The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia.
PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTRr) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance.
We demonstrated that several lines of highly MTRr G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTRr Giardia. However, reduction of flavins is suppressed in highly MTRr cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTRr Trichomonas vaginalis.
These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.
metronidazole; ronidazole; tinidazole; Blastocystis; NADPH oxidase
The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated using laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.
The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE3MtrC6), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.
Antibiotics; Bacteria; Membrane Proteins; Multidrug Transporters; Protein Assembly; Neisseria gonorrhoeae
CoA inhibits the oxidation of 2-propanol and the reduction of acetaldehyde, acetone and a yet unidentified “background” substrate by ADH-1.
•Trichomonas vaginalis NADPH-dependent alcohol dehydrogenase-1 (ADH-1) reduces acetaldehyde and acetone, and oxidizes 2-propanol.•In addition to its canonical function, a strong reducing background activity was observed.•All reactions catalyzed by ADH-1 are strongly inhibited by CoA.•These observations also apply for the parasites Entamoeba histolytica and Tritrichomonas foetus, but not for Giardia lamblia which lacks ADH-1.
Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase.
Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension.
Trichomonas vaginalis; NADP-dependent alcohol dehydrogenase-1; Background activity; CoA
Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (Ki, 2 to 10 μM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO2) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and 1H nuclear magnetic resonance analyses determined that the spectral shift (A418 to A351) of NTZ was due to protonation of the anion (NTZ−) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pKa = 6.18). We propose that NTZ− intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the “activated cofactor” of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.
MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of the mtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand how MtrA activates mtrCDE expression, we successfully purified MtrA and found that it could bind specifically to the mtrCDE promoter region. The affinity of MtrA for the mtrCDE promoter increased 2-fold in the presence of a known effector and substrate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition with MtrR, the transcriptional repressor of mtrCDE, MtrA was found to bind with apparent lower affinity than MtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increased MtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important in MtrA binding) defined the MtrA-binding site 20–30 bp upstream of the known MtrR-binding site. Collectively, these results suggest that the MtrA and MtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for the mtrCDE promoter such that MtrR binding is negatively impacted. Our results provide a mechanism for transcriptional activation of mtrCDE by MtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci.
Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a “superbug.” One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump has been done on the transcriptional repressor, MtrR. This study is the first one to purify and define the DNA-binding ability of the transcriptional activator, MtrA. Understanding how levels of the MtrCDE efflux pump are regulated increases our knowledge of gonococcal biology and how the gonococcus can respond to various stresses, including antimicrobials.
Apply ultrasound (US) imaging techniques to better describe the characteristics of myofascial trigger points (MTrPs) and the immediately adjacent soft tissue.
Descriptive (exploratory) study.
Biomedical research center.
9 subjects meeting Travell and Simons’s criteria for MTrPs in a taut band in the upper trapezius.
Main Outcome Measures
MTrPs were evaluated by 1) physical examination, 2) pressure algometry, and 3) three types of ultrasound imaging including grayscale (2D US), vibration sonoelastography (VSE), and Doppler.
Four sites in each patient were labeled based on physical examination as either active MTrP (spontaneously-painful, A-MTrP), latent MTrP (non-painful, L-MTrP), or normal myofascial tissue. US examination was performed on each subject by a team blinded to the physical findings. A 12-5 MHz US transducer was used. VSE was performed by color Doppler variance imaging while simultaneously inducing vibrations (~92Hz) with a handheld massage vibrator. Each site was assigned a tissue imaging score (TIS) as follows: 0 = uniform echogenicity and stiffness; 1 = focal hypoechoic region with stiff nodule; 2 = multiple hypoechoic regions with stiff nodules. Blood flow in the neighborhood of MTrPs was assessed using Doppler imaging. Each site was assigned a blood flow waveform score (BFS) as follows: 0 = normal arterial flow in muscle; 1 = elevated diastolic flow; 2 = high-resistance flow waveform with retrograde diastolic flow.
