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-complementation tests. Moreover, the exact redox cofactor(s) participating in metronidazole activation need to be identified.

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis.

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

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 development. This has potential implications for metronidazole resistance in clinical isolates.

Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene.

Bacteroides fragilis is a commensal of the human gut but can also cause severe infections when reaching other body sites, especially after surgery or intestinal trauma. Bacteroides fragilis is an anaerobe innately susceptible to metronidazole, a 5-nitroimidazole drug that is prescribed against the majority of infections caused by anaerobic bacteria. In most of the cases, metronidazole treatment is effective but a fraction of B. fragilis is resistant to even very high doses of metronidazole. Metronidazole resistance is still poorly understood, but the so-called nim genes have been described as resistance determinants. They have been suggested to encode nitroreductases which reduce the nitro group of metronidazole to a non-toxic aminoimidazole. More recent research, however, showed that expression levels of nim genes are widely independent of the degree of resistance observed. In the search for an alternative model for nim-mediated metronidazole resistance, we screened a strain carrying an episomal nimA gene and its parental strain 638R without a nim gene for physiological differences. Indeed, the 638R daughter strain with the nimA gene had a far higher pyruvate-ferredoxin oxidoreductase (PFOR) activity than the parental strain. High PFOR activity was also observed in metronidazole-resistant clinical isolates, either with or without a nim gene. Moreover, the strain carrying a nimA gene fully retained PFOR activity and other enzyme activities such as thioredoxin reductase (TrxR) after resistance had been induced. In the parental strain 638R, these were lost or very strongly downregulated during the development of resistance. Further, after induction of high-level metronidazole resistance, parental strain 638R was highly susceptible to oxygen whereas the daughter strain with a nimA gene was hardly affected. Ensuing RT-qPCR measurements showed that a pathway for iron import via hemin uptake is downregulated in 638R with induced resistance but not in the resistant nimA daughter strain. We propose that nimA primes B. fragilis toward an alternative pathway of metronidazole resistance by enabling the preservation of normal iron levels in the cell.

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-2,4-dinitrobenzene as substrates but cannot reduce metronidazole. CONCLUSION: TrxR shows an increase in activity under the conditions of oxidative stress and exerts nitroreductase activity.

Haemin deprivation renders Bacteroides fragilis hypersusceptible to metronidazole and cancels high-level metronidazole resistance.

BACKGROUND: Infections with Bacteroides fragilis are routinely treated with metronidazole, a 5-nitroimidazole antibiotic that is active against most anaerobic microorganisms. Metronidazole has remained a reliable treatment option, but resistance does occur, including in B. fragilis. OBJECTIVES: In this study we tested whether haemin, a growth supplement for B. fragilis in vivo and in vitro, had an influence on the susceptibility of resistant B. fragilis strains to metronidazole. We further tested whether haemin-deprived B. fragilis would be more susceptible to oxygen and oxidative stress. Metronidazole has been described to cause oxidative stress, which we argued would be exacerbated in haemin-deprived B. fragilis because the bacteria harness haemin, and the iron released from it, in antioxidant enzymes such as catalase and superoxide dismutase. METHODS: Haemin was omitted from growth media and the effect on metronidazole susceptibility was monitored in susceptible and resistant B. fragilis strains. Further, haemin-deprived B. fragilis were tested for resistance to aeration and hydrogen peroxide and the capacity for the removal of oxygen. RESULTS: Omission of haemin from the growth medium rendered metronidazole-resistant B. fragilis strains, including an MDR isolate from the UK, highly susceptible to metronidazole. Haemin deprivation further rendered B. fragilis highly susceptible to oxygen, which was further exacerbated in resistant strains. B. fragilis was incapable of scavenging oxygen when haemin was omitted. CONCLUSIONS: We propose that haemin deprivation overrules resistance mechanisms by rendering B. fragilis hypersusceptible to metronidazole due to a compromised antioxidant defence. Monitoring of haemin concentrations is imperative when conducting metronidazole susceptibility testing in B. fragilis.

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

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.

A Bibliometric Review of the Keap1/Nrf2 Pathway and its Related Antioxidant Compounds.

Nrf2 is a master transcriptional regulator of antioxidant and cytoprotective pathways. Currently in its third decade, research on Nrf2 has expanded to encompass not only basic but also clinical studies. In the present bibliometric review, we employed the VOSviewer tool to describe the existing Nrf2 literature landscape. As of July 2019, 11,931 papers on Nrf2 were listed in the "Web of Science" database, with more than 1000 new papers published each year. As expected, terms related to oxidative stress and antioxidant molecules occur very often in the Nrf2 literature throughout the years. Interestingly, there is also a gradual increase in the occurrence of terms related to diseases or to natural compounds, the most prominent being sulforaphane, curcumin, and resveratrol that modulate the Nrf2 pathway. Going beyond molecular biology/biochemistry and related fields, Nrf2 research has begun to spread into more clinical areas like endocrinology/metabolism, cardiology, and nephrology, likely reflecting an increased interest in clinical applications of Nrf2 pathway activators. China has become the most prolific producer of Nrf2 papers the last five years followed by the USA and Japan, a reverse pattern compared to the past. In conclusion, Nrf2 is the subject of a globally active research field that keeps growing and extends from bench to bedside.

