SodA Contributes to the Virulence of Avian PathogenicEscherichia coliO2 Strain E058 in Experimentally Infected Chickens

ABSTRACTStrains of avian pathogenicEscherichia coli(APEC), the common pathogen of avian colibacillosis, encounter reactive oxygen species (ROS) during the infection process. Superoxide dismutases (SODs), acting as antioxidant factors, can protect against ROS-mediated host defenses. Our previous reports showed that thesodAgene (encoding a Mn-cofactor-containing SOD [MnSOD]) is highly expressed during the septicemic infection process of APEC.sodAhas been proven to be a virulence factor of certain pathogens, but its role in the pathogenicity of APEC has not been fully identified. In this study, we deleted thesodAgene from the virulent APEC O2 strain E058 and examined thein vitroandin vivophenotypes of the mutant. ThesodAmutant was more sensitive to hydrogen peroxide in terms of both its growth and viability than was the wild type. The ability to form a biofilm was weakened in thesodAmutant. ThesodAmutant was significantly more easily phagocytosed by chicken macrophages than was the wild-type strain. Chicken infection assays revealed significantly attenuated virulence of thesodAmutant compared with the wild type at 24 h postinfection. The virulence phenotype was restored by complementation of thesodAgene. Quantitative real-time reverse transcription-PCR revealed that the inactivation ofsodAreduced the expression of oxidative stress response geneskatE,perR, andosmCbut did not affect the expression ofsodBandsodC. Taken together, our studies indicate that SodA is important for oxidative resistance and virulence of APEC E058.IMPORTANCEAvian colibacillosis, caused by strains of avian pathogenicEscherichia coli, is a major bacterial disease of severe economic significance to the poultry industry worldwide. The virulence mechanisms of APEC are not completely understood. This study investigated the influence of an antioxidant protein, SodA, on the phenotype and pathogenicity of APEC O2 strain E058. This is the first report demonstrating that SodA plays an important role in protecting a specific APEC strain against hydrogen peroxide-induced oxidative stress and contributes to the virulence of this pathotype strain. Identification of this virulence factor will enhance our knowledge of APEC pathogenic mechanisms, which is crucial for designing successful strategies against associated infections and transmission.

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Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.02282-14 ◽

2014 ◽

Vol 197 (3) ◽

pp. 431-440 ◽

Cited By ~ 41

Author(s):

Lu Zhang ◽

James R. Alfano ◽

Donald F. Becker

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Hydrogen Peroxide ◽

Mutant Strain ◽

Stress Resistance ◽

Proline Metabolism ◽

Wild Type ◽

Content Type ◽

Oxidative Stress Resistance ◽

Proline Utilization

The oxidation ofl-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type andputAmutant strains ofEscherichia coli. Initial stress assays revealed that theputAmutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in theputAmutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded bykatG) expression and activity. Furthermore, the ΔkatGstrain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression ofkatGalong with several other genes involved in oxidative stress defense. In addition tokatG, proline increased the expression ofgrxA(glutaredoxin 1) andtrxC(thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance inE. colivia a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.

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Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽

10.1093/genetics/151.2.439 ◽

1999 ◽

Vol 151 (2) ◽

pp. 439-446 ◽

Cited By ~ 2

Author(s):

Masaaki Onda ◽

Katsuhiro Hanada ◽

Hirokazu Kawachi ◽

Hideo Ikeda

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Hydrogen Peroxide ◽

Dna Damage ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Recombination Analysis ◽

Wild Type ◽

Strand Breaks ◽

In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

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Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

Infection and Immunity ◽

10.1128/iai.01138-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 187-193 ◽

Cited By ~ 12

Author(s):

Renu Verma ◽

Thaís Cabrera Galvão Rojas ◽

Renato Pariz Maluta ◽

Janaína Luisa Leite ◽

Livia Pilatti Mendes da Silva ◽

...

Keyword(s):

Escherichia Coli ◽

Soft Agar ◽

Pleiotropic Effect ◽

Biological Characteristics ◽

Wild Type ◽

Content Type ◽

Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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Inactivation ofclpBin the Pathogen Leptospira interrogans Reduces Virulence and Resistance to Stress Conditions

Infection and Immunity ◽

10.1128/iai.05168-11 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3711-3717 ◽

Cited By ~ 69

Author(s):

Kristel Lourdault ◽

Gustavo M. Cerqueira ◽

Elsio A. Wunder ◽

Mathieu Picardeau

Keyword(s):

Oxidative Stress ◽

Stress Conditions ◽

Leptospira Interrogans ◽

Wild Type ◽

Content Type ◽

General Stress Response ◽

Growth Defects ◽

Resistance To Stress ◽

Increased Susceptibility

ABSTRACTLeptospira interrogansis the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze aclpBmutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. TheclpBmutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetically complemented strain was restored in ClpB expression andin vitrowild-type growth. We also showed that theclpBmutant was attenuated in virulence in an animal model of acute leptospirosis. Our findings demonstrate that ClpB is involved in the general stress response. The chaperone is also necessary, either directly or indirectly, for the virulence of the pathogenL. interrogans.

