Identification of the NADH-oxidase gene in Trichomonas vaginalis

Lamien-Meda, Aline; Leitsch, David

doi:10.1007/s00436-019-06572-8

Cited by 9 publications

(11 citation statements)

References 17 publications

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“…Since T. vaginalis is a microaerophilic microorganism, it requires redox and antioxidant systems to counter the harmful effects of oxygen and to express a wide range of genes encoding for defense molecules, including peroxiredoxins, thioredoxin reductases, superoxide dismutase, and rubrerythrin [15]. Two main activities have been identified, whereby NADPH oxidase and NADH oxidase eliminate oxygen present in the cytoplasm [16].…”

Section: Introductionmentioning

confidence: 99%

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Morales-Luna

Hernández-Ochoa

Ramírez-Nava

et al. 2020

IJMS

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This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

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Section: Introductionmentioning

confidence: 99%

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Morales-Luna

Hernández-Ochoa

Ramírez-Nava

et al. 2020

IJMS

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“…Flavin reductase and NADH oxidase activites of purified recombinant flavin reductases were measured similarly as described before ( Leitsch et al, 2014 ; Lamien-Meda and Leitsch 2020 ), but in 100 mM potassium phosphate pH 6.8 instead of pH 5.5. FMN, FAD, riboflavin, NADPH, and NADH were all purchased from Sigma Aldrich.…”

Section: Methodsmentioning

confidence: 99%

“…foetus and T . vaginalis cell extracts were measured as described before ( Leitsch et al, 2012 ; Lamien-Meda and Leitsch, 2020 ) but a larger amount of cell extract (an equivalent of 50 μg of protein instead of 20 μg) was used. Enzymatic parameters were calculated using GraphPad Prism 8 software.…”

Section: Methodsmentioning

confidence: 99%

“…Flavin reductase and NADH oxidase activities in KV1 cell extracts were visualized through enzymatic reduction of NBT (0.2%) in SDS-free PAA gels (10%) after gel electrophoresis according to an earlier published protocol ( Leitsch et al, 2014 ; Lamien-Meda and Leitsch, 2020 ). Either NADPH (0.5 mM) or NADH (0.5 mM) was used, depending on the enzyme to be visualized.…”

Section: Methodsmentioning

confidence: 99%

“…It has remained unknown if a homolog of FR1 also exists in T . foetus but both parasites have an NADH oxidase ( Linstead and Bradley, 1988 ; Cerkasovová and Cerkasov, 1974 ; Lamien-Meda and Leitsch, 2020 ) which reduces oxygen to water. Interestingly, however, this enzyme activity could not be linked to 5-nitroimidazole resistance so far.…”

Section: Introductionmentioning

confidence: 99%

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A reassessment of the role of oxygen scavenging enzymes in the emergence of metronidazole resistance in trichomonads

Gehl

Paunkov

Leitsch

2021

International Journal for Parasitology: Drugs and Drug Resistan

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Trichomonads are an order of parasitic protists which infect a wide range of hosts. The human parasite Trichomonas vaginalis and the bovine parasite Tritrichomonas foetus which also infects cats and swine are of considerable medical and veterinary importance, respectively. Since trichomonads are microaerophiles/anaerobes they are susceptible to 5-nitroimidazoles such as metronidazole. 5-nitroimidazoles are exclusively toxic to microaerophilic/anaerobic organisms because reduction, i.e. activation, of the drug can only occur in a highly reductive environment. 5-nitroimidazoles have remained a reliable treatment option throughout the last decades but drug resistance can be a problem. Clinical resistance to 5-nitroimidazoles has been studied in more detail in T . vaginalis and has been ascribed to defective oxygen scavenging mechanisms which lead to higher intracellular oxygen concentrations and, consequently, to less drug being reduced. Two enzymes, flavin reductase (FR) and NADH oxidase have been suggested to be the major oxygen scavenging enzymes in T . vaginalis . The loss, or at least an impairment of FR which reduces oxygen to hydrogen peroxide, has been proposed as the central mechanism that enables the emergence of 5-nitroimidazole resistance. In this study we explored if T . foetus also encodes a homolog of FR and if it is, likewise, involved in resistance. T. foetus was indeed found to express a FR but it was only weakly active as compared to the T . vaginalis homolog. Further, activity of FR in T . foetus was unchanged in metronidazole-resistant cell lines, ruling out that it has a role in metronidazole resistance. Finally, we measured oxygen scavenging rates in metronidazole-sensitive and –resistant cell lines and found that NADH oxidase and FR are not the major oxygen scavenging enzymes in trichomonads and that oxygen scavenging is possibly a consequence, rather than a cause of metronidazole resistance.

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Oxygen levels are key to understanding “Anaerobic” protozoan pathogens with micro-aerophilic lifestyles

Lloyd

Chapman

Ellis³

et al. 2021

Advances in Microbial Physiology

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Identification of the NADH-oxidase gene in Trichomonas vaginalis

Cited by 9 publications

References 17 publications

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

A reassessment of the role of oxygen scavenging enzymes in the emergence of metronidazole resistance in trichomonads

Oxygen levels are key to understanding “Anaerobic” protozoan pathogens with micro-aerophilic lifestyles

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