Modeling the effect of oxidative stress on Bordetella pertussis fermentations

A mathematical model is proposed for Bordetella pertussis with the main goal to better understand and describe the relation between cell growth, oxidative stress and NADPH levels under different oxidative conditions. The model is validated with flask experiments conducted under different conditions of oxidative stress induced by high initial glutamate concentrations, low initial inoculum and secondary culturing following exposure to starvation. The model exhibited good accuracy when calibrated and validated for the different experimental conditions. From comparisons of model predictions to data with different model mechanisms, it was concluded that intracellular reactive oxidative species only have an indirect effect on growth rate by reacting with NADPH and thereby reducing the amount of NADPH that is available for growth.     

Improvement of Photorhabdus temperata bioinsecticides production in low‐cost media through adequate fermentation technology

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H₂O₂ was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO‐shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10⁸, 1.15 × 10⁸ cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Monitoring of an antigen manufacturing process

Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production. Then, partial least square (PLS) was employed to correlate the fluorescence spectra obtained from pure PRN samples and the final protein content measured by a Kjeldahl test from these samples. In view that a statistically significant correlation was found between fluorescence and PRN levels, this approach could be further used as a method to predict the final protein content.     

Isolation and characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants with enhanced tolerance to oxidative stress

We have previously reported a method for isolation of mutants with enhanced tolerance to the fungal AAL toxin and given a detailed characterization of atr1 (AAL toxin resistant, Gechev et al. in Biochem Biophys Res Commun 375:639–644, 2008). Herewith, we report eight more mutants with enhanced tolerance to the AAL toxin. Phenotypic analysis showed that six of the mutants were reduced in size compared with their original background loh2. Furthermore, atr2 showed delayed flowering and senescence. The mutants were also evaluated for oxidative stress tolerance by growing them on ROS-inducing media supplemented with either aminotriazole or paraquat, generating, respectively, H₂O₂ or superoxide radicals. Oxidative stress, confirmed by induction of the marker genes, HIGH AFFINITY NITRATE TRANSPORTER At1G08090 and HEAT SHOCK PROTEIN 17 At3G46230, inhibited growth of all lines. However, while the original background loh2 developed necrotic lesions and died rapidly on ROS-inducing plant growth media, atr1, atr2, atr7 and atr9 remained green and viable. The tolerance against oxidative stress-induced cell death was confirmed by fresh weight and chlorophyll measurements. Real-time PCR analysis revealed that the expression of the EXTENSIN gene At5G46890, previously shown to be downregulated by aminotriazole in atr1, was repressed in all lines, consistent with the growth inhibition induced by oxidative stress. Taken together, the data indicate a complex link between growth, development and oxidative stress tolerance and indicates that growth inhibition can be uncoupled from oxidative stress-induced cell death.     

Shotgun proteome analysis of Bordetella pertussis reveals a distinct influence of iron availability on the bacterial metabolism,virulence, and defense response

One of the mechanisms involved in host immunity is the limitation of iron accessibility to pathogens, which in turn provokes the corresponding physiological adaptation of pathogens. This study reports a gel‐free nanoLC‐MS/MS‐based comparative proteome analysis of Bordetella pertussis grown under iron‐excess and iron‐depleted conditions. Out of the 926 proteins covered 98 displayed a shift in their abundance in response to low iron availability. Forty‐seven of them were found to be increased in level while 58 were found with decreased protein levels under iron starvation. In addition to proteins previously reported to be influenced by iron in B. pertussis, we observed changes in metabolic proteins involved in fatty acid utilization and poly‐hydroxybutyrate production. Additionally, many bacterial virulence factors regulated by the BvgAS two‐component system were found at decreased levels in response to iron limitation. These results, together with the increased production of proteins potentially involved in oxidative stress resistance, seem to indicate that iron starvation provokes changes in B. pertussis phenotype that might shape host–pathogen interaction.     

Comparative proteomics of oxidative stress response of Lactobacillus acidophilus NCFM reveals effects on DNA repair and cysteine de novo synthesis

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H₂O_2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H₂O₂ were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide‐reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein‐refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability.     

Impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from CHO cell cultures

Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452–461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP ⁺)/NADPH-dependent manner.     

