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Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
SUMMARY INTRODUCTION EPIDEMIOLOGY, BIOLOGY, AND DISEASE Entamoeba histolytica Giardia intestinalis Trichomonas vaginalis ENERGY METABOLISM OF ''AMITOCHONDRIATE'' PARASITES Divergence of Mitochondrion-Related Organelles Diversity in the Conversion of Phosphoenolpyruvate to Acetyl Coenzyme A via Pyruvate Predominant End Products of Energy Metabolism Three Major Iron-Sulfur Proteins That Play an Essential Role in Energy Metabolism Pyruvate:ferredoxin oxidoreductase. Ferredoxin. Hydrogenase. CURRENT CHEMOTHERAPEUTICS Current Chemotherapeutics for E. histolytica Infection Current Chemotherapeutics for G. intestinalis Infection Current Chemotherapeutics for T. vaginalis Infection DRUG TARGETS AND MECHANISMS OF RESISTANCE Mechanism of Action of 5-Nitroimidazole and Benzimidazoles Drug Resistance in E. histolytica Drug Resistance in G. intestinalis Drug Resistance in Trichomonas SULFUR-CONTAINING-AMINO-ACID METABOLISM AS A NOVEL DRUG TARGET Metabolic Pathways in Protozoan Parasites under Investigation To Explore as Targets for Drug Development Physiological Importance of Cysteine and Fe-S Cluster Biosynthesis Heterogeneity of Fe-S Cluster Biosynthesis Unique Aspects of Sulfur-Containing-Amino-Acid Metabolism Degradation of Sulfur-Containing Amino Acids Structural Differences between Protozoan MGL and Mammalian CGL Drug Development Targeting Methionine gamma-Lyase Sulfur-Assimilatory De Novo Cysteine Biosynthesis Cysteine Synthase as a Drug Target CONCLUSIONS REFERENCES
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-lyase-mediated catabolism of sulfur-containing amino acids, which are present in T. vaginalis and E. histolytica but absent in G. intestinalis. These pathways are potentially exploitable for development of drugs against amoebiasis and trichomoniasis. For instance, L-trifluoromethionine, which is catalyzed by methionine -lyase and produces a toxic product, is effective against T. vaginalis and E. histolytica parasites in vitro and in vivo and may represent a good lead compound. In this review, we summarize the biology of these microaerophilic parasites, their clinical manifestation and epidemiology of disease, chemotherapeutics, the modes of action of representative drugs, and problems related to these drugs, including drug resistance. We further discuss our approach to exploit unique sulfur-containing-amino-acid metabolism, focusing on development of drugs against E. histolytica. | INTRODUCTION |
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Recent molecular and biochemical characterization of the sulfur-containing-amino-acid metabolism in these organisms revealed that metabolic pathways for sulfur-containing amino acids in E. histolytica, G. intestinalis, and T. vaginalis are distinct from those of their mammalian hosts in several ways. First, they lack part of both the forward and reverse transsulfuration pathways and thus are unable to complete transsulfuration sequences in either direction between methionine and cysteine. Second, they lack the enzymes responsible for cysteine and homocysteine degradation in mammals. Instead, E. histolytica and T. vaginalis possess a unique enzyme for the degradation of methionine, homocysteine, and cysteine called methionine -lyase. Third, E. histolytica and T. vaginalis are capable of sulfur-assimilatory de novo cysteine biosynthesis. Since aspects of sulfur-containing-amino-acid metabolism differ significantly between parasites and their mammalian hosts, molecular dissection and characterization of the unique properties of the sulfur-containing-amino-acid metabolism of these "amitochondriate" parasites should lead to the exploitation of new chemotherapeutic agents against infections caused by these pathogens. This review discusses the current therapeutic agents against infections by these "amitochondriate" protozoan parasites, their targets and mode of action, and the molecular mechanism of drug resistance. We also summarize various aspects of the unique sulfur-containing-amino-acid metabolism in these protozoa and discuss how these metabolic pathways could be exploited as novel targets for development of drugs against these infections.
| EPIDEMIOLOGY, BIOLOGY, AND DISEASE |
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E. histolytica has a simple life cycle consisting of two stages, an infective cyst stage and a proliferating trophozoite form. Human and certain nonhuman primates are its only natural hosts. Infection of the host occurs upon ingestion of water or food contaminated with cysts. E. histolytica cysts are round, usually 10 to 15 µm in diameter, and surrounded by a refractive wall containing chitin. After ingestion, the cyst excysts in the small intestine and forms the amoeboid trophozoite, which then colonizes the large intestine of the host. Colonization by E. histolytica trophozoites often results in an asymptomatic intestinal infection similar to that resulting from E. dispar (67). Unlike the inert cysts, E. histolytica trophozoites are highly motile, with polymorphic shapes and sizes varying from 10 to 50 µm in diameter. Trophozoites reproduce by binary fission, ingest bacteria and food particles, and adhere to and destroy epithelial cells in the bowel. The destruction of the epithelial tissue causes disease and symptoms. After penetration into the blood vessels, trophozoites are occasionally transported to extraintestinal organs, including the liver, lung, brain, and skin, and produce abscesses, often with lethal outcomes. Trophozoites transform into dormant and infectious cysts which are excreted into the environment. Although signals leading to encystation and excystation of E. histolytica are still poorly understood, osmolality changes, nutrient depletion (278), and adherence via galactose-binding lectin (79) are likely involved in encystation. In vitro encystation of the related reptilian species Entamoeba invadens was established by manipulating osmotic and nutrient conditions of axenic cultures (20, 278, 317). However, encystation has not been achieved with an axenic E. histolytica line.