Impact of Antioxidant Natural Compounds on the Thyroid Gland and Implication of the Keap1/Nrf2 Signaling Pathway.

BACKGROUND: Natural compounds with potential antioxidant properties have been used in the form of food supplements or extracts with the intent to prevent or treat various diseases. Many of these compounds can activate the cytoprotective Nrf2 pathway. Besides, some of them are known to impact the thyroid gland, often with potential side-effects, but in other instances, with potential utility in the treatment of thyroid disorders. OBJECTIVE: In view of recent data regarding the multiple roles of Nrf2 in the thyroid, this review summarizes the current bibliography on natural compounds that can have an effect on thyroid gland physiology and pathophysiology, and it discusses the potential implication of the Nrf2 system in the respective mechanisms. METHODS & RESULTS: Literature searches for articles from 1950 to 2018 were performed in PubMed and Google Scholar using relevant keywords about phytochemicals, Nrf2 and thyroid. Natural substances were categorized into phenolic compounds, sulfur-containing compounds, quinones, terpenoids, or under the general category of plant extracts. For individual compounds in each category, respective data were summarized, as derived from in vitro (cell lines), preclinical (animal models) and clinical studies. The main emerging themes were as follows: phenolic compounds often showed potential to affect the production of thyroid hormones; sulfur-containing compounds impacted the pathogenesis of goiter and the proliferation of thyroid cancer cells; while quinones and terpenoids modified Nrf2 signaling in thyroid cell lines. CONCLUSION: Natural compounds that modify the activity of the Nrf2 pathway should be evaluated carefully, not only for their potential to be used as therapeutic agents for thyroid disorders, but also for their thyroidal safety when used for the prevention and treatment of non-thyroidal diseases.

Dopamine type 1- and 2-like signaling in the modulation of spatial reference learning and memory.

Spatial reference memory is known to be modulated by the dopaminergic system involving different brain regions. Here, we sought to identify the contribution of D1 (D1R) and D2 (D2R)-like dopamine receptor signaling on learning and memory in a food rewarded hole-board task by intracerebroventricular infusing D1R- and D2R- like receptor agonists (SKF-81297 and Sumanirole) and antagonists (SCH 23390 and Remoxipride) once 30 min prior to daily training sessions. D1R agonism induced persistent enhancement of performance, whereas D1R antagonism impaired reference memory formation. D2R agonist and antagonist exerted no effects. Phase specific comparisons revealed an enhancement of spatial acquisition in the presence of the D1R but not D2R agonism on acquisition, but not during retention. Since task difficulty might skew dopamine-induced improvements in learning and memory, we tested the D1R agonist in the hole-board task with increased difficulty. Drug treated animals performed significantly better during all training phases, with results better resolved than in the easy task. Additionally, proteomic analysis of the prefrontal cortex revealed ninety six proteins to be regulated by D1R agonism, from which 35 were correlated with behavioral performance. Obtained targets were grouped by function, showing synaptic transmission, synaptic remodeling, and dendritic spine morphology as the major functional classes affected. In sum, we find that activation of D1R signaling during spatial acquisition and retention improved reference memory index, depended on the task difficulty, and altered the proteome landscape of the prefrontal cortex indicative of massive organizational synaptic restructuring.

Proteomic Studies on the Swim Bladder of the European Eel (Anguilla anguilla).

The swim bladder of a fish is a vital organ that with gas gland cells in the swim bladder wall enables key physiological functions including buoyancy regulation in the face of different hydrostatic pressures. Specific gas gland cells produce and secrete acidic metabolites into the blood in order to reduce the physical solubility of gases and blood gas transport capacity for regulating the volume of the swim bladder. Transcriptomic analyses have provided evidence at the RNA level but no specific studies at the protein level have been carried out so far. Herein, it was the aim of the study to show swim bladder proteins of the yellow stage European eel by label-free LCMS (Q-Exactive Plus) that resulted in the identification of 6223 protein groups. Neurotransmitter receptors and transporters were enriched in the membrane fraction and enzymes for acid production were observed. The list of identified proteins may represent a useful tool for further proteomics experiments on this organ. All MS proteomics data are available at the PRIDE repository with the dataset identifier PXD007850.