Oxidative and nitrosative stress defence systems in Escherichia coli and Pseudomonas aeruginosa: a model organism of study versus a human opportunistic pathogen.

ABSTRACT Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, ΔnirS) did not disperse, whereas a NO reductase mutant (ΔnorCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

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Targeted Genome Reduction of Pseudomonas aeruginosa Strain PAO1 Led to the Development of Hypovirulent and Hypersusceptible rDNA Hosts

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.640450 ◽

2021 ◽

Vol 9 ◽

Author(s):

Mélanie Grosjean ◽

Sophie Guénard ◽

Caroline Giraud ◽

Cédric Muller ◽

Patrick Plésiat ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Recombinant Dna ◽

Model Organism ◽

Opportunistic Pathogen ◽

Natural Resistance ◽

Biotechnological Applications ◽

Strain Pao1 ◽

Metabolic Versatility ◽

Wide Range

Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections, which is largely used as a model organism to study antibiotic resistance and pathogenesis. As other species of the genus, its wide metabolic versatility appears to be attractive to study biotechnological applications. However, its natural resistance to antibiotics and its capacity to produce a wide range of virulence factors argue against its biotechnological potential. By reducing the genome of the reference strain PAO1, we explored the development of four hypovirulent and hypersusceptible recombinant DNA hosts (rDNA hosts). Despite deleting up to 0.8% of the core genome, any of the developed strains presented alterations of fitness when cultured under standard laboratory conditions. Other features such as antibiotic susceptibility, cytotoxicity, in vivo pathogenesis, and expression of heterologous peptides were also explored to highlight the potential applications of these models. This work stands as the first stage of the development of a safe-platform strain of Pseudomonas aeruginosa that will be further optimized for biotechnological applications.

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Expressed Soybean Leghemoglobin: Effect on Escherichia coli at Oxidative and Nitrosative Stress

Molecules ◽

10.3390/molecules26237207 ◽

2021 ◽

Vol 26 (23) ◽

pp. 7207

Author(s):

Olga V. Kosmachevskaya ◽

Elvira I. Nasybullina ◽

Konstantin B. Shumaev ◽

Alexey F. Topunov

Keyword(s):

Escherichia Coli ◽

Energy Demand ◽

Nitrosative Stress ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Model Systems ◽

Resonance Spectroscopy ◽

Oxygen Binding ◽

Oxidative And Nitrosative Stress ◽

E Coli ◽

Soybean Leghemoglobin

Leghemoglobin (Lb) is an oxygen-binding plant hemoglobin of legume nodules, which participates in the symbiotic nitrogen fixation process. Another way to obtain Lb is its expression in bacteria, yeasts, or other organisms. This is promising for both obtaining Lb in the necessary quantity and scrutinizing it in model systems, e.g., its interaction with reactive oxygen (ROS) and nitrogen (RNS) species. The main goal of the work was to study how Lb expression affected the ability of Escherichia coli cells to tolerate oxidative and nitrosative stress. The bacterium E. coli with the embedded gene of soybean leghemoglobin a contains this protein in an active oxygenated state. The interaction of the expressed Lb with oxidative and nitrosative stress inducers (nitrosoglutathione, tert-butyl hydroperoxide, and benzylviologen) was studied by enzymatic methods and spectrophotometry. Lb formed NO complexes with heme-nitrosylLb or nonheme iron-dinitrosyl iron complexes (DNICs). The formation of Lb-bound DNICs was also detected by low-temperature electron paramagnetic resonance spectroscopy. Lb displayed peroxidase activity and catalyzed the reduction of organic peroxides. Despite this, E. coli-synthesized Lb were more sensitive to stress inducers. This might be due to the energy demand required by the Lb synthesis, as an alien protein consumes bacterial resources and thereby decreases adaptive potential of E. coli.