Many individuals infected with E. histolytica have no symptoms and clear their infection without disease. However, up to 10% of asymptomatic infected individuals develop disease within a year of being infected (293). Clinical symptoms of amoebic colitis include bloody diarrhea and abdominal pain and tenderness. Multiple mucoid stools are common and are almost invariably heme positive. Fever is unusual except in cases with concurrent amoebic liver abscess. Fulminant amoebic colitis characterized by profuse bloody diarrhea, fever, pronounced leucocytosis, and severe abdominal pain is occasionally seen in individuals at risk, including pregnant women, immunocompromised individuals, patients receiving corticosteroids, and individuals with diabetes and alcoholism. Liver abscess is the most common extraintestinal manifestation of amoebic infection, most likely caused by hematogenous spread of amoebic trophozoites from the colon. Symptoms associated with amoebic liver abscess are fever, right upper quadrant pain, and hepatic tenderness and sometimes include cough, anorexia, and weight loss. Pleuropulmonary amoebiasis may also develop when an amoebic liver abscess is ruptured through the diaphragm. Patients with pleuropulmonary amoebiasis have chest pain, pleural effusions, atelectasis, and respiratory distress. Rupture into the peritoneum occurs occasionally, leading to peritonitis and shock in some individuals with amoebic liver abscess. Patients with amoebic liver abscess infrequently develop rupture into the pericardium, leading to pericarditis, dyspnoea, tachycardia, and cardiac tamponade. Amoebic brain abscess, although very rare, may also occur with concomitant amoebic liver abscess. Clinical symptoms include headache, vomiting, and seizures. The onset and progression of amoebic brain abscess is very rapid, and outcomes are often lethal. Comprehensive reviews of the pathophysiology and clinical signs and symptoms are found elsewhere (122, 293).
E. histolytica is considered to be anaerobic or microaerophilic because it is sensitive to oxygen yet is able to reduce it to water (296). E. histolytica can grow only under reduced oxygen tension in vitro (325). Toxic reactive oxygen derivatives, including superoxide, are produced by the electron transfer that occurs during energy metabolism. E. histolytica possesses iron-containing superoxide dismutase (SOD) (39) for the detoxification of reactive oxygen intermediates, but it lacks catalase, peroxidase, and enzymes for glutathione synthesis and metabolism (92, 205, 325), which are almost ubiquitously present in aerobes and are responsible for the removal of hydrogen peroxide. However, E. histolytica possesses alternative mechanisms for detoxification of the reactive oxygen species. The alternative mechanism depends on reducing agents (thiols), especially cysteine. In glutathione-depleted medium, cysteine was the main thiol compound in E. histolytica and was present largely in the thiol rather than the disulfide form (92). As mentioned above, extracellular cysteine is required to maintain a reducing environment and is essential for growth, attachment, and survival of E. histolytica trophozoites. It is conceivable that cysteine and other, as-yet-uncharacterized thiols are involved in the regulation of antioxidative defense mechanisms that likely consist of SOD (39, 297), peroxiredoxin (a thiol-specific antioxidant) (323), p34 NADPH oxidoreductase (38), and rubrerythrin (185, 205). However, several key components of the pathway have not been identified, e.g., a reducing system for peroxiredoxin and rubrerythrin.
The life cycle of G. intestinalis consists of two stages, an infective cyst and a proliferating trophozoite form. The cyst is the infective form of the parasite and can survive in water for several months (320). The ingested cyst excysts in the duodenum and then becomes a motile trophozoite after passing through the acidic environment of the stomach. The trophozoite reproduces by binary fission and attaches to the duodenal or jejunal mucosa (7, 8, 88), where it causes symptoms. Environmental stimuli, including cholic acid and low cholesterol, induce trophozoites to encyst (111, 189). However, the biochemical and physiological significance of cholesterol deprivation and the role of bile salts and fatty acids in the encystation process remain controversial. For the related issues of encystation and excystation of G. intestinalis and E. histolytica, recent reviews should be consulted (7, 78).
A range of symptoms, including nausea, vomiting, stomach cramps, and diarrhea, occur after an incubation period of 1 to 2 weeks. The acute phase of infection usually lasts 3 to 4 days, but symptoms sometimes persist for a longer period (8). In children, a failure-to-thrive syndrome caused by infection with high parasite burdens and malnutrition occasionally results in severe loss of body weight (up to 20%) (95). Thus, in the developing world, giardiasis is considered to be an important cause of morbidity (43). In the small intestine, infestation with the organism reduces the number of microvilli and the surface area at the brush border, causing atrophy of the villi and enterocyte immaturity. It also decreases disaccharidase and other luminal enzymes and causes malabsorption of electrolytes (43). A number of other symptoms are associated with giardiasis (43), but it is unlikely that these symptoms are causally connected to the infection. Consult recent reviews for updates on the pathogenesis, pathology, and cell biology of giardiasis (280).
The pathogenesis of giardiasis is partially attributable to the enzymes produced by the parasite, including serine and cysteine proteases which disrupt the epithelial barrier in the intestine, causing inflammation and an immune response in the host. Giardia trophozoites induce apoptosis of enterocytes, which is associated with disruption of the cytoskeleton and tight junctions. It has also been reported that disruption of cellular F-actin and ZO-1 in tight junctions and the concurrent increase in enterocyte permeability are modulated by caspase-3 and myosin light chain kinase. Giardia proteases are proposed to activate protease-activated receptor 1, a unique class of G protein-coupled signaling receptors which modulate apoptosis and increase enterocyte permeability (280).
The T. vaginalis life cycle consists of a single dividing trophozoite stage; no dormant (e.g., cyst) stage has been identified in this parasite. The trophozoite varies in size and shape, with an average length and width of 10 µm and 7 µm, respectively (134). In axenic culture, the shape of the trophozoite tends to be more uniform, e.g., pear or oval shaped, but the parasite takes on a more amoeboid appearance when attached to vaginal epithelial cells (19, 124). The trophozoite has four free anterior flagella [9(2) + 2 arrangement] and a single recurrent flagellum incorporated into an undulating membrane which is supported by a noncontractile costa (282). The trophozoite divides by binary fission (247).
T. vaginalis infection in women ranges from an asymptomatic carrier state to profound acute inflammatory disease (261). The parasite generally infects the squamous epithelium of the genital tract, but it is occasionally recovered from the urethra, fallopian tubes, and pelvis (117, 247). The incubation period is 4 to 28 days in about 50% of infected individuals (247). The clinical picture in acute infection includes vaginal discharge, odor, and edema or erythema. The discharge is classically described as frothy, but actually it is frothy in only 10% of patients (282). Small punctuate hemorrhagic spots may be found on the vaginal and cervical mucosae in 2 to 3% of patients. These signs and symptoms are cyclic and worsen around the time of menses. Other complications associated with trichomoniasis include adnexitis, pyosalpinx, and endometritis (260); infertility and low birth weight (123); and cervical erosion (204). In males, T. vaginalis infection is often asymptomatic but occasionally causes urethritis and prostatitis (166). Urogenital trichomoniasis in men is categorized into three groups: an asymptomatic carrier state identified through an investigation of the sexual contacts of infected women, acute trichomoniasis characterized by profuse purulent urethritis, and mild symptomatic disease which is clinically indistinguishable from other causes of nongonococcal urethritis. The duration of infection is 10 days or less in most male patients. In symptomatic men, common complaints include scanty clear to mucopurulent discharge, dysuria, and mild pruritus or a burning sensation immediately after sexual intercourse (166). Complications associated with trichomoniasis include nongonococcal urethritis, prostatitis, balanoposthitis, urethral disease, and infertility (131, 167, 197). Pneumonia, bronchitis, and oral infection caused by T. vaginalis have also been documented (132). In rare cases, respiratory infections are acquired perinatally from infected mothers (132, 158). In children, T. vaginalis can infect the urinary tract as well as the vagina. It has been suggested that T. vaginalis infection is associated with sterility, but there is no unequivocal report available. In contrast, there is an established causal relationship between T. vaginalis infection and adverse pregnancy outcomes (9). Similarly, T. vaginalis infection was associated with low birth weight and preterm delivery in 40% of black women (57, 279). In that study, in which 14,000 American women were examined, Trichomonas was also associated with high infant mortality. In cattle, Trichomonas foetus infection causes infertility, which results in a tremendous economic loss (55, 133). It was suggested that T. vaginalis infection, as well as other STDs, increases susceptibility to HIV infection due to local inflammation and microscopic breaches in mucosal barriers (282). It was also suggested that T. vaginalis infection predisposes HIV carriers to symptomatic AIDS (211, 292). However, this issue is still debatable. Conversely, immunosuppression from HIV infection increases susceptibility to STDs (173, 211, 250, 282, 283, 292).
| ENERGY METABOLISM OF "AMITOCHONDRIATE" PARASITES |
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The conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) is catalyzed by pyruvate:ferredoxin oxidoreductase (PFOR), which utilizes ferredoxin rather than NAD+ as an electron acceptor in E. histolytica (257), G. intestinalis (307, 308), and T. vaginalis (330) in place of the pyruvate dehydrogenase complex found in aerobic bacteria and eukaryotes. Pyruvate dehydrogenase in the mitochondria of aerobic eukaryotes catalyzes an oxidative decarboxylation to form acetyl-CoA and NADH. Energy metabolism in these protists was recently reviewed in more detail (112, 217).
In Trichomonas, where part of the energy metabolism is compartmentalized in the hydrogenosome, electrons produced by the oxidation of pyruvate catalyzed by PFOR are donated to an Fe-hydrogenase via ferredoxin, which produces hydrogen gas (H2) by transferring electrons to hydrogen ions (Fig. 2). ATP is generated exclusively by substrate-level phosphorylation in the hydrogenosomes (75). In wild-type T. vaginalis, the major end product from pyruvate is lactate produced by lactate dehydrogenase in the cytosol; lactate dehydrogenase is present only in T. vaginalis. The preferred end product also differs between Trichomonas species and between wild-type and drug-resistant strains (168) (see below). In metronidazole-susceptible T. foetus, a Trichomonas species that affects cattle, either succinate or ethanol is the major or minor metabolic end product, respectively, in the cytosol (168), because lactate dehydrogenase is absent in this organism. In the hydrogenosomes, acetate is the only end product of both metronidazole-susceptible T. vaginalis and T. foetus strains. In contrast, major end products in the cytosol of metronidazole-resistant T. vaginalis and T. foetus strains are lactate and ethanol, respectively.
