Nitroreductase Activites in Giardia lamblia: ORF 17150 Encodes a Quinone Reductase with Nitroreductase Activity

The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea. Current treatments are based on nitro drugs, especially metronidazole. Nitro compounds are activated by reduction, yielding toxic intermediates. The enzymatic systems responsible for this activation are not completely understood. By fractionating cell free crude extracts by size exclusion chromatography followed by mass spectrometry, enzymes with nitroreductase (NR) activities are identified. The protein encoded by ORF 17150 found in two pools with NR activities is overexpressed and characterized. In pools of fractions with main NR activities, previously-known NRs are identified, as well as a previously uncharacterized protein encoded by ORF 17150. Recombinant protein 17150 is a flavoprotein with NADPH-dependent quinone reductase and NR activities. Besides a set of previously identified NRs, we have identified a novel enzyme with NR activity.

A Chalcone-based Fluorescent Responsive Probe for Selective Detection of Nitroreductase Activity in Bacteria

A new chalcone-based fluorescent turn-on probe (3c) responsive to the nitroreductase (NTR) activity and its application toward the detection of bacteria are presented. The probe exhibits turn-on fluorescence at 535...

Detection of Microbial Nitroreductase Activity by Monitoring Exogenous Volatile Organic Compound Production Using HS-SPME-GC-MS

Development of a rapid approach for universal microbial detection is required in the healthcare, food and environmental sectors to aid with medical intervention, food safety and environmental protection. This research investigates the use of enzymatic hydrolysis of a substrate by a microorganism to generate a volatile organic compound (VOC). One such enzyme activity that can be used in this context is nitroreductase as such activity is prevalent across a range of microorganisms. A study was developed to evaluate a panel of 51 microorganisms of clinical interest for their nitroreductase activity. Two enzyme substrates, nitrobenzene and 1-fluoro-2-nitrobenzene, were evaluated for this purpose with evolution, after incubation, of the VOCs aniline and 2-fluoroaniline, respectively. Detection of the VOCs was done using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) with obtained limits of quantitation (LOQ) of 0.17 and 0.03 µg/mL for aniline and 2-fluoroaniline, respectively. The results indicated that both enzyme substrates were reduced by the same 84.3% of microorganisms producing the corresponding volatile anilines which were detected using HS-SPME-GC-MS. It was found that nitroreductase activity could be detected after 6–8 h of incubation for the selected pathogenic bacteria investigated. This approach shows promise as a rapid universal microbial detection system.

Rv3131, a gene encoding nitroreductase, is essential for metronidazole activation in Mycobacterium tuberculosis under hypoxic condition

ObjectivesThe impressive potency of metronidazole (MTZ) against anaerobic bacteria indicates the potential for killing anaerobic Mtb. However, how MTZ is activated in Mtb still remains unknown. We aimed to characterize the endogenous nitroreductase responsible for MTZ activation in anaerobic Mtb.MethodsThe minimal inhibitory concentrations (MICs) of Mtb isolates against MTZ were determined by microplate Alamar Blue assay. Intracellular anti-TB activities of MTZ and pyrazinamide (PZA) were tested in THP-1 cells infected by Mycobacterium tuberculosis (Mtb) H37Rv with a multiplicity of infection (MOI) of 10. The nitroreductase activity of purified wild-type Rv3131 and mutants were measured under anaerobic conditions generated by glucose oxidase/catalase system. Two-tailed unpaired Student’s t test was used to compare the difference between various groups.Results180 Mtb isolates (81.8%, 180/220) had MIC values higher than 16 μg/mL, and 40 had MIC values of 16 μg/mL, demonstrating high-level resistance to MTZ under aerobic condition. The number of viable bacteria in macrophages treated with MTZ was dramatically decreased by 71.3% after 5-day MTZ treatment, indicating significant inhibition of MTZ against anaerobic Mtb. In vitro biochemical analysis demonstrated that Rv3131 exhibited the NADPH oxidase activity under anaerobic condition. The substitutions of Cys75Ser and Cys279Ser could maintain 41.7% and 71.1% of enzyme activity compared to wild-type protein, respectively.ConclusionsOur data demonstrate that MTZ has more potent efficacy against intracellular Mtb than PZA. Rv3131 is identified as a nitroreductase enzyme in the activation of MTZ, and Cys75 of Rv3131 is the major active residue for nitroreductase activity.

Precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity with a targetable and activatable fluorescent probe

A probe with a targetable feature and ratiometric fluorescence as well as NIR emission light-up response to nitroreductase is presented for the precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity.