4 research outputs found
Characterization of the components of the thioredoxin system in Bacteroides fragilis and evaluation of its activity during oxidative stress
Objectives: Bacteroides fragilis has a pronounced ability to survive prolonged exposure to atmospheric oxygen. The major objective of this study was to biochemically characterize the components of the thioredoxin system in B. fragilis. The nitroreductase activity of TrxR was also assayed. Methods: Components of the thioredoxin system were expressed in E. coli and used in a disulfide reductase activity assay. Activity of TrxR was measured with purified recombinant enzyme or with cell extracts after or without exposure to oxygen or hydrogen peroxide, respectively. Results: Of all six thioredoxins tested, only thioredoxins A, D, and F were reduced by recombinant TrxR and natural TrxR present in B. fragilis cell extracts. Exposure to oxygen and hydrogen peroxide increased the activity of TrxR. Further, B. fragilis TrxR acts as a nitroreductase with furazolidone or 1-Chloro-2,4dinitrobenzene as substrates but cannot reduce metronidazole. Conclusion: TrxR shows an increase in activity under the conditions of oxidative stress and exerts nitroreductase activity. (c) 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Initial expression levels of nimA are decisive for protection against metronidazole in Bacteroides fragilis
Objectives: In the genus Bacteroides, the nim genes are resistance determinants for metronidazole, a nitroimidazole drug widely used against anaerobic pathogens. The Nim proteins are considered to act as nitroreductases. However, data from several studies suggest that the expression levels of Nim do not increase with increasing resistance which is conflicting with this notion. The impact of Nim protein levels on low-level metronidazole resistance, however, representing the early stage of induced resistance in the laboratory, has not been assessed as yet.Methods: The nimA gene was cloned into two different plasmids and introduced into B. fragilis strain 638R. Expression levels of nimA mRNA were measured by RT-qPCR and compared to those in strain 638R harbouring plasmid pI417, the original clinical plasmid harbouring IS element IS1168 with the nimA gene. Further, metronidazole susceptibility was assessed by Etest and the activity of pyruvate:ferredoxin oxidoreductase (PFOR) was measured in all strains after induction of high-level metronidazole resistance.Results: The level of protection against metronidazole by nimA correleated with the level of expression of nimA mRNA. Further, the activity of PFOR in highly-resistant B. fragilis 638R was only preserved when expression levels of nimA were high.Conclusions: Although the development of high-level metronidazole resistance in B. fragilis strains with a nimA gene is not caused by an increase of nimA expression as compared to the less resistant parent strains, nimA expression levels might be of decisive importance in the early stage of resistance devel-opment. This has potential implications for metronidazole resistance in clinical isolates.(c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Proteomics-Based RT-qPCR and Functional Analysis of 18 Genes in Metronidazole Resistance of Bacteroides fragilis
Previously, we reported that metronidazole MICs are not dependent on the expression levels of nim genes in B. fragilis strains and we compared the proteomes of metronidazole-resistant laboratory B. fragilis strains to those of their susceptible parent strains. Here, we used RT-qPCR to correlate the expression levels of 18 candidate genes in a panel of selected, clinical nim gene-positive and -negative B. fragilis strains to their metronidazole MICs. Metronidazole MICs were correlated with the expression of certain tested genes. Specifically, lactate dehydrogenase expression correlated positively, whereas cytochrome fumarate reductase/succinate dehydrogenase, malate dehydrogenase, phosphoglycerate kinase redox and gat (GCN5-like acetyltransferase), and relA (stringent response) regulatory gene expressions correlated negatively with metronidazole MICs. This result provides evidence for the involvement of carbohydrate catabolic enzymes in metronidazole resistance in B. fragilis. This result was supported by direct substrate utilization tests. However, the exact roles of these genes/proteins should be determined in deletion鈥揷omplementation tests. Moreover, the exact redox cofactor(s) participating in metronidazole activation need to be identified
Proteomics-Based RT-qPCR and Functional Analysis of 18 Genes in Metronidazole Resistance of <i>Bacteroides fragilis</i>
Previously, we reported that metronidazole MICs are not dependent on the expression levels of nim genes in B. fragilis strains and we compared the proteomes of metronidazole-resistant laboratory B. fragilis strains to those of their susceptible parent strains. Here, we used RT-qPCR to correlate the expression levels of 18 candidate genes in a panel of selected, clinical nim gene-positive and -negative B. fragilis strains to their metronidazole MICs. Metronidazole MICs were correlated with the expression of certain tested genes. Specifically, lactate dehydrogenase expression correlated positively, whereas cytochrome fumarate reductase/succinate dehydrogenase, malate dehydrogenase, phosphoglycerate kinase redox and gat (GCN5-like acetyltransferase), and relA (stringent response) regulatory gene expressions correlated negatively with metronidazole MICs. This result provides evidence for the involvement of carbohydrate catabolic enzymes in metronidazole resistance in B. fragilis. This result was supported by direct substrate utilization tests. However, the exact roles of these genes/proteins should be determined in deletion鈥揷omplementation tests. Moreover, the exact redox cofactor(s) participating in metronidazole activation need to be identified