Recently the NADH dehydrogenase module (also called NADH:ubiquinone oxidoreductase) of complex I was identified in T. vaginalis (75, 140, 141). Similar to mitochondrial respiratory complex I, NADH dehydrogenase can reduce a variety of electron carriers, including ubiquinone. Unlike the mitochondrial enzyme, ferredoxin is used as an electron carrier for hydrogen production in a reaction catalyzed by ferredoxin-dependent Fe-hydrogenase. Malate is one of the major hydrogenosomal substrates and is oxidatively decarboxylated to form pyruvate and CO2 by a NAD-dependent malic enzyme. The electrons are transferred from NADH to ferredoxin by an NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I. Thus, the discovery of an NADH dehydrogenase module of complex I solved the long-lasting conundrum of how Trichomonas regenerates NAD+, which is essential for malate oxidation in the hydrogenosome (Fig. 2).
It was previously shown that T. vaginalis possesses two additional 2-keto acid oxidoreductases besides PFOR for energy production (34). These two 2-keto acid oxidoreductases, KOR1 and KOR2, prefer indolepyruvate as a substrate. KOR1 is present in both metronidazole-sensitive and -resistant strains, while KOR2 is present only in metronidazole-resistant strains of T. vaginalis (34, 73, 313). It was reported that 2-keto acid oxidoreductase activity increased in the metronidazole-resistant strain (34). However, neither KOR1 nor KOR2 could donate electrons to ferredoxin in the metronidazole-resistant line, because no detectable ferredoxin was produced in this strain (73) (see below).
200 to 300 kDa containing 2[4Fe-4S] clusters and thiamine phosphate. Its structure is similar to that of PFORs from a wide range of eubacteria (138); e.g., PFOR from the eubacterium Clostridium acetobutylicum is a homodimer of 123-kDa subunits (207). PFOR from the thermophilic archaebacterium Pyrococcus furiosus is composed of four fragmented subunits (29, 161), all of which share significant homology to PFORs from eubacteria, fungi, and protists (138). PFORs from E. histolytica, G. intestinalis, and T. vaginalis are homodimers of 240 to 280 kDa containing 2[4Fe-4S] clusters and thiamine phosphate (257, 264, 308, 330). Despite the similar overall structure and the Fe-S clusters of PFORs in these "amitochondriate" protists, the intracellular localization of PFOR is divergent. While PFORs from both archaea and eubacteria are cytosolic, biochemical characterization (179, 330) has established that PFOR is localized to hydrogenosomes and is associated with the hydrogenosome membrane in T. vaginalis (330). In addition, a 120-kDa surface glycoprotein that shares cross-reacting epitopes with PFOR is involved in the adhesion of T. vaginalis trophozoites (213). This may suggest that PFOR or a PFOR-related protein is associated with hydrogenosomes and the plasma membrane, but this premise is still a matter of debate and needs to be independently verified. The PFORs from both G. intestinalis (81, 308) and E. histolytica (263, 274) were also suggested to be associated with the plasma membrane. PFOR was shown to be associated with the plasma membrane and with a cytoplasmic structure in E. histolytica that appeared as a ring form or a compact small body (263). However, the majority of PFOR activity was detected in an 80,000 x g supernatant fraction (257), suggesting that the proposed membrane association of E. histolytica PFOR (263, 274) might be transient and/or weak. At the primary sequence level, T. vaginalis PFOR possesses the putative mitochondrial targeting peptide at the amino terminus (142). In contrast, neither E. histolytica nor G. intestinalis PFOR possesses the mitochondrial targeting sequence. Since pyruvate metabolism is not compartmentalized in either of these organisms like it is in T. vaginalis, the physiological significance of the possible membrane association of PFOR in G. intestinalis and E. histolytica is not well understood.
Ferredoxin.
Ferredoxin is another important class of proteins containing Fe-S clusters. Ferredoxins in the three microaerophilic/anaerobic parasitic protists differ in the nature of the Fe-S clusters they contain and their intracellular localization, i.e., cytosolic or hydrogenosomal. The E. histolytica genome encodes at least three ferredoxins (185), all of which contain either 2[4Fe-4S] clusters or [4Fe-4S] and [3Fe-4S] clusters, which correspond to a molecular mass of 6 kDa, as previously characterized (256). E. histolytica apparently lacks [2Fe-2S] ferredoxin (15, 17), but ferredoxins containing these clusters are present in T. vaginalis and G. intestinalis. A larger ferredoxin from Trichomonas with one [2Fe-2S] cluster corresponding to a molecular mass of 10 kDa was biochemically characterized and was localized in hydrogenosomes (116). The genome of G. intestinalis encodes at least four ferredoxins: one ferredoxin containing one [2Fe-2S] cluster (ferredoxin I) and three ferredoxins containing either 2[4Fe-4S] clusters or one [4Fe-4S] cluster and one [3Fe-4S] cluster (ferredoxins 1 to 3) (229). Among these ferredoxins, only ferredoxin I was shown to accept electrons from PFOR in vitro (308). The physiological role of [4Fe-4S] ferredoxins in G. intestinalis remains unclear. The small 2[4Fe-4S] ferredoxin of E. histolytica is evolutionarily closest to ferredoxin from anaerobic bacteria (143), while the larger [2Fe-2S] ferredoxin of Trichomonas hydrogenosomes shows a close kinship to ferredoxins of the cytochrome P450-linked mixed-function oxidase systems of bacterial and vertebrate mitochondria (154).
