Leishmania amazonensis: metabolic adaptations induced by resistance to an ABC transporter blocker

We compared growth rate, cell glucose turnover and expression of ATP-binding-cassette (ABC) transporters in Leishmania amazonensis (LTB0016; LTB) versus LTB(160) selected for resistance against the ABC transporter blocker glibenclamide. Additionally, we evaluated the influence of drug-resistance on Leishmania sensitivity against 2-mercaptoacetate and 2-deoxyglucose. Our data demonstrate that (1) LTB(160) and LTB constitutively express ABC transporters for neutral substrates, (2) glibenclamide resistance induces the expression of organic anion ABC transporters, members of the drug resistance associated transporters subfamily, (3) LTB(160) parasites use less glucose as energy substrate and exhibit a slower glucose uptake than LTB cells, and (4) LTB(160) parasites are less sensitive to 2-mercaptoacetate and 2-deoxyglucose than the glibenclamide-sensitive Leishmania LTB. Together these and previous results indicate that the metabolic adaptations expressed in drug-resistant LTB(160) differ from those described for mammalian drug resistant cells and constitute general mechanisms that underlie drug resistance in Leishmania and may be helpful for identifying alternative strategies to circumvent drug resistance in leishmaniasis.     

ABC transporters in the protozoan parasite Leishmania

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.     

Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.     

The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase

The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to glutathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein were fused at the COOH terminus of PGPA. Immunofluorescence, confocal, and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PGPAHA transfectants was performed to further characterize this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmol/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transports thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.     

Incidence of symptomatic and asymptomatic Leishmania donovani infections in high-endemic foci in India and Nepal: a prospective study

Incidence of Leishmania donovani infection and Visceral Leishmaniasis (VL) was assessed in a prospective study in Indian and Nepalese high-endemic villages. DAT-seroconversion was used as marker of incident infection in 3 yearly surveys. The study population was followed up to month 30 to identify incident clinical cases. In a cohort of 9034 DAT-negative individuals with neither active signs nor history of VL at baseline, 42 VL cases and 375 asymptomatic seroconversions were recorded in the first year, giving an infection:disease ratio of 8.9 to 1. In the 18 months' follow-up, 7 extra cases of VL were observed in the seroconverters group (N=375), against 14 VL cases among the individuals who had not seroconverted in the first year (N=8570) (RR=11.5(4.5相似文献   

Glibenclamide modulates glucantime activity and disposition in Leishmania major

A source of chemotherapeutic failure in anti-infective therapies is the active movement of drugs across membranes, through ATP-binding cassette (ABC) transporters. In fact, simultaneous administration of therapeutic drugs with ABC transporter blockers has been invoked to be the way to actively prevent the emergence of drug resistance. Herein, we demonstrate that glucantime’s efficacy in decreasing the infection rate of Leishmania-infected macrophages is strongly enhanced when used in combination with glibenclamide, a specific blocker of ABC transporters. Intracellular ABC transporters mediate glucantime sequestration in intracellular organelles. Their selective inhibition may effectively increase the cytoplasmic concentration of glucantime and its leishmanicidal activity. Our results reveal for the first time that glibenclamide targets in Leishmania major a compartment associated with a multivesicular system that is simultaneously labeled by the acidic marker LysoTracker-red and may represent the organelle where antimonials are sequestered. These results constitute a proof of concept that conclusively demonstrates the potential value that combination therapy with an ABC transporter blocker may have for leishmaniasis therapy.     

Efficacy of human beta-casein fragment (54-59) and its synthetic analogue compound 89/215 against Leishmania donovani in hamsters

The characteristic feature of visceral leishmaniasis (VL) is the profound impairment of immune system of the infected host, which contributes significantly to the partial success of antileishmanial chemotherapy. Since in VL, cure is the combinatorial effect of drug and immune status of the host, the rationale approach towards antileishmanial chemotherapy would be to potentiate the immune functioning of the host to extract desired results. Towards this direction several rationally designed analogues of human beta-casein fragment (54-59) were evaluated for their ability to stimulate the non-specific resistance in hamsters against Leishmania donovani infection. By virtue of being derived from the food protein casein derivatives may be devoid of unwanted side effects associated with the substances of microbial origin, e.g. muramyl dipeptide (MDP). Out of this one peptide Val-Glu-Gly-Ile-Pro-Tyr (compound 89/215) had been reported to have such activity. In this communication, the prophylactic and therapeutic efficacy of the peptide along with its natural sequence has been evaluated in detail against experimental VL in hamsters. Their use as an adjunct to chemotherapy was also explored. Human beta-casein fragment, compound 89/215 and MDP were tested in vivo at various dose levels wherein compound 89/215 showed superiority over MDP at 3 mg/kg x 2 given intraperitoneally (i.p.). Compound 89/215 sensitized peritoneal macrophages acquired considerable resistance and only 24% of the cells were found infected in comparison to control peritoneal macrophages where 76.4% of the cells were found infected. Similarly, the efficacy of sodium antimony gluconate (SAG) in hamsters pretreated with compound 89/215 enhanced significantly (P < 0.001). This peptide also exhibited considerably good therapeutic efficacy when evaluated either alone or in combination with SAG in established infection of L. donovani.     