Dietary antioxidants provide differential subcellular protection in epithelial cells

Abstract

This study aimed to evaluate the organelle-specific antioxidant/pro-oxidant actions of clinically important dietary antioxidants against oxidative stress. An in vitro cellular model was employed to investigate the antioxidant/pro-oxidant effects of various concentrations (1, 10 and 100 μM) of ascorbic acid, α-tocopherol and β-carotene during H₂O₂-induced oxidative stress. Damage to nuclear and mitochondrial genomes was analyzed by quantitative polymerase chain reaction and oxidation of membrane lipids was measured via colorimetric assays. The key findings were: (i) dietary antioxidants conferred a dose-dependent protective effect (with a pro-oxidant shift at higher concentrations); (ii) the protection conferred to different sub-cellular organelles is highly specific to the dietary antioxidant; (iii) the mtDNA is highly sensitive to oxidative attack compared to nDNA (P < 0.05); and (iv) mtDNA protection conferred by dietary antioxidants was required to improve protection against oxidative-induced cell death. This study shows that antioxidant-induced protection of mtDNA is an important target for future oxidative stress therapies.     

A whole-organ perfusion model of bordetella pertussis adherence to mouse tracheal epithelium

Summary A whole-organ perfusion system was used to culture tracheas from adult Swiss mice and test this system's adaptability for use in adherence assays for virulentBordetella pertussis. Culture medium and bacterial suspensions flowed readily through the tracheal lumen, ciliary activity was maintained throughout the culture period, and scanning electron microscopy revealed retention of normal surface morphology. The number of adherent colony-forming units (cfu) per trachea was determined for all threeBordetella species every 30 min over a 3.5-h incubation period and the resultant adherence patterns were reproducible. Adherent cfu were dependent on the concentration of microorganisms in the infecting inoculum.Bordetella pertussis did not demonstrate a preferential adherence to either the dorsal or ventral surface of the tracheal epithelium nor did it demonstrate a preference for adherence to the laryngeal or bronchial end of the trachea. Static growth conditions did alter the adherence pattern ofB. pertussis from that observed when the organism was grown with constant agitation. This work was presented in part at the 1984 annual meeting of the American Society for Microbiology, St. Louis, MO.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica

The Bordetella pertussis wlb locus (wlb_pe, formerly bpl ) is required for the biosynthesis of a trisaccharide that, when attached to the B. pertussis lipopolysaccharide (LPS) core (band B), generates band A LPS. The equivalent loci in Bordetella bronchiseptica (wlb_br) and Bordetella parapertussis (wlb_pa) were identified and cloned. The wlb_br and wlb_pa loci differ from wlb_pe in that they lack the insertion sequence that defines the right-hand terminus of wlb_pe. Deletion of 12 kb of DNA containing the whole wlb locus (Δwlb) by allelic exchange in each of the three bordetellae had no effect on band B biosynthesis, whereas band A biosynthesis was prevented in B. pertussis and B. bronchiseptica. In B. bronchiseptica and B. parapertussis, Δwlb mutants also lacked O-antigen. Reintroduction of the wlb_pe or wlb_br loci on a shuttle vector into the three Δwlb mutants restored the wild-type LPS phenotype in the B. pertussis and B. bronchiseptica mutants. In the case of B. parapertussis, which normally does not synthesize an apparent band A structure, introduction of the wlb_pe or wlb_br loci now enabled the generation of band A. This suggests that the attachment point for band A trisaccharide on the LPS core is present in B. parapertussis, and further suggests that the wild-type wlb_pa locus is not fully functional. Introduction of the wlb_pa locus into the Δwlb_pe, Δwlb_br and Δwlb_pa mutants had interesting consequences. The B. bronchiseptica and B. parapertussis recipients were now able to biosynthesize O-antigen, but no band A was generated. In the B. pertussis recipient, a truncated band A was expressed consistent with a mutation in the wlbH gene, but on Western blotting the expression of a small amount of full-length band A was also seen. Evidence that the wlbH_pa protein is not fully functional was provided by the failure of the wlb_pa locus to fully complement a B. pertussis wlbH (ΔwlbH_pe) mutant. This was supported by DNA sequence data showing that a single amino acid, conserved between homologous proteins from a range of bacteria, is altered in the B. parapertussis WlbH protein.     

