-phase microextraction and gas chromatography with either fluorine-induced chemiluminescence or mass spectrometric detection. Differences in the electron impact fragmentation pattern of the mixed sulfur/selenide compounds allow the tentative differentiation between the symmetric and asymmetric isomers in this bacterium's headspace in favor of the asymmetric CH3SeSeSCH3 isomer.

Biological interactions of selenocyanate: bioprocessing, detection and toxicity.

The selenocyanate anion, SeCN(-), has been reported in wastewater from refineries whose petroleum comes from Se-rich marine shales. A metalloid-resistant bacterium was exposed to aqueous solutions of SeCN(-) to examine the relative toxicity of SeCN(-), and the results were compared with the toxicity of selenate and selenite and another G16 metalloid oxyanion, tellurite. We also determined the volatile organo-selenium species produced by bacterial cultures amended with selenocyanate anion, and we investigated a solid phase preconcentration technique for collecting SeCN(-) from aqueous samples with different ionic strengths and subsequent detection using capillary electrophoresis. The relative toxicity of SeCN(-) is comparable to that of selenate and selenite using the metalloid-resistant bacterium LHVE as the test organism. Tellurite was more toxic at all concentrations examined than all three selenium-containing anions, SeO4(2-), SeO3(2-), SeCN(-). Live cultures of LHVE amended with 1 mM NaSeCN produced volatile organo-sulphides and organo-selenides that could be collected in headspace using a solid phase microextraction fibre. The bioprocessing, i.e. the reduction and methylation of SeCN(-), is similar to that of selenate and selenite by other metalloid-resistant bacteria. An aqueous 1.0 mM solution of SeCN(-) could be captured from solution on solid-phase extraction (SPE) cartridges using an aminopropyl-based stationary phase. Selenocyanate anions, slowly pumped into a wetted SPE cartridge, were trapped on the cartridge's solid phase and were subsequently eluted, thereby providing an increase in concentration above that of the original SeCN(-)-containing solution. Preconcentration factors of 3.9 were achieved using a mixed sodium hydroxide/methanol elution solvent and by adding NaCl to aqueous SeCN(-) before loading on the SPE cartridge.

Tellurite-mediated disabling of [4Fe-4S] clusters of Escherichia coli dehydratases.

The tellurium oxyanion tellurite is toxic for most organisms and it seems to alter a number of intracellular targets. In this work the toxic effects of tellurite upon Escherichia coli [4Fe-4S] cluster-containing dehydratases was studied. Reactive oxygen species (ROS)-sensitive fumarase A (FumA) and aconitase B (AcnB) as well as ROS-resistant fumarase C (FumC) and aconitase A (AcnA) were assayed in cell-free extracts from tellurite-exposed cells in both the presence and absence of oxygen. While over 90 % of FumA and AcnB activities were lost in the presence of oxygen, no enzyme inactivation was observed in anaerobiosis. This result was not dependent upon protein biosynthesis, as determined using translation-arrested cells. Enzyme activity of purified FumA and AcnB was inhibited when exposed to an in vitro superoxide-generating, tellurite-reducing system (ITRS). No inhibitory effect was observed when tellurite was omitted from the ITRS. In vivo and in vitro reconstitution experiments with tellurite-damaged FumA and AcnB suggested that tellurite effects involve [Fe-S] cluster disabling. In fact, after exposing FumA to ITRS, released ferrous ion from the enzyme was demonstrated by spectroscopic analysis using the specific Fe(2+) chelator 2,2'-bipyridyl. Subsequent spectroscopic paramagnetic resonance analysis of FumA exposed to ITRS showed the characteristic signal of an oxidatively inactivated [3Fe-4S](+) cluster. These results suggest that tellurite inactivates enzymes of this kind via a superoxide-dependent disabling of their [4Fe-4S] catalytic clusters.

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli.

Potassium tellurite (K(2)TeO(3)) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

Escherichia coli YqhD exhibits aldehyde reductase activity and protects from the harmful effect of lipid peroxidation-derived aldehydes.

Evidence that Escherichia coli YqhD is involved in bacterial response to compounds that generate membrane lipid peroxidation is presented. Overexpression of yqhD results in increased resistance to the reactive oxygen species-generating compounds hydrogen peroxide, paraquat, chromate, and potassium tellurite. Increased tolerance was also observed for the lipid peroxidation-derived aldehydes butanaldehyde, propanaldehyde, acrolein, and malondialdehyde and the membrane-peroxidizing compound tert-butylhydroperoxide. Expression of yqhD was also associated with changes in the concentration of intracellular peroxides and cytoplasmic protein carbonyl content and with a reduction in intracellular acrolein levels. When compared with the wild type strain, an yqhD mutant exhibited a sensitive phenotype to all these compounds and also augmented levels of thiobarbituric acid-reactive substances, which may indicate an increased level of lipid peroxidation. Purified YqhD catalyzes the in vitro reduction of acetaldehyde, malondialdehyde, propanaldehyde, butanaldehyde, and acrolein in a NADPH-dependent reaction. Finally, yqhD transcription was induced in cells that had been exposed to conditions favoring lipid peroxidation. Taken together these results indicate that this enzyme may have a physiological function by protecting the cell against the toxic effect of aldehydes derived from lipid oxidation. We speculate that in Escherichia coli YqhD is part of a glutathione-independent, NADPH-dependent response mechanism to lipid peroxidation.

Capillary electrophoretic determination of selenocyanate and selenium and tellurium oxyanions in bacterial cultures.

A simple capillary zone electrophoretic method for the determination of biospherically important oxyanions of selenium (Se) and tellurium and another Se-containing anion, selenocyanate, has been developed. The method uses direct UV absorption detection. Time course experiments with time slices as short as 6 min are possible. This method's detection limits and linear range compare well with other methods involving samples containing complex biological matrices. The metalloid-containing anions examined were selenocyanate, selenite, selenate, tellurite, and tellurate. We applied this method to live cultures of two different bacteria in two different growth media in time course experiments following the changes in metalloid-containing anion concentrations. The results show that this method is a useful means of following the biological processing of these analytes in bacterial cultures.

Bacterial toxicity of potassium tellurite: unveiling an ancient enigma.

Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2-)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2-) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium.

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host .zntA gene product.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host ·zntA gene product.