MTrPs appeared as focal, hypoechoic regions on 2D US, indicating local changes in tissue echogenicity, and as focal regions of reduced vibration amplitude on VSE, indicating a localized stiff nodule. MTrPs were elliptical in shape, with a size of 0.16 ± 0.11 cm2. There were no significant differences in size between A-MTrPs and L-MTrPs. Sites containing MTrPs were more likely to have higher TIS compared to normal myofascial tissue (p<0.002). Small arteries (or enlarged arterioles) near A-MTrPs showed retrograde flow in diastole indicating a highly resistive vascular bed. A-MTrP sites were more likely to have higher BFS compared to L-MTrPs (p<0.021).
Preliminary findings show that, under the conditions of this investigation, US imaging techniques can be used to distinguish myofascial tissue containing MTrPs from normal myofascial tissue (lacking trigger points). Ultrasound enables visualization and some characterization of MTrPs and adjacent soft tissue.
Ultrasonography; Sonoelastography; Rehabilitation; Myofascial Pain Syndromes; Trigger Points; Myofascia
The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.
Antibiotics; Bacteria; Drug Resistance; Membrane; Multidrug Transporters
•Mithramycin (MTR) binds to core histones but not to linker histone H1.•Unlike MTR–DNA interaction, MTR–histone association is metal independent.•MTR alters H3K18 acetylation in vitro and ex vivo.•MTR is a dual binder (binds to both DNA and histones) in the chromatin context.
Mithramycin (MTR) is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin – histone proteins – as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode). The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR–DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg2+. As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo. Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.
MTR, mithramycin; HD, Huntington’s disease; NIH, National Institutes of Health; EWS-FLI1, transcription factor with a DNA binding domain FLI1 and a transcription enhancer domain EWS; HAT, histone acetyltransferase; CBP, CREB-binding protein; H3K18Ac, histone H3 lysine 18 acetylation; SGR, sanguinarine; ITC, isothermal titration calorimetry; CD, circular dichroism; EM, electron microscopy; FACS, fluorescence activated cell sorting; BAC, benzalkonium chloride; TCA, trichloroacetic acid; TBST, Tris-buffered saline Tween-20; MTT, 3-(4-5 dimethylthiazol-2-yl) 2-5diphenyl-tetrazolium bromide; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PTM, post-translational modification; M2+, bivalent metal ion such as Mg2+; Mithramycin; Core histones; Dual binding mode; Epigenetic modulator; H3K18 acetylation
The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via SRN1 and subsequent reactions, were assayed against the protozoan parasites Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica. Two metronidazole-sensitive lines and two metronidazole-resistant lines of Giardia and one line each of metronidazole-sensitive and -resistant Trichomonas were tested. All except one of the compounds were as effective or more effective than metronidazole against Giardia and Trichomonas, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against Entamoeba. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of Giardia and Trichomonas. However, some drugs were lethal to metronidazole-resistant Giardia and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.
Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.
Giardia lamblia is both the most common intestinal parasite in the United States and a frequent cause of diarrheal illness throughout the world. In spite of its recognition as an important human pathogen, there have been relatively few agents used in therapy. This paper discusses each class of drugs used in treatment, along with their mechanism of action, in vitro and clinical efficacy, and side effects and contraindications. Recommendations are made for the preferred treatment in different clinical situations. The greatest clinical experience is with the nitroimidazole drugs, i.e., metronidazole, tinidazole, and ornidazole, which are highly effective. A 5- to 7-day course of metronidazole can be expected to cure over 90% of individuals, and a single dose of tinidazole or ornidazole will cure a similar number. Quinacrine, which is no longer produced in the United States, has excellent efficacy but may be poorly tolerated, especially in children. Furazolidone is an effective alternative but must be administered four times a day for 7 to 10 days. Paromomycin may be used during early pregnancy, because it is not systematically absorbed, but it is not always effective. Patients who have resistant infection can usually be cured by a prolonged course of treatment with a combination of a nitroimidazole with quinacrine.