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Biofilm of Pseudomonas aeruginosa in Nosocomial Infection

Journal of Molecular Biology Research ◽

10.5539/jmbr.v7n1p29 ◽

2017 ◽

Vol 7 (1) ◽

pp. 29

Author(s):

Z. Mahmmudi ◽

A. A. Gorzin

Keyword(s):

Pseudomonas Aeruginosa ◽

Pathogenic Bacteria ◽

Molecular Mechanisms ◽

Matrix Material ◽

Model Organism ◽

Opportunistic Pathogen ◽

Chronic Infections ◽

Microbial Biofilms ◽

Alternative Measures ◽

Opportunistic Pathogenic

Bacteria in natural, industrial and clinical settings predominantly live in biofilms, i.e., sessile structured microbial communities encased in self-produced extracellular matrix material. One of the most important characteristics of microbial biofilms is that the resident bacteria display a remarkable increased tolerance toward antimicrobial attack. Biofilms formed by opportunistic pathogenic bacteria are involved in devastating persistent medical device-associated infections, and chronic infections in individuals who are immune-compromised or otherwise impaired in the host defense. Because the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially, biofilm infections where P. aeruginosa plays an important role are described. Subsequently, current insights into the molecular mechanisms involved in P. aeruginosa biofilm formation and the associated antimicrobial tolerance are reviewed. And finally, based on our knowledge about molecular biofilm biology, a number of therapeutic strategies for combat of P. aeruginosa biofilm infections are presented.

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Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1

10.21203/rs.3.rs-154442/v1 ◽

2021 ◽

Author(s):

Michele Castro ◽

Graciela Maria Dias ◽

Tiago Salles ◽

Núbia Cabral ◽

Danielly Mariano ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutant Strain ◽

Biofilm Formation ◽

Dna Microarrays ◽

Model Organism ◽

Opportunistic Pathogen ◽

Wild Type ◽

Swarming Motility ◽

Or Gene ◽

Flagellar Genes

Abstract Background: Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role on biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids’ modulation of those group behaviors. Results: Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the non-motile phenotype of the rhlA-minus strain. Swarming motility was restored with the addition of exogenous rhamnolipids obtained from the wild-type strain. Conclusions: Our results show significant evidence that rhamnolipids and/or their precursors, 3-(3-hydroxyalkanoyloxy) alkanoic acids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa. Swarming motility assays support this hypothesis, since the non-motile rhlA-mutant strain had its swarming ability restored by the addition of exogenous rhamnolipids.

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Roles of RcsA, an AhpD Family Protein, in Reactive Chlorine Stress Resistance and Virulence in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.01480-20 ◽

2020 ◽

Vol 86 (20) ◽

Author(s):

Benya Nontaleerak ◽

Jintana Duang-nkern ◽

Lampet Wongsaroj ◽

Wachareeporn Trinachartvanit ◽

Adisak Romsang ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Hypochlorous Acid ◽

Ectopic Expression ◽

Fruit Fly ◽

Opportunistic Pathogen ◽

Model Systems ◽

Harsh Environments ◽

Core Motif ◽

Content Type

ABSTRACT Reactive chlorine species (RCS), particularly hypochlorous acid (HOCl), are powerful antimicrobial oxidants generated by biological pathways and chemical syntheses. Pseudomonas aeruginosa is an important opportunistic pathogen that has adapted mechanisms for protection and survival in harsh environments, including RCS exposure. Based on previous transcriptomic studies of HOCl exposure in P. aeruginosa, we found that the expression of PA0565, or rcsA, which encodes an alkyl hydroperoxidase D-like protein, exhibited the highest induction among the RCS-induced genes. In this study, rcsA expression was dominant under HOCl stress and greatly increased under HOCl-related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid residues Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After allelic exchange mutagenesis in the P. aeruginosa rcsA, the P. aeruginosa rcsA deletion mutant showed significantly decreased HOCl resistance. Ectopic expression of P. aeruginosa rcsA led to significantly increased NaOCl resistance in Escherichia coli. Moreover, the pathogenicity of the rcsA mutant decreased dramatically in both Caenorhabditis elegans and Drosophila melanogaster host model systems compared to the wild type (WT). Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful at complementing phenotypes of the rcsA mutant. Overall, our data indicate the importance of P. aeruginosa RcsA in defense against HOCl stress under disinfections and during infections of hosts, which involves the catalytic Cys60, Cys63, and His67 residues. IMPORTANCE Pseudomonas aeruginosa is a common pathogen that is a major cause of serious infections in many hosts. Hypochlorous acid (HOCl) is a potent antimicrobial agent found in household bleach and is a widely used disinfectant. P. aeruginosa has evolved adaptive mechanisms for protection and survival during HOCl exposure. We identified P. aeruginosa rcsA as a HOCl-responsive gene encoding an antioxidant protein that may be involved in HOCl degradation. RcsA has a distinguishing core motif containing functional Cys60, Cys63, and His67 residues. P. aeruginosa rcsA plays an important role in bleach tolerance, with expression of P. aeruginosa rcsA in Escherichia coli also conferring HOCl resistance. Interestingly, RcsA is required for full virulence in worm and fruit fly infection models, indicating a correlation between mechanisms of bleach toxicity and host immunity during infection. This provides new insights into the mechanisms used by P. aeruginosa to persist in harsh environments such as hospitals.