A gp63 based vaccine candidate against Visceral Leishmaniasis

Visceral leishmaniasis is a macrophage associated disorder which leads to a profound decrease in the natural immunotherapeutic potential of the infected subjects to combat the disease. The major surface glycoprotein gp63 has been found to be a significant vaccine candidate against visceral leishmaniasis. The current study addresses the levels of similarity and identity in the gp63 obtained from different species of Leishmania viz donovoni, chagasi and infantum linked to the cause of visceral leishmaniasis. The results from BLAST, Phylogram and Cladogram studies indicate significant identity, similarity and conservation of important residues in the protein which lead us to conclude that a common gp63 based vaccine can be used as a therapeutical tool against visceral leishmaniasis caused by different species strains of leishmania.     

Canine visceral leishmaniasis in Rio de Janeiro, Brazil. Clinical, parasitological, therapeutical and epidemiological findings (1977-1983)

Forty dogs from the periphery of the city of Rio de Janeiro were studied. All dogs where diagnosed as positive for leishmaniasis either parasitologically and/or serologically. Among them, 19 came from areas where only Visceral Leishmaniasis (VL) occurs (Realengo, Bangu, Senador Camará). Clinical signs of the disease were seen in 36.8% of the cases, including emaciation - 100%, lymphadenopathy and depilation - 85.7%. The other 21 dogs came from an area (Campo Grande) where both diseases (VL, and American Cutaneous Leishmaniasis - ACL) occur. Clinical signs of the disease, mainly cutaneous or mucocutaneous ulcers were seen in 76.2% of the cases. Leishmania parasites were found in 39 cases: 22% in viscera, 42.5% in viscera and normal skin and 35% in cutaneous or mucocutaneous ulcers. All the Leishmania stocks isolated from dogs which came from Realengo, Bangu, Senador Camará (VL area), and from Campo Grande (VL + ACL area) were characterized as L. donovani (except in one case) according to their schizodeme, zymodeme and serodeme. The only stock characterized as L. b. braziliensis, was isolated from the lymph node of a dog from Campo Grande with visceral disease and without skin lesions. Antimony therapy attempted in eight Leishmania donovani positive dogs was unsuccessful.     

Case Report: No Response to Liposomal Daunorubicin in a Patient with Drug-Resistant HIV-Associated Visceral Leishmaniasis

Visceral leishmaniasis (VL) in patients with HIV co-infection presents a significant therapeutic challenge due to the lessened chance of achieving long-term cure. We report a case of VL in a 60-year-old man with HIV infection who became refractory to anti-leishmania treatment due to multi-drug resistance. In the face of a worsening clinical situation, and with no other options available, he was treated with an experimental regimen of liposomal daunorubicin, which has previously been shown to have in vitro activity against Leishmania donovani and to be effective treatment of VL in animal studies. To our knowledge, he was the first patient with VL and HIV co-infection to have this treatment evaluated. We report on the lack of response to this treatment and possible causes for its failure.     

The diversity of ABC transporter genes across the Phylum Nematoda

It is estimated that one billion people globally are infected by parasitic nematodes, with children, pregnant women, and the elderly particularly susceptible to morbidity from infection. Control methods are limited to de-worming, which is hampered by rapid re-infection and the inevitable development of anthelmintic resistance. One family of proteins that has been implicated in nematode anthelmintic resistance are the ATP binding cassette (ABC) transporters. ABC transporters are characterized by a highly conserved ATP-binding domain and variable transmembrane regions. A growing number of studies have associated ABC transporters in anthelmintic resistance through a protective mechanism of drug efflux. Genetic deletion of P glycoprotein type ABC transporters in Caenorhabditis elegans demonstrated increased sensitivity to anthelmintics, while in the livestock parasite, Haemonchus contortus, anthelmintic use has been shown to increase the expression of ATP transporter genes. These studies as well as others, provide evidence for a potential role of ABC transporters in drug resistance in nematodes. In order to understand more about the family of ABC transporters, we used hidden Markov models to predict ABC transporter proteins from 108 species across the phylum Nematoda and use these data to analyze patterns of diversification and loss in diverse nematode species. We also examined temporal patterns of expression for the ABC transporter family within the filarial nematode Brugia malayi and identify cases of differential expression across diverse life-cycle stages. Taken together, our data provide a comprehensive overview of ABC transporters in diverse nematode species and identify examples of gene loss and diversification in nematodes based on lifestyle and taxonomy.     