In addition, G. intestinalis possesses two genes, and E. histolytica possesses one gene, encoding a putative ferredoxin nitroreductase which consists of an amino-terminal 2[4Fe-4S] ferredoxin domain and a carboxyl-terminal nitroreductase domain (229). This ferredoxin-nitroreductase may be a physiological acceptor of electrons from ferredoxin or PFOR and may be responsible for the activation of metronidazole. A ferredoxin-nitroreductase with a similar overall structure was also discovered in the whole-genome sequence of Clostridium acetobutylicum (231).
T. vaginalis ferredoxin possesses a [2Fe-2S] cluster and an 8-amino-acid putative mitochondrial targeting sequence at the amino terminus (154), similar to that of G. intestinalis [2Fe-2S] ferredoxin. Recently, the mitosome-targeting signal of G. intestinalis [2Fe-2S] ferredoxin was shown to target ferredoxin to the hydrogenosome of T. vaginalis, suggesting that the mitosome and the hydrogenosome share a common mode of protein targeting similar to the protein import machinery of mitochondria (69). Similarly, the mitochondrial targeting signal from Trypanosoma cruzi was also shown to be interchangeable with that of E. histolytica (303, 316). While import of ferredoxin and IscU, a scaffolding protein for iron-sulfur biosynthesis, relies on the amino-terminal signal, the targeting of other mitosomal proteins (e.g., the catalytic component of iron-sulfur cluster biosynthesis IscS, Cpn60, and mitochondrial Hsp70) into the mitosome does not require this presequence in G. intestinalis (258). The underlying mechanisms of mitosome import of the latter proteins are not clear, but they likely possess cryptic internal signals, as demonstrated in the ADP-ATP carrier protein localized in the hydrogenosome of T. vaginalis (74) and other proteins found in the inner membrane of mitochondria (248). Mitosome import of Cpn60 relies on the presequence in E. histolytica (303), suggesting organism dependence in the targeting mechanisms. It also remains to be determined which ferredoxin(s) among the three types is involved in energy metabolism and redox regulation as well as the activation of metronidazole in E. histolytica and G. intestinalis. It is also unknown why different types of ferredoxins are retained throughout the evolution of these parasites.
Hydrogenase. Hydrogenases are enzymes responsible for producing hydrogen gas by transferring electrons to two protons. Hydrogenases are oxygen sensitive and are present in a wide range of anaerobic bacteria and hydrogenosomes from certain microaerophilic/anaerobic protozoan parasites, ciliates, fungi, and chytrids (86, 87, 136, 251). Two types of hydrogenases are known; heterodimeric NiFe-hydrogenase and monomeric Fe-hydrogenase. The heteromeric NiFe-hydrogenases consist of a large subunit containing a bimetalic NiFe cluster at the hydrogen-activating site and a small subunit containing up to three Fe-S clusters that transport electrons to the physiological acceptors. NiFe-hydrogenases are generally involved in H2 uptake in archaebacteria and eubacteria (46). In contrast, the mostly monomeric Fe-hydrogenases usually catalyze H2 evolution (98). In some bacteria, e.g., the hyperthermophilic bacterium Thermotoga martima and the anaerobic bacterium Desulfovibrio fructosovorans, Fe-hydrogenase is composed of two or three subunits sharing homology with monomeric Fe-hydrogenases (113, 318). T. vaginalis possesses at least three Fe-hydrogenase genes (42, 137). There are two types of Fe-hydrogenases, referred as "short-form" (50-kDa) and "long-form" (64-kDa) Fe-hydrogenases (86). These Fe-hydrogenases, which differ in size due to the heterogeneity at the amino-terminal end, have been well studied (42, 137). An overlapping region of 450 amino acids of the longer T. vaginalis form of Fe-hydrogenases showed 47% amino acid identity to short-form Fe-hydrogenases (137). While the 50-kDa Fe-hydrogenase contains 2[4Fe-4S] clusters and lacks the mitochondrion/hydrogenosome-targeting sequence, the 64-kDa Fe-hydrogenase possesses an amino-terminal extension containing additional [4Fe-4S] and [2Fe-2S] clusters and the mitochondrion-targeting signal (137), suggesting that long-form Fe-hydrogenase is fully active and accepts electrons from ferredoxin (86).
An Fe-hydrogenase gene was also identified in G. intestinalis and E. histolytica (137, 228), and its enzymatic activity and/or mRNA expression was demonstrated in cultured E. histolytica and G. intestinalis (228). However, a recent report suggests that Giardia produces hydrogen at a 10-fold-lower rate than T. vaginalis does (183). The physiological role of Fe-hydrogenase in E. histolytica and G. intestinalis has not been clearly demonstrated as in T. vaginalis, where hydrogenase plays an indispensable role in the final transfer of electrons to protons (218). Recently, auxiliary Fe-hydrogenase maturases (S-adenosylmethionine-dependent HydG, HydE, and the small GTPase HydF) were identified from the T. vaginalis hydrogenosomes by proteomic analysis and from the genome database (251), which further elucidates the molecular mechanisms of biosynthesis and maturation of Fe-hydrogenase in this parasite.
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Various nonimidazole drugs, including nitazoxanide, paromomycin, and niridazole, reportedly have the potential to be used for treatment of microaerophilic protozoan parasite infections (62, 73). Nitazoxanide, a nitrothiazoyl-salicylamide derivative, could be used as the first-line agent against amoebiasis and other intestinal parasitic diseases in the future (246). Nitazoxanide has broad-spectrum antiparasitic activity, including activity against the protozoans E. histolytica, G. intestinalis, T. vaginalis, Cryptosporidium parvum, and Isospora belli and the helminths Ascaris lumbricoides, Ancylostoma duodenale, Trichuris trichiura, Taenia saginata, Hymenolepsis nana, and Fasciola hepatica (267). The mode of action for nitazoxanide is unproven, but it is predicted to inhibit PFOR. Clinical efficacy was also demonstrated in a randomized, double-blind, placebo-controlled study (267).