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In Vivo Fitness Adaptations of Colistin-Resistant Acinetobacter baumannii Isolates to Oxidative Stress

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00598-16 ◽

2016 ◽

Vol 61 (3) ◽

Cited By ~ 8

Author(s):

Crystal L. Jones ◽

Shweta S. Singh ◽

Yonas Alamneh ◽

Leila G. Casella ◽

Robert K. Ernst ◽

...

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Acinetobacter Baumannii ◽

Catalase Activity ◽

Content Type ◽

Increased Susceptibility

ABSTRACT The loss of fitness in colistin-resistant (CR) Acinetobacter baumannii was investigated using longitudinal isolates from the same patient. Early CR isolates were outcompeted by late CR isolates for growth in broth and survival in the lungs of mice. Fitness loss was associated with an increased susceptibility to oxidative stress since early CR strains had reduced in vitro survival in the presence of hydrogen peroxide and decreased catalase activity compared to that of late CR and colistin-susceptible (CS) strains.

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Inactivation of Escherichia coli by Polychromatic Simulated Sunlight: Evidence for and Implications of a Fenton Mechanism Involving Iron, Hydrogen Peroxide, and Superoxide

Applied and Environmental Microbiology ◽

10.1128/aem.02419-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 935-942 ◽

Cited By ~ 17

Author(s):

Michael B. Fisher ◽

Kara L. Nelson

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Enteric Bacteria ◽

Growth Conditions ◽

Simulated Sunlight ◽

Wild Type ◽

Intracellular Iron ◽

Content Type ◽

E Coli ◽

Degree Of Sensitization

ABSTRACTSunlight inactivation ofEscherichia colihas previously been shown to accelerate in the presence of oxygen, exogenously added hydrogen peroxide, and bioavailable forms of exogenously added iron. In this study, mutants unable to effectively scavenge hydrogen peroxide or superoxide were found to be more sensitive to polychromatic simulated sunlight (without UVB wavelengths) than wild-type cells, while wild-type cells grown under low-iron conditions were less sensitive than cells grown in the presence of abundant iron. Furthermore, prior exposure to simulated sunlight was found to sensitize cells to subsequent hydrogen peroxide exposure in the dark, but this effect was attenuated for cells grown with low iron. Mutants deficient in recombination DNA repair were sensitized to simulated sunlight (without UVB wavelengths), but growth in the presence of iron chelators reduced the degree of sensitization conferred by this mutation. These findings support the hypothesis that hydrogen peroxide, superoxide, and intracellular iron all participate in the photoinactivation ofE. coliand further suggest that the inactivation rate of enteric bacteria in the environment may be strongly dependent on iron availability and growth conditions.

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Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.00369-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3220-3231 ◽

Cited By ~ 9

Author(s):

Kumiko Kurabayashi ◽

Tomohiro Agata ◽

Hirofumi Asano ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Antimicrobial Agents ◽

Host Cells ◽

Type I ◽

Wild Type ◽

Content Type ◽

Type I Fimbriae ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

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Antimicrobial Activity Evaluation of Tebipenem (SPR859), an Orally Available Carbapenem, against a Global Set of Enterobacteriaceae Isolates, Including a Challenge Set of Organisms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02618-18 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 3

Author(s):

S. J. Ryan Arends ◽

Paul R. Rhomberg ◽

Nicole Cotroneo ◽

Aileen Rubio ◽

Robert K. Flamm ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Broth Microdilution ◽

Wild Type ◽

Content Type ◽

E Coli

ABSTRACT The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum β-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.

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ISKpn40-Mediated Mobilization of the Colistin Resistance Gene mcr-3.11 in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00851-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Yu-Zhang He ◽

Teng-Fei Long ◽

Cai-Ping Chen ◽

Bing He ◽

Xing-Ping Li ◽

...

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Wild Type ◽

Colistin Resistance ◽

Content Type ◽

First Report ◽

E Coli ◽

Target Sites

ABSTRACT The mobile colistin resistance gene mcr-3 has globally disseminated since it was first reported in 2017 in Escherichia coli. In vitro mobilization assays in this study demonstrate the functionality of the composite transposon structure ISKpn40-mcr-3.11-dgkA-ISKpn40 in wild-type and recA− E. coli strains. These transpositions generated 4-bp duplications at the target sites. This is the first report demonstrating the mobility of the mcr-3.11 gene by transposition.

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