Transport proteins of the ABC systems superfamily and their role in drug action and resistance in nematodes

The completion of a number of nematode genomes has provided significant information on ABC systems in these organisms. Nematodes have more ABC systems genes and greater diversity than do mammalian species. Class 1 and class 2 ABC systems, more commonly known as ABC transporters, are present. As in other organisms, nematode ABC systems are characterized by a highly conserved ATP-binding domain (ABC_2) and a less conserved transmembrane domain (ABC_TM1/TM1F). Studies of drug resistance in nematodes have suggested that ABC transporters are part of the resistance mechanism. Evidence in support of this has been obtained from genetic studies where an association between anthelmintic selection and ABC transporters was shown by comparisons between unselected and drug selected, or resistant, populations of parasitic nematodes. In drug resistant populations, genetic polymorphism and diversity, genotype patterns, and linkage disequilibrium were disrupted. Multidrug resistance (MDR) reversing agents that inhibit ABC function improve efficacy in sensitive nematode populations and restore sensitivity in resistant populations. Similar to the situation in clinical oncology, overexpression of ABC systems occurs in drug resistant and sensitive populations following drug exposure, particularly those in the P-glycoprotein (PGP) subfamily. Deletion or disruption of ABC genes, particularly PGP and the multidrug resistance associated protein (MRP), increases sensitivity to some drugs, particularly ivermectin. These studies provide evidence that ABC transporters play a role in drug action and resistance in nematodes.     

A survey for American cutaneous and visceral leishmaniasis among 1,342 dogs from areas in Rio de Janeiro (Brazil) where the human diseases occur

There are areas in the periphery of Rio de Janeiro city where human cases of Visceral and/or Cutaneous Leishmaniasis occur. The parasites have been identified as Leishmania donovani and Leishmania braziliensis braziliensis respectively. A survey for Leishmaniasis was done among 1,342 dogs from those areas using an indirect immunofluorescent test. From the dogs, 616 came from areas where only human cases of Visceral Leishmaniasis occurred, 373 from an area where all human cases were of Cutaneous Leishmaniasis and 353 from a third area (Campo Grande) where both visceral and cutaneous human cases were detected. The prevalence of parasite antibody titers among dogs from areas of Cutaneous Leishmaniasis was significantly higher than that of Visceral Leishmaniasis (8.6% vs. 4.3%, p less than 0.02). The highest prevalence was observed among dogs from the area where both diseases are present (12.7%).     

A novel mechanism for an old drug: amphotericin B in the treatment of visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by various species of the genus Leishmania. Internalization of Leishmania into host cells is facilitated by a large number of receptors, and therefore no panacea is available for the treatment of leishmaniasis. We previously demonstrated the requirement of host membrane cholesterol in the entry of Leishmania into macrophages by cholesterol depletion using methyl-β-cyclodextrin (MβCD). We recently showed that leishmanial infection is inhibited upon sequestration of host membrane cholesterol using amphotericin B (AmB), considered as the best existing drug against VL. The reason for the antileishmanial activity of AmB is generally believed to be its ability to bind ergosterol in parasite membranes. Our recent results offer the opportunity to reexamine the mechanism behind the effectiveness of current AmB-based therapeutic strategies to treat leishmaniasis. We propose here a novel mechanism in which the effectiveness of AmB treatment could be partly based on its ability to sequester cholesterol in the host membrane, thereby abrogating macrophage-parasite interaction.     

Visceral leishmaniasis mimicking autoimmune hepatitis, primary biliary cirrhosis, and systemic lupus erythematosus overlap

Visceral leishmaniasis (VL) is a life-threatening infection caused by Leishmania species. In addition to typical clinical findings as fever, hepatosplenomegaly, and cachexia, VL is associated with autoimmune phenomena. To date, VL mimicking or exacerbating various autoimmune diseases have been described, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and autoimmune hepatitis (AIH). Herein, we presented a patient with VL who had overlapping clinical features with SLE, AIH, as well as antimitochondrial antibody (AMA-M2) positive primary biliary cirrhosis.     