The disadvantages of the current antiamoebic drugs, besides their relative ineffectiveness against luminal cysts, include various side effects. Adverse effects of metronidazole include anorexia, nausea, vomiting, diarrhea, abdominal discomfort, disulfiram-like alcohol intolerance, and hypersensitivity (62, 122). Neurological side effects include dizziness, vertigo, paresthesias, and, rarely, encephalopathy or convulsions which warrant discontinuation of the drug (246). Neutrocytopenia is also associated with metronidazole. Metronidazole is also known to be mutagenic in bacteria and carcinogenic in rodents, making teratogenicity a concern (28, 44, 47, 51, 265). Metronidazole is also known to cross the placental barrier. The Food and Drug Administration (FDA) classified metronidazole as a class B risk factor for pregnancy. Accordingly, use of metronidazole for amoebiasis in pregnant women is not currently recommended (58, 59), although a causal connection between metronidazole exposure during pregnancy and birth defects has not been established (45, 63, 94). Emetine and dehydroemetine hydrochloride have serious side effects, including nausea, vomiting, cardiotoxicity, local pain, and tenderness (162). These drugs are poorly excreted into the gut and urine and accumulate at high concentrations in the liver, heart, and other tissues. Side effects of nitazoxanide include diarrhea, nausea, vomiting, abdominal pain, and flatulence (246).
Albendazole, currently used as an anthelminthic, was first successfully used against giardiasis (340, 341) with other benzimidazoles (fenbendazole and mebendazole), with variable success. The first large-scale human study of albendazole, conducted in Bangladesh, showed a lower average efficacy than for metronidazole (120), suggesting that single high doses of metronidazole or tinidazole are advantageous with regard to patient compliance. A recent study demonstrated that combinations of albendazole and phenyl-carbamate derivatives were more effective against albendazole-resistant G. intestinalis strains (150). Nitazoxanide was also effective for treatment of giardiasis cases resistant to metronidazole and albendazole therapy (1). The side effects of benzimidazoles are similar to those of metronidazole (anorexia, vomiting, and metal taste) but of lower intensity (155).
Although metronidazole is still the drug of choice (153), drug-resistant cases of trichomoniasis are considerably more common than in infections with G. intestinalis or E. histolytica. The second drug of choice for treatment of trichomoniasis is tinidazole. A recent study using >100 clinical isolates demonstrated that they showed slightly higher sensitivity to tinidazole (minimum lethal concentration [MLC], 1.0 ± 1.3 mM) than to metronidazole (MLC, 2.6 ± 1.9 mM) under aerobic conditions. However, there was no significant difference in MLCs between these nitroimidazoles under anaerobic conditions (61). The efficacies of a number of nitroimidazole derivatives other than metronidazole and tinidazole for the treatment of T. vaginalis infection have been investigated. Although the modes of action of these derivatives are similar, the pharmacokinetics, tissue distributions, trichomonicidal activities, and toxicities are variable for these compounds (62). Ornidazole and secnidazole are similar to tinidazole in that they have longer half-lives but lower rates of efficacy to cure infection than metronidazole. In contrast, nimorazole, a nitrothiazole derivative, is converted into two major metabolites that possess significantly higher trichomonicidal activity than metronidazole (177). Nimorazole was active against both metronidazole-sensitive and -resistant T. vaginalis strains (334, 335).
Intravaginal application of paromomycin was successfully used to treat recurrent trichomoniasis. However, severe side effects, including pain and mucosal ulceration, made it an unlikely candidate for clinical therapy (249). Nitazoxanide was as active against T. vaginalis as metronidazole (6, 48), similar to the case for infections caused by E. histolytica and G. intestinalis as described above and for C. parvum and Blastocystis (6, 100). Hamycin, an aromatic polyene related to amphotericin B, was shown to induce cell death in T. vaginalis and other eukaryotes by binding to ergosterols in the plasma membrane and forming pores. Although lower concentrations of hamycin effectively killed both metronizole-sensitive and -resistant T. vaginalis strains, toxicity both in vitro and in vivo is likely to make hamycin inapplicable for clinical use (190). Sodium nitrite, sodium nitroprusside, and Roussinn's black salt, traditionally used to prevent food contamination, exhibited trichomonicidal activity against both sensitive and resistant T. vaginalis strains (268). Sulfimidazole, which has two functional groups, sulfonamide and 5-nitroimidazole, exhibited activity comparable to that of metronidazole against metronidazole-sensitive strains (MLC, 10 µg/ml) and was more effective against the metronidazole-resistant strains (MLC, 40 to 60 µg/ml) (73, 196).