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Modulation of oxidative and nitrosative stress attenuates microvascular hyperpermeability in ovine model of Pseudomonas aeruginosa sepsis

Scientific Reports ◽

10.1038/s41598-021-03320-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Satoshi Fukuda ◽

Yosuke Niimi ◽

Yasutaka Hirasawa ◽

Ennert R. Manyeza ◽

C. Edwin Garner ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Nitrosative Stress ◽

Lung Injury Score ◽

Vascular Endothelial ◽

Organ Function ◽

Oxidative And Nitrosative Stress ◽

Arterial Lactate ◽

Tissue Edema ◽

Fluid Requirement ◽

Endothelial Growth Factor

AbstractIn sepsis, microvascular hyperpermeability caused by oxidative/nitrosative stress (O&NS) plays an important role in tissue edema leading to multi-organ dysfunctions and increased mortality. We hypothesized that a novel compound R-107, a modulator of O&NS, effectively ameliorates the severity of microvascular hyperpermeability and preserves multi-organ function in ovine sepsis model. Sepsis was induced in twenty-two adult female Merino sheep by intravenous infusion of Pseudomonas aeruginosa (PA) (1 × 1010 CFUs). The animals were allocated into: 1) Control (n = 13): intramuscular injection (IM) of saline; and 2) Treatment (n = 9): IM of 50 mg/kg R-107. The treatment was given after the PA injection, and monitored for 24-h. R-107 treatment significantly reduced fluid requirement (15–24 h, P < 0.05), net fluid balance (9–24 h, P < 0.05), and water content in lung/heart/kidney (P = 0.02/0.04/0.01) compared to control. R-107 treatment significantly decreased lung injury score/modified sheep SOFA score at 24-h (P = 0.01/0.04), significantly lowered arterial lactate (21–24 h, P < 0.05), shed syndecan-1 (3–6 h, P < 0.05), interleukin-6 (6–12 h, P < 0.05) levels in plasma, and significantly attenuated lung tissue 3-nitrotyrosine and vascular endothelial growth factor-A expressions (P = 0.03/0.002) compared to control. There was no adverse effect in R-107 treatment. In conclusion, modulation of O&NS by R-107 reduced hyperpermeability markers and improved multi-organ function.

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lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 Encode Membrane-Associated Proteins and Are Required for Expression of 2,6-Dideoxy-2-Acetamidino-l-Galactose in Lipopolysaccharide O Antigen

Journal of Bacteriology ◽

10.1128/jb.01708-07 ◽

2007 ◽

Vol 190 (5) ◽

pp. 1671-1679 ◽

Cited By ~ 19

Author(s):

Jerry D. King ◽

Erin F. Mulrooney ◽

Evgeny Vinogradov ◽

Bernd Kneidinger ◽

Kristen Mead ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Cell Surface ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Nuclear Magnetic Resonance Analysis ◽

O Antigen ◽

O Antigens

ABSTRACT The rare sugar 2,6-dideoxy-2-acetamidino-l-galactose (l-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of l-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in l-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-l-galactose (l-FucNAc) in place of l-FucNAm. The mutation could be complemented by expression of either His6-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His6 tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.