The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump–component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux.
► Metronidazole and tinidazole bind Giardia proteins, including thioredoxin reductase. ► 5-Nitroimidazoles cause degradation of translation elongation factor-1γ (EF-1 γ). ► Nitroimidazoles deplete intracellular thiol levels. ► Nitroimidazoles affect Giardia differently than other parasites.
Giardia lamblia (syn. duodenalis, intestinalis) is a globally occurring micro-aerophilic human parasite that causes gastrointestinal disease. Standard treatment of G. lamblia infections is based on the 5-nitroimidazole drugs metronidazole and tinidazole. In two other micro-aerophilic parasites, Entamoeba histolytica and Trichomonas vaginalis, 5-nitroimidazole drugs bind to proteins involved in the thioredoxin-mediated redox network and disrupt the redox equilibrium by inhibiting thioredoxin reductase and depleting intracellular thiol pools. The major aim of this study was to assess whether nitroimidazoles exert a similar toxic effect on G. lamblia physiology.
The 5-nitroimidazoles metronidazole and tinidazole were found to bind to the same subset of proteins including thioredoxin reductase. However, in contrast to E. histolytica and T. vaginalis, none of the other proteins bound are candidates for being involved in the thioredoxin-mediated redox network. Translation elongation factor EF-1γ, an essential factor in protein synthesis, was widely degraded upon treatment with 5-nitroimidazoles. 2-Nitroimidazole (azomycin) and the 5-nitroimidazole ronidazole did not bind to any G. lamblia proteins, which is in contrast to previous findings in E. histolytica and T. vaginalis. All nitroimidazoles tested reduced intracellular thiol pools in G. lamblia, but metronidazole, also in contrast to the situation in the other two parasites, had the slightest effect. Taken together, our results suggest that nitroimidazole drugs affect G. lamblia in a fundamentally different way than E. histolytica and T. vaginalis.
Nitroimidazoles; Protein binding; Translation elongation factor-1γ; Thiol depletion
The 79-amino-acid (79-aa) open reading frame (UL111a) gene within morphological transforming region II (mtrII) of human cytomegalovirus strain Towne has been shown to transform rodent cells in vitro (J. Thompson, J. Doniger, and L. J. Rosenthal, Arch. Virol. 136:161-172, 1994). Moreover, a translation termination linker (TTL) mutant of mtrII that coded for the first 49 aa of mtrII oncoprotein (designated TTL49) was sufficient for malignant transformation, whereas a TTL mutant that coded for the first 24 aa (designated TTL24) was not. The current study demonstrates the binding of mtrII oncoprotein to the tumor suppressor protein p53 both in vivo using transiently transfected cells and in vitro using labeled proteins. Furthermore, the C-terminally truncated mtrII protein TTL49, but not truncated protein TTL24, bound to p53. The mtrII binding domain mapped to the N-terminal region of p53, residues 1 to 106, with a critical region from aa 27 to 44, whereas the p53 binding domain of mtrII protein was the first 49 aa. Furthermore, mtrII inhibited p53-activated transcription, indicating its ability to alter p53-directed cellular regulatory mechanisms. mtrII oncoprotein was detected both in stably transfected NIH 3T3 cell lines and human cytomegalovirus-infected HEL 299 cells (as early as 12 h after infection) in the perinuclear region and in the nucleus. mtrII-transformed cell lines, at both early and late passage, exhibited high levels of p53 with a 15-fold-extended half-life. However, p53-activated transcription was suppressed in these cells in spite of the increased p53 levels. Finally, the results with wild-type mtrII and its TTL mutants with respect to p53 binding, p53-activated transcription, and transforming ability suggest that the mechanism of mtrII transformation is linked to both p53 binding and disruption of p53 cell regulation.