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-hydroxyethyl-2-methyl-5-nitroimidazole), a synthetic 5-nitroimidazole (56), enters the cell and its organelles via passive diffusion. Metronidazole is relatively inert until its 5-nitro group is reduced within the cell or organelle by an appropriate electron donor such as ferredoxin. Importantly, metronidazole activation occurs only under strong reducing conditions. As oxygen is an efficient electron acceptor, increased levels of oxygen result in the impaired reduction and activation of metronidazole which could theoretically result in metronidazole resistance. Thus, sensitivity to metronidazole is influenced by oxygen tension. Metronidazole is usually activated through the acceptance of electrons from ferredoxin or flavodoxin that is reduced by PFOR (178, 218-220, 264, 308). Among microorganisms, there is a strong correlation between the presence of PFOR activity and metronidazole sensitivity (138, 218, 219, 266, 275, 308). In humans, where PFOR is absent, pyruvate dehydrogenase catalyzes the oxidative decarboxylation to form acetyl-CoA and NADH in mitochondria (see above). It was directly demonstrated by using purified PFOR and ferredoxin from G. intestinalis trophozoites that these Fe-S cluster-containing proteins activate metronidazole in vitro (306-308, 314). The reduction of metronidazole results in the nitro-radical form, which binds transiently to DNA to disrupt or break the nucleotide strands, leading to cell death (65, 76, 77, 182). Studies with Escherichia coli showed that the damage caused by the direct binding of activated metronidazole to DNA is repaired by the excision repair pathway, which was not activated in T. vaginalis trophozoites exposed to metronidazole (172). DNA damage may be the basis for the carcinogenicity of metronidazole in animals, although carcinogenicity of metronidazole in humans has not been demonstrated. The cellular compartments where metronidazole is activated differ among the "amitochondriate" protists. It was proposed that metronidazole is activated in E. histolytica by ferredoxin in the cytosol (256). As described above, ferredoxin nitroreductase, a fusion protein of 2[4Fe-4S] ferredoxin and nitroreductase, may be a target of metronidazole in E. histolytica and G. intestinalis (229). G. intestinalis also has, in addition to ferredoxin-nitroreductases, an oxygen-insensitive nitroreductase that lacks the ferredoxin domain and which is also present in archaea and bacteria (229). This nitroreductase reduces and activates the nitro groups of metronidazole and furazolidone in Helicobacter pylori and E. coli, respectively. Bacterial mutants lacking this nitroreductase activity were resistant to a corresponding drug (115, 163, 327). Although a physiological electron acceptor from reduced ferredoxin in E. histolytica has not been unequivocally demonstrated, oxygen competes with metronidazole for electrons from the physiological electron donor. Therefore, it is conceivable that the metronidazole sensitivity of E. histolytica is influenced by oxygen tension, as demonstrated for Trichomonas (see below). In G. intestinalis, the terminal oxidase (NADH oxidase), which converts oxygen directly to water to scavenge for oxygen and protect the anaerobic PFOR and ferredoxins (37), seems to be a physiological acceptor of electrons from ferredoxin in the cytosol. In contrast, in T. vaginalis, metronidazole is metabolized and activated in the hydrogenosomes. Metronidazole competes with the terminal enzyme hydrogenase for electrons from ferredoxin (218).
The underlying mode of action of benzimidazoles, including albendazole, fenbendazole, and mebendazole, has been extensively studied in the parasitic nematode Haemonchus contortus (171, 187, 188, 226). Benzimidine binds to the ß-tubulin monomer prior to dimerization with -tubulin to block microtubule formation (171). More specifically, benzimidazoles bind to the high-affinity binding site on the ß-tubulin monomer (187). The effect of benzimidazoles on in vitro assembly of microtubules was investigated in benzimidazole binding assays using recombinant - or ß-tubulin and dimeric tubulin (ß-tubulin) from G. intestinalis, Encephalitozoon intestinalis, and C. parvum (192).
An emetine-resistant E. histolytica strain was developed in vitro (21, 276, 277). This emetine-resistant E. histolytica line overexpressed P-glycoprotein and showed some features of the multidrug-resistant phenotype (21). The accumulation of emetine by the mutant amoebae was 50% lower than in wild-type amoebae (21). However, the rate of drug entry and efflux from the parasite per se was not examined in that study. Emetine resistance was reversed by the calcium channel blocker verapamil (21, 276). It was hypothesized that E. histolytica actively expelled hydrophobic drugs, including emetine, by P-glycoprotein as described for multidrug-resistant tumor cells (97), because the emetine-resistant E. histolytica line was cross-resistant to other hydrophobic drugs such as iodoquinol and diloxanide but not to nonpolar drugs such as chloroquine and metronidazole (277). Also, the resistant strain released radiolabeled emetine more rapidly than the susceptible strain, suggesting that P-glycoprotein overexpression was responsible for emetine resistance in E. histolytica (277).
PFOR expression was down-regulated fivefold in a metronidazole-resistant G. intestinalis line (308), which is consistent with the premise that PFOR is the primary target of metronidazole and that a decreased amount of the target is the mode of resistance in G. intestinalis. Recently, antisense inhibition of PFOR caused metronidazole resistance in G. intestinalis (64). In contrast to the case for metronidazole-resistant lines, PFOR did not change in a furazolidone- and quinacrine-cross-resistant G. intestinalis line (314). The activity of the next electron acceptor in the transport chain, ferredoxin I, decreased by sevenfold, while the amount of ferredoxin I decreased by only twofold (181, 314), suggesting that another layer of regulation influenced the acceptor activity of ferredoxin. In an independent study, a furazolidone-resistant line showed increased activity of NADH oxidase, which activates the drug to its free radical state (35, 37). That study concluded that furazolidone resistance is due to the reduced expression of ferredoxin and increased NADH oxidase activity, which differs from metronidazole resistance. In contrast, albendazole-resistant G. intestinalis strains revealed changes in the cytoskeleton, especially for ß-tubulin (309), suggesting that qualitative changes in ß-tubulin lead to decreased sensitivity to albendazole (191). In that study, benzimidazole analogues showed higher affinity for monomeric ß- and heterodimeric ß-tubulin derived from benzimidazole-sensitive parasites than for those from benzimidazole-insensitive organisms (191).