Identification of antimony resistance markers in Leishmania tropica field isolates through a cDNA-AFLP approach

Pentavalent antimonial compounds have been the first line therapy for leishmaniasis; unfortunately the rate of treatment failure of anthroponotic cutaneous leishmaniasis (ACL) is increasing due to emerging of drug resistance. Elucidation of the molecular mechanisms operating in antimony resistance is critical for development of new strategies for treatment. Here, we used a cDNA-AFLP approach to identify gene(s) which are differentially expressed in resistant and sensitive Leishmania tropica field isolates. We identified five genes, aquaglyceroporin (AQP1) acts in drug uptake, ATP-binding cassette (ABC) transporter (MRPA) involved in sequestration of drug, phosphoglycerate kinase (PGK) implicated in glycolysis metabolism, mitogen activated protein kinase (MAPK) and protein tyrosine phosphatase (PTP) responsible for phosphorylation pathway. The results were confirmed using real time RT-PCR which revealed an upregulation of MRPA, PTP and PGK genes and downregulation of AQP1 and MAPK genes in resistant isolate. To our knowledge, this is the first report of identification of PTP and PGK genes potentially implicated in resistance to antimonials. Our findings support the idea that distinct biomolecules might be involved in antimony resistance in L. tropica field isolates.     

Risk Factors for Visceral Leishmaniasis and Asymptomatic Leishmania donovani Infection in India and Nepal

There is increasing interest in the role of asymptomatic infection in transmission of Visceral Leishmaniasis (VL). We studied the individual, household and environmental factors associated with asymptomatic Leishmania donovani infected individuals and VL. 7,538 individuals living in VL endemic villages in India and Nepal were divided into three mutually exclusive groups based on their VL history and Direct Agglutination Test (DAT) results in yearly serosurveys over a two-year period. The groups were (1) VL cases, (2) asymptomatically infected individuals (seroconverters) and (3) seronegative individuals. VL cases and seroconverters were compared to seronegative individuals in mixed logistic regression models. The risk of seroconversion and disease was significantly increased in individuals aged 14 to 24 years old and by the presence of other DAT-positive, asymptomatically infected individuals and VL cases in the house. The risk of seroconversion was higher in Indian than in Nepalese villages and it increased significantly with age, but not so for VL. This study demonstrates that, when risk factors for leishmanial infection and VL disease are evaluated in the same population, epidemiological determinants for asymptomatic infection and VL are largely similar.     

Evolution of ABC transporters by gene duplication and their role in human disease

The ATP-binding cassette (ABC) transporter genes represent the largest family of transporters and these genes are abundant in the genome of all vertebrates. Through analysis of the genome sequence databases we have characterized the full complement of ABC genes from several mammals and other vertebrates. Multiple gene duplication and deletion events were identified in ABC genes in different lineages indicating that the process of gene evolution is still ongoing. Gene duplication resulting in either gene birth or gene death plays a major role in the evolution of the vertebrate ABC genes. The understanding of this mechanism is important in the context of human health because these ABC genes are associated with human disease, involving nearly all organ systems of the body. In addition, ABC genes play an important role in the development of drug resistance in cancer cells. Future genetic, functional, and evolutionary studies of ABC transporters will provide important insight into human and animal biology.     

A novel ATP-binding cassette transporter from Leishmania is involved in transport of phosphatidylcholine analogues and resistance to alkyl-phospholipids

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present work reports the characterization of the first ABC subfamily G (ABCG)-like transporter, LiABCG4, in the protozoan parasite Leishmania. LiABCG4 localized mainly to the parasite plasma membrane. Overexpression of this half-transporter reduced the accumulation of phosphatidylcholine analogues and conferred resistance to alkyl-phospholipids. Likewise, when expressed in Saccharomyces cerevisiae, the protein localized to the yeast plasma membrane and conferred resistance to alkyl-phospholipids. Post-Golgi secretory vesicles isolated from a LiABCG4-overexpressing yeast mutant contained the leishmanial ABC transporter and exhibited ATP-dependent, vanadate-sensitive transport of phosphatidylcholine analogues from the cytosolic to the lumenal leaflet of the vesicle membrane. Cross-linking showed dimerization of LiABCG4. These results suggest that LiABCG4 is involved in the active transport of phosphatidylcholine and resistance to alkyl-phospholipids in Leishmania.     

The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.