Phycobiliproteins from <Emphasis Type="Italic">Pseudanabaena tenuis</Emphasis> rich in c-phycoerythrin protect against HgCl<Subscript>2</Subscript>-caused oxidative stress and cellular damage in the kidney

Our objective was to study if the phycobiliproteins of the cyanobacterium Pseudanabanea tenuis rich in phycoerythrin protect renal cells against mercury-caused oxidative stress and cellular damage in the kidney. We used 40 male mice that were assigned into five groups: a control group that received phosphate buffer (PB) and saline and four treatment groups which received either PB+HgCl₂, PB+phycobiliproteins, or HgCl₂+phycobiliproteins. The kidneys of the mice were used to determine lipid peroxidation and quantification of reactive oxygen species, oxidized glutathione, and peroxidase activities (catalase and glutathione peroxidase) and were also examined histologically. Our results demonstrated that HgCl₂ causes oxidative stress and cellular damage and that all doses of phycobiliproteins prevented the increase of oxidative markers and partially protected against HgCl₂-caused cell damage. This is the first report which applied phycobiliproteins of P. tenuis rich in c-phycoerythrin, like antioxidants against mercury chloride-caused oxidative stress and renal damage.     

降香通过抗氧化应激改善后负荷增加型心衰小鼠的心脏功能

目的:研究降香对后负荷增加引起的的心脏功能下降的保护作用及其机制。方法:雄性C57小鼠30只,随机分为三组,分别给予假手术(sham)、主动脉弓结扎(Transverse aortic constriction,TAC)手术和主动脉弓结扎手术降香治疗(TAC+DO)处理。通过灌胃给药4周,随后超声检测心脏功能、四腔心切片观察心肌重构,RT-PCR检测左心室αMHC、βMHC的m RNA表达、相应试剂盒心肌总抗氧化能力(TAOC)和丙二醇(MDA)含量。结果:同sham组相比,TAC组射血分数(EF),αMHC m RNA水平和TOAC均显著降低,且左室舒张末内径(LVIDd)、左室舒张期后壁厚度(LVPWd)、左室质量(LV mass)、心肌质量/胫骨长度(HW/TL)及β及β度、MDA均显著增加。同TAC组相比,DO组射血分数(EF),αMHC m RNA水平和TOAC均显著增加,且左室舒张末内径(LVIDd)、舒张末室间隔厚度(IVSd)、左室质量(LV mass)、心肌质量/胫骨长度(HW/TL)及βMHC、MDA均显著下降。在离体培养的心肌细胞,H_2O_2可显著增加细胞内ROS含量,给予降香或TEMPOL处理均可减轻H_2O_2诱导的氧化应激并增加心肌细胞存活率。结论:降香可通过降低氧化应激抑制线粒体分裂并改善后负荷增加型心衰的心脏功能。     

<Emphasis Type="Italic">Chlorella vulgaris</Emphasis> administration prevents HgCl<Subscript>2</Subscript>-caused oxidative stress and cellular damage in the kidney

Chlorella vulgaris is a unicellular green alga resistant to heavy metals. Chlorella is a rich nutritional ingredient because it contains high levels of antioxidants. The objective of this research was to study if C. vulgaris protect renal cells against mercury-chloride-caused oxidative stress and cellular damage in the kidney. Our results demonstrated that HgCl₂ causes oxidative stress and cellular damage, and that C. vulgaris administration prevents oxidative stress and cellular damage in kidney     

A novel F420‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F₄₂₀‐dependent anti‐oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F₄₂₀‐deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD⁺ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F₄₂₀^? mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin‐dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F₄₂₀H₂‐dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F₄₂₀H₂‐specific obligate two‐electron reduction of endogenous quinones, thereby competing with the one‐electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F₄₂₀ biosynthesis pathway or Fqr‐class proteins as a mechanism to potentiate the action of bactericidal agents.     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

RpoS and oxidative stress conditions regulate succinyl‐CoA: 3‐ketoacid‐coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei

Burkholderia pseudomallei, a pathogenic gram‐negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild‐type B. pseudomallei following exposure to H₂O₂ and between wild‐type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down‐regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT‐PCR that these genes are indeed co‐transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down‐regulation of SCOT expression in response to oxidative stress in B. pseudomallei.     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.