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The Genetics of Prey Susceptibility to Myxobacterial Predation: A Review, Including an Investigation into Pseudomonas aeruginosa Mutations Affecting Predation by Myxococcus xanthus

Microbial Physiology ◽

10.1159/000515546 ◽

2021 ◽

pp. 1-10

Author(s):

Natashia Sydney ◽

Martin T. Swain ◽

Jeffery M.T. So ◽

Egbert Hoiczyk ◽

Nicholas P. Tucker ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Myxococcus Xanthus ◽

Hydrolytic Enzymes ◽

Model Organism ◽

Opportunistic Pathogen ◽

World Health Organisation ◽

World Health ◽

Stress System ◽

Extracellular Milieu ◽

A Genome

Bacterial predation is a ubiquitous and fundamental biological process, which influences the community composition of microbial ecosystems. Among the best characterised bacterial predators are the myxobacteria, which include the model organism Myxococcus xanthus. Predation by M. xanthus involves the secretion of antibiotic metabolites and hydrolytic enzymes, which results in the lysis of prey organisms and release of prey nutrients into the extracellular milieu. Due to the generalist nature of this predatory mechanism, M. xanthus has a broad prey range, being able to kill and consume Gram-negative/positive bacteria and fungi. Potential prey organisms have evolved a range of behaviours which protect themselves from attack by predators. In recent years, several investigations have studied the molecular responses of a broad variety of prey organisms to M. xanthus predation. It seems that the diverse mechanisms employed by prey belong to a much smaller number of general “predation resistance” strategies. In this mini-review, we present the current state of knowledge regarding M. xanthus predation, and how prey organisms resist predation. As previous molecular studies of prey susceptibility have focussed on individual genes/metabolites, we have also undertaken a genome-wide screen for genes of Pseudomonas aeruginosa which contribute to its ability to resist predation. P. aeruginosa is a World Health Organisation priority 1 antibiotic-resistant pathogen. It is metabolically versatile and has an array of pathogenic mechanisms, leading to its prevalence as an opportunistic pathogen. Using a library of nearly 5,500 defined transposon insertion mutants, we screened for “prey genes”, which when mutated allowed increased predation by a fluorescent strain of M. xanthus. A set of candidate “prey proteins” were identified, which shared common functional roles and whose nature suggested that predation resistance by P. aeruginosa requires an effective metal/oxidative stress system, an intact motility system, and mechanisms for de-toxifying antimicrobial peptides.

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Modulation of Oxidative and Nitrosative Stress Attenuates Microvascular Hyperpermeability in Ovine Model of Pseudomonas Aeruginosa Sepsis

10.21203/rs.3.rs-421490/v2 ◽

2021 ◽

Author(s):

Satoshi Fukuda ◽

Yosuke Niimi ◽

Yasutaka Hirasawa ◽

Ennert Manyeza ◽

C. Garner ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Nitrosative Stress ◽

Lung Injury Score ◽

Vascular Endothelial ◽

Organ Function ◽

Oxidative And Nitrosative Stress ◽

Arterial Lactate ◽

Tissue Edema ◽

Fluid Requirement ◽

Endothelial Growth Factor

Abstract In sepsis, microvascular hyperpermeability caused by oxidative/nitrosative stress (O&NS) plays an important role in tissue edema leading to multi-organ dysfunctions and increased mortality. We hypothesized that a novel compound R-107, a modulator of O&NS, effectively ameliorates the severity of microvascular hyperpermeability and preserves multi-organ function in ovine sepsis model. Sepsis was induced in twenty-two adult female Merino sheep by intravenous infusion of Pseudomonas aeruginosa (PA) (1x1010 CFUs). The animals were allocated into: 1) Control (n = 13): intramuscular injection (IM) of saline; and 2) Treatment (n = 9): IM of 50 mg/kg R-107. The treatment was given after the PA injection, and monitored for 24-hour. R-107 treatment significantly reduced fluid requirement (15–24 hours, p < 0.05), net fluid balance (9–24 hours, p < 0.05), and water content in lung/heart/kidney (p = 0.02/0.04/0.01) compared to control. R-107 treatment significantly decreased lung injury score/modified sheep SOFA score at 24-hour (p = 0.01/0.04), significantly lowered arterial lactate (21–24 hours, p < 0.05), shed syndecan-1 (3–6 hours, p < 0.05), interleukin-6 (6–12 hours, p < 0.05) levels in plasma, and significantly attenuated lung tissue 3-nitrotyrosine and vascular endothelial growth factor-A expressions (p = 0.03/0.002) compared to control. There was no adverse effect in R-107 treatment. In conclusion, modulation of O&NS by R-107 reduced hyperpermeability markers and improved multi-organ function.

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