Metronidazole is the most commonly used drug for the treatment of giardiasis in humans. In spite of its therapeutic efficacy for giardiasis, low patient compliance, especially in children, side effects, and the emergence of metronidazole-resistant strains may restrict its use. Albendazole has been used to treat Giardia duodenalis infections in recent years. However, efficacy studies in vivo and in vitro have produced diverse results as to its effectiveness. A moderately benign side effect profile, combined with established efficacy against many helminths, renders it promising for treatment of giardiasis in humans.
Methodology and Principal Findings
We performed a search in the PubMed, Scopus, EMBASE, the ISI Web of Science, LILIACS, and Cochrane Controlled Trials Register for trials published before February 2010 as well as in references of relevant research and review articles. Eight randomized clinical trials (including 900 patients) comparing the effectiveness of albendazole with that of metronidazole were included in meta-analysis. After extracting and validating the data, the pooled risk ratio (RR) was calculated using an inverse-variance random-effects model. Albendazole was found to be equally as effective as metronidazole in the treatment of giardiasis in humans (RR 0.97; 95% CI, 0.93, 1.01). In addition, safety analysis suggested that patients treated with albendazole had a lower risk of adverse effects compared with those who received metronidazole (RR 0.36; 95% CI, 0.10, 1.34), but limitations of the sample size precluded a definite conclusion.
The effectiveness of albendazole, when given as a single dose of 400 mg/day for 5 days, was comparable to that of metronidazole. Patients treated with albendazole tended to have fewer side effects compared with those who took metronidazole. Given the safety, effectiveness, and low costs of albendazole, this drug could be potentially used as an alternative and/or a replacement for the existing metronidazole therapy protocols in the treatment of giardiasis in humans.
Giardiasis is one of the most common intestinal protozoal infections worldwide. Although metronidazole is the most common drug used to treat giardiasis in humans, its use is associated with a variety of side effects. Poor compliance and the emergence of metronidazole-resistant strains may restrict use of the drug. Albendazole is an orally administered broad-spectrum anthelmintic agent. The use of albendazole has fewer side effects than metronidazole. The anthelmintic has been used against Giardia duodenalis both in vivo and in vitro with different results. However, the current meta-analysis assessed the effectiveness and safety of albendazole compared with metronidazole for the treatment of giardiasis in humans. After searching different databases, eight comparative randomized clinical trials, including 900 patients, met our criteria and were selected for the current meta-analysis. Results showed that albendazole was as effective as metronidazole for the treatment of giardiasis in humans and people receiving the drug tended to have fewer side effects compared with those who received metronidazole. Given the safety, effectiveness, and low costs of albendazole, this drug may be considered a potential alternative and/or a replacement for the existing widely used metronidazole in the treatment of giardiasis in humans.
The MtrR transcriptional-regulatory protein is known to repress transcription of the mtrCDE operon, which encodes a multidrug efflux pump possessed by Neisseria gonorrhoeae that is important in the ability of gonococci to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to determine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated. rpoH, which encodes the general stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by MtrR, as it was found to bind to a DNA sequence upstream of rpoH that included sites within the rpoH promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely indirect and a reflection of MtrR control of rpoH expression. Inducible expression of MtrR was found to repress rpoH expression and to increase gonococcal susceptibility to hydrogen peroxide (H2O2) and an antibiotic (erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability to control the expression of the mtrCDE-encoded efflux pump operon and, as a consequence, levels of gonococcal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability of MtrR to regulate the expression levels of rpoH and RpoH-regulated genes also modulates levels of gonococcal susceptibility to H2O2.
The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z’ factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.
Carbamate kinase; Giardia; high throughput screening; assay development.
