Flavohemoglobin Hmp Affords Inducible Protection for Escherichia coli Respiration, Catalyzed by Cytochromesbo′ or bd, from Nitric Oxide

Stevanin, Tânia M.; Ioannidis, Nikolaos; Mills, Catherine E.; Kim, Sung Oog; Hughes, Martin N.; Poole, Robert K.

doi:10.1074/jbc.m002471200

Cited by 150 publications

(153 citation statements)

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“…Respiratory inhibition in response to NO is well established in non-pathogenic E. coli [3,6,43]. Respiration of UPEC was also significantly inhibited by NO and the inhibition was initiated momentarily upon NO addition and resumed after the NO levels decreased.…”

Section: Discussionmentioning

confidence: 90%

“…The samples were boiled for 5 min and 5 µg of total protein was separated on a 12% NuPAGE pre-cast gel (Invitrogen Life Technologies, Carlsbad, CA) and transferred onto a Hybond ™ P+ membrane (Amersham Biosciences). The membrane was incubated with a rabbit polyclonal antibody against E. coli Hmp (1:10 000) [3]. After incubation with a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:10 000; Stressgene Biotechnologies, San Diego, CA) detection was performed using ECL plus Western Blotting detection system (Amersham Biosciences).…”

Section: Western Blot Analysismentioning

confidence: 99%

“…The method was adapted but slightly modified from previous studies [3,18]. Cultures in LB were grown aerobically until reaching an OD 600 of 0.7-1.0, harvested by centrifugation and resuspended in 1 ml PBS.…”

Section: No Consumption and Respiration Rate In J96wt J96∆hmp And J9mentioning

confidence: 99%

“…Microbial targets of RNI include thiols, metal centers, lipids and DNA. NO itself is considered to be bacteriostatic and NO-related toxicity includes inhibition of respiration [3] and reduced cell division by disrupting DNA replication [4]. In approximately 80 % of the community-acquired urinary tract infections (UTIs) the cause of the infection is a uropathogenic Escherichia coli (UPEC) strain.…”

mentioning

confidence: 99%

“…In addition, detrimental effects of NO and RNI on non-pathogenic E. coli K-12 strains are also well established [3,7,10,22,23]. Studies in humans and mice have shown that urinary neutrophils and urinary tract epithelial cells are able to express iNOS in response to UPEC infection [24,25,26] and NO levels have been documented to increase in the urinary bladder in patients suffering from UTI [27].…”

mentioning

confidence: 99%

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Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coli – Implications for urinary tract infection

Svensson

Poljakovic

Säve

et al. 2010

Microbial Pathogenesis

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Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coliimplications for urinary tract infection. During the course of urinary tract infection (UTI) nitric oxide (NO) is generated as part of host response. The aim of this study was to investigate the significance of the NO-detoxifying enzymes flavohemoglobin (Hmp) and flavorubredoxin (Norv) in protection of uropathogenic Escherichia coli (UPEC) against nitrosative stress. Hmp (J96∆hmp) and norV (J96∆norV) knockout mutants of UPEC strain J96 were constructed using a single-gene deletion strategy. Bacterial tolerance and expression of hmp and norV in response to the NO-donor DETA/NO was evaluated in Luria broth and urine from healthy volunteers. Bacterial NO consumption and respiratory inhibition were assessed when exposed to NO. Expression of hmp and norV from E. coli originating from patients with UTI was evaluated using real-time PCR. The colonizing ability of J96 wild-type (wt) compared to an hmp-deficient mutant was assessed using a competition-based mouse UTI model. The viability of J96∆hmp and J96∆norV was significantly reduced compared to the wildtype strain after exposure to DETA/NO. The hmp expression in DETA/NO-exposed cultures was similar in J96wt and J96∆norV, while J96∆hmp showed an increased norV expression compared to J96wt. The NO consumption in J96∆hmp, but not in J96∆norV, was significantly impaired compared to J96wt. An up-regulation of hmp expression was found in E. coli isolated from all UTIpatients while norV expression increased in 50% of the patients. In the mouse UTI model, the hmp-mutant strain was significantly out-competed by the wild-type strain in the bladder and kidney. Hmp and NorV contribute to the protection of UPEC against NOmediated toxicity in vitro. Screening UPEC isolates from UTI patients revealed an increased hmp expression in all patients which confirms that hmp expression occurs in vivo in the infected human urinary tract. The ability to colonize the mouse urinary tract was impaired in the hmp-deficient mutant compared to the wild-type strain. NO-detoxification by Hmp is suggested to be an important characteristic for UPEC in protection against nitrosative stress and may be a virulence-facilitating factor.

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Section: Discussionmentioning

confidence: 90%

Section: Western Blot Analysismentioning

confidence: 99%

Section: No Consumption and Respiration Rate In J96wt J96∆hmp And J9mentioning

confidence: 99%

mentioning

confidence: 99%