Because oxygen is an efficient electron acceptor, increased levels of cellular oxygen in hydrogenosomes result in the impaired reduction and activation of metronidazole. Oxygen concentrations in resistant strains were much higher than those in susceptible strains (82). The high oxygen concentrations likely inhibit accumulation of the drug, because oxygen competes with metronidazole for electrons from reduced ferredoxin. If metronidazole is not reduced, there is no concentration gradient of the drug across the plasma membrane to allow extracellular metronidazole to enter the cell. In addition, the reduced nitro free radical is oxidized back to the original compound by oxygen and in turn produces a superoxide anion (62, 245). This process is known as futile cycling and results in only limited damage to the cells via superoxide anions in comparison to cell death due to reactive nitro radicals. While "aerobic resistance" phenotypes were frequently observed in clinical isolates, anaerobic resistance is found mostly in in vitro-induced metronidazole-resistant lines. Metronidazole-resistant T. vaginalis strains artificially developed by increasing the drug concentration in vitro showed either reduced or absent PFOR and hydrogenase activities (169). Unfortunately, the current view of metabolic mechanisms giving rise to metronidazole-resistant trichomoniasis, both in clinical settings and in vitro, is not completely elucidated by the "aerobic" versus "anaerobic" models.
Clinical drug-resistant T. vaginalis isolates show various biochemical changes, e.g., decreased expression of PFOR, ferredoxin, and hydrogenase activities (62, 73, 168, 253); oxygen resistance (80, 336); and decreased oxidase activity (336). In addition, highly resistant T. vaginalis clinical isolates possess neither detectable PFOR activity nor PFOR and ferredoxin mRNAs (34, 73, 169, 175, 253). Earlier work suggested that hydrogenosomes could be lost in drug-resistant parasites. However, it was reported later that the organelle remained, but in a modified form (168). Although structural changes of hydrogenosomes were reported to occur in a metronidazole-resistant T. vaginalis strain, it is unclear whether these are primary events or are secondary to the advent of resistance (153). Also, metronidazole-resistant strains simultaneously lost multiple hydrogenosomal proteins, including ferredoxin, PFOR, malic enzyme, and hydrogenase, due to inactivation of hydrogenosomes (140, 254). The metronidazole-resistant T. vaginalis trophozoites showed enhanced lactate fermentation as they lost PFOR activity, PFOR mRNA, and ferredoxin mRNA and thus the pyruvate-oxidizing pathway in the hydrogenosomes (34, 73). Hrdy et al. (140) demonstrated that metronidazole is activated by electrons from ferredoxin that originate not from PFOR but from malate in this metronidazole-resistant strain. These data support the notion that trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from hydrogenosomes. These lines of evidence also suggested that "aerobic" and "anaerobic" resistance mechanisms are not mutually exclusive.
One previous study provided contradictory evidence that there was no significant change in PFOR activity, anaerobic fermentation, and intracellular accumulation of metronidazole between metronidazole-resistant and -susceptible isolates (220). However, accumulation of [14C]metronidazole was more inhibited under aerobic conditions in resistant isolates than in susceptible strains. Thus, they concluded that the production of electrons was not hampered but that the activation of metronidazole was reduced in the resistant isolates (220). This observation was also consistent with the premise that the metronidazole-resistant strain had reduced electron transport ability, and this therefore was classified as "aerobic resistance."
One line of evidence suggests that ferredoxin plays a major role in metronidazole resistance. The major ferredoxin purified from T. vaginalis trophozoites was indeed reduced by PFOR in vitro, as detected by electron paramagnetic resonance spectroscopy (116). This ferredoxin could interact with metronidazole and accept electrons from PFOR (116). Changes in the upstream transcriptional regulatory regions (nucleotide –239 upstream of the transcription initiation site) of the ferredoxin gene were also demonstrated in metronidazole-resistant strains (253). The mRNA and protein levels of ferredoxin decreased by 50% in these strains. A recent study showed that ferredoxin gene replacement in T. vaginalis did not lead to in vitro metronidazole resistance (176). Thus, it remains unclear to what extent ferredoxins are involved in metronidazole resistance. The T. vaginalis genome project indicates that the genome is 160 to 180 Mb in size and highly repetitive. These data indicate that knockout of a single gene may not be deleterious for the parasite and that an alternative ferredoxin or flavodoxin might also be responsible for metronidazole activation in T. vaginalis.
Cross-resistance between different nitroimidazoles was also reported (208, 225). For instance, metronidazole-resistant clinical isolates showed partial cross-resistance to tinidazole (61, 319). However, metronidazole-resistant isolates do not always coexist with cross-resistance against other nitroimidazole compounds (61). Metronidazole-resistant isolates described by Narcisi and Secor were sensitive to a nonnitroimidazole nitrofuran, furazolidone (225), which is consistent with the notion that the mechanism of metronidazole resistance in T. vaginalis differs from that in G. intestinalis (314). Further studies need to be conducted to clarify molecular mechanisms of cross-resistance.
| SULFUR-CONTAINING-AMINO-ACID METABOLISM AS A NOVEL DRUG TARGET |
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