We have applied a model that permits the estimation of the sensitivity of flux through branch point enzymes (D. C. LaPorte, K. Walsh, and D. E. Koshland, J. Biol. Chem. 259:14068-14075, 1984) in order to analyze the control of flux through the lactate-acetate branch point of Selenomonas ruminantium grown in glucose-limited continuous culture. At this branch point, pyruvate is the substrate of both the NAD-dependent L-(+)-lactate dehydrogenase (LDH) and the pyruvate:ferredoxin oxidoreductase (PFOR). The LDH was purified, and it exhibited positive cooperativity for the binding of pyruvate. The LDH had an [S].5 for pyruvate of 0.43 mM, a Hill coefficient of 2.4, and a K' equal to 0.13 mM. The PFOR, assayed in cell extracts, exhibited Michaelis-Menten kinetics for pyruvate, with a Km of 0.49 mM. Carbon flux through the LDH and the PFOR increased 80-fold and 3-fold, respectively, as the dilution rate was increased from 0.07 to 0.52 h-1 in glucose-limited continuous culture. There was nearly a twofold increase, from 6.5 to 11.2 mumol min-1 mg of protein-1 in the specific activity (i.e., maximum velocity) of the LDH at dilution rates of 0.11 and 0.52 h-1, respectively. A flux equation was used to calculate the intracellular concentration of pyruvate; a fourfold increase in pyruvate, from 0.023 to 0.093 mM, was thereby predicted as the dilution rate was increased from 0.07 to 0.52 h-1. When these calculated values of intracellular pyruvate concentration were inserted into the flux equation, the predicted values of flux through the LDH and the PFOR were found to match closely the flux actually measured in the chemostat-grown cells. Thus, the 80-fold increase in flux through the LDH was due to a twofold increase in the maximum velocity of the LDH and a fourfold increase in the intracellular pyruvate concentration. In addition, the flux through the LDH exhibited ultrasensitivity to changes in both the maximum velocity of the LDH and the intracellular concentration of pyruvate. The flux through the PFOR exhibited ultrasensitivity to changes in the maximum velocity of the LDH and hyperbolic sensitivity to changes in the intracellular concentration of pyruvate.
Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.
Metronidazole (MTZ) is often used in combination therapies to treat infections caused by the gastric pathogen Helicobacter pylori. Resistance to MTZ results from loss-of-function mutations in genes encoding RdxA and FrxA nitroreductases. MTZ-resistant strains, when cultured at sub-MICs of MTZ (5 to 20 μg/ml), show dose-dependent defects in bacterial growth; depressed activities of many Krebs cycle enzymes, including pyruvate:ferredoxin oxidoreductase (PFOR); and low transcript levels of porGDAB (primer extension), phenotypes consistent with an involvement of a transcriptional regulator. Using a combination of protein purification steps, electrophoretic mobility shift assays (EMSAs), and mass spectrometry analyses of proteins bound to porG promoter sequences, we identified HP1043, an essential homeostatic global regulator (HsrA [for homeostatic stress regulator]). Competition EMSAs and supershift analyses with HsrA-enriched protein fractions confirmed specific binding to porGDAB and hsrA promoter sequences. Exposure to MTZ resulted in >10-fold decreases in levels of HsrA and in levels of the HsrA-regulated gene products PFOR and TlpB. Exposure to paraquat (PQ), hydrogen peroxide, or organic peroxides showed near equivalence with MTZ, revealing a common oxidative stress response pathway. Finally, direct superoxide dismutase (SOD) assays showed an inverse relationship between HsrA levels and SOD activity, suggesting that HsrA may serve as a repressor of sodB. As a homeostatic sentinel, HsrA appears to be ideally positioned to enable rapid shutdown of genes associated with metabolism and growth while activating (directly or indirectly) oxidative defense genes in response to low levels of toxic metabolites (MTZ or oxygen) before they reach DNA-damaging levels.
Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.
Shoulder pain is reported to be highly prevalent and tends to be recurrent or persistent despite medical treatment. The pathophysiological mechanisms of shoulder pain are poorly understood. Furthermore, there is little evidence supporting the effectiveness of current treatment protocols. Although myofascial trigger points (MTrPs) are rarely mentioned in relation to shoulder pain, they may present an alternative underlying mechanism, which would provide new treatment targets through MTrP inactivation. While previous research has demonstrated that trained physiotherapists can reliably identify MTrPs in patients with shoulder pain, the percentage of patients who actually have MTrPs remains unclear. The aim of this observational study was to assess the prevalence of muscles with MTrPs and the association between MTrPs and the severity of pain and functioning in patients with chronic non-traumatic unilateral shoulder pain.
An observational study was conducted. Subjects were recruited from patients participating in a controlled trial studying the effectiveness of physical therapy on patients with unilateral non-traumatic shoulder pain. Sociodemographic and patient-reported symptom scores, including the Disabilities of the Arm, Shoulder, and Hand (DASH) Questionnaire, and Visual Analogue Scales for Pain were compared with other studies. To test for differences in age, gender distribution, and education level between the current study population and the populations from Dutch shoulder studies, the one sample T-test was used. One observer examined all subjects (n = 72) for the presence of MTrPs. Frequency distributions, means, medians, standard deviations, and 95% confidence intervals were calculated for descriptive purposes. The Spearman's rank-order correlation (ρ) was used to test for association between variables.
MTrPs were identified in all subjects. The median number of muscles with MTrPs per subject was 6 (active MTrPs) and 4 (latent MTrPs). Active MTrPs were most prevalent in the infraspinatus (77%) and the upper trapezius muscles (58%), whereas latent MTrPs were most prevalent in the teres major (49%) and anterior deltoid muscles (38%). The number of muscles with active MTrPs was only moderately correlated with the DASH score.
The prevalence of muscles containing active and latent MTrPs in a sample of patients with chronic non-traumatic shoulder pain was high.
myofascial; pain; trigger points; prevalence; shoulder
The human methionine synthase gene (MTR) is located on chromosome 1q43; it is of 105.24 kb and is made up of 33 exons. Methionine synthase is a cytoplasmic enzyme that requires methylcobalamin for activity and catalyzes the remethylation of homocysteine to methionine. In this reaction, the methyl group of 5-methyltetrahydrofolate is transferred to the enzyme bond cob(I) alamin to generate methylcobalamin, followed by the transfer of the methyl group to homocysteine to reform methionine.
MATERIALS AND METHODS:
The frequencies of the polymorphisms of MTR 2756A>G and MTR 2758C>G have been determined in this study in a sample of 491 individuals collected from all regions of Jordan and representing the Jordanian population. The different alleles and genotypes at the two polymorphic sites were identified using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) analysis.
Showed that the percentages of the polymorphic alleles at the MTR 2756 position in the north, middle and south regions were 90.38, 92.65 and 83.69%, respectively, for the MTR 2756A allele, and were 9.61, 7.34 and 16.30%, respectively, for the MTR 2756G allele, with overall percentages in the whole Jordanian population of 90.73 and 9.27% for the MTR 2756A and MTR 2756G alleles, respectively. The percentages of the genotype MTR 2756AA were 82.90% in the northern region, 86.72% in the middle region and 71.73% in the southern region, and an overall percentage of MTR 2756AA in the whole Jordanian population was 83.50%. The frequencies of MTR 2756AG genotype in the northern, middle and southern regions were 14.95, 11.84 and 23.91%, respectively, with an overall percentage of 14.46% in the whole Jordanian population. The percentages of the genotype MTR 2756GG in the northern, middle and southern regions were 2.13, 1.42 and 4.34%, respectively, with an overall percentage of 2.04% in the whole Jordanian population. Only the wild type allele (C) of the MTR 2758C>G polymorphism was detected in this study. In addition, the association of MTR 2756A>G and MTR 2758C>G polymorphisms with the development of neural tube defects (NTDs) was examined using 17 cases of mothers from the northern part of Jordan, who gave birth to NTD affected children during the period of this study. Results showed no association between these two examined polymorphisms and the increase in maternal risk for giving birth to NTD children.
results of this study recommend that examination should be done on larger populations to arrive at better conclusions. Also, more studies on gene–gene interaction should be done to examine the associations with NTDs.
Jordan; MTR; neural tube defects; polymorphism
Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a kcat of 122 s−1, and an apparent Km of 14 μM for menadione and 26 μM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO2, and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO2 fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO2-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO2 fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.