ABSTRACT
Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.
Subject(s)
Drug Resistance , Leishmania donovani , Leishmaniasis, Visceral , Sterol 14-Demethylase , Leishmania donovani/enzymology , Sterol 14-Demethylase/metabolism , Sterol 14-Demethylase/genetics , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/drug therapy , Amphotericin B/pharmacology , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , NADPH-Ferrihemoprotein Reductase/genetics , Antiprotozoal Agents/pharmacology , Humans , Ergosterol/metabolismABSTRACT
OBJECTIVE: We contribute to the understanding of the transmission dynamics of Leishmania infantum suggesting the involvement of rabbits as wild reservoirs. RESULTS: The prevalence of infection was 86.0% (270/314 wild rabbits) ranging from 18.2% to 100% in natural geographical regions. The estimated average parasite load was 324.8 [CI 95% 95.3-554.3] parasites per mg of ear lobe ranging from 0 to 91,597 parasites/mg per tissue section. CONCLUSIONS: A positive correlation was found between skin parasite load in wild rabbits and human incidence with evidence of the presence of the same L. infantum genotypes in rabbits and humans, providing new epidemiological and biological basis for the consideration of wild rabbits as a relevant L. infantum wild reservoir. Molecular parasite surveillance reflects the great genotypic variability of the parasite population in wild rabbits. Most of these genotypes have also been found to infect humans, dogs and sandflies in the region. Our findings also highlight that direct genotyping of the parasite in host tissues should be used for molecular surveillance of the parasite instead of cultured isolates.
Subject(s)
Disease Reservoirs , Leishmania infantum , Leishmaniasis, Visceral , Animals , Leishmania infantum/genetics , Leishmania infantum/isolation & purification , Rabbits/parasitology , Spain/epidemiology , Disease Reservoirs/veterinary , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/transmission , Leishmaniasis, Visceral/parasitology , Humans , Animals, Wild/parasitology , Prevalence , GenotypeABSTRACT
Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.
Subject(s)
Antiprotozoal Agents , Leishmania major , Leishmania major/enzymology , Leishmania major/drug effects , Leishmania major/genetics , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/chemistry , Protozoan Proteins/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Protozoan Proteins/chemistry , Animals , Humans , Leishmania donovani/enzymology , Leishmania donovani/drug effects , Leishmania donovani/geneticsABSTRACT
While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.
Subject(s)
Cytostatic Agents , Trypanocidal Agents , Trypanosoma brucei brucei , Trypanosomiasis, African , Animals , Humans , Trypanosomiasis, African/drug therapy , Trypanocidal Agents/therapeutic use , Trypanocidal Agents/pharmacokinetics , Cytostatic Agents/therapeutic use , Blood-Brain BarrierABSTRACT
Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014-2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spread of L. infantum in Morocco by determining the density, abundance and vector role of the species of the P. perniciosus complex identified correctly.
Subject(s)
Leishmania infantum , Phlebotomus , Psychodidae , Female , Animals , Phlebotomus/parasitology , Leishmania infantum/genetics , Morocco/epidemiology , Phylogeny , Psychodidae/parasitologyABSTRACT
Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.
Subject(s)
Antimalarials , Malaria, Falciparum , Humans , Plasmodium falciparum/metabolism , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Antimalarials/pharmacology , Antimalarials/therapeutic use , Mutation , Ligases/metabolismABSTRACT
Canine leishmaniosis is a challenge in veterinary medicine and no drug to date has achieved parasite clearance in dogs. Histone deacetylase inhibitors are a drug class widely used in cancer chemotherapy. We have successfully used O-alkyl hydroxamates (vorinostat derivatives) in the treatment of a laboratory model of visceral leishmaniasis without showing toxicity. In order to test the effectiveness of a particular compound, MTC-305, a parallel-group, randomized, single-centre, exploratory study was designed in naturally infected dogs. In this clinical trial, 18 dogs were allocated into 3 groups and were treated with either meglumine antimoniate (104 mg SbV/kg), MTC-305 (3.75 mg/kg) or a combination of both using a lower MTC-305 dose (1.5 mg/kg) through a subcutaneous route for 2 treatment courses of 30 days, separated by a 30-day rest period. After treatment, a follow-up time of 4 months was established. Parasite burden in bone marrow, lymph node and peripheral blood were quantified through qPCR. Antibody titres were determined through an immunofluorescence antibody test, and cytokine expression values were calculated through RT-qPCR. Treatment safety was evaluated through the assessment of haematological and biochemical parameters in blood, weight, and gastrointestinal alterations. Assessment was carried out before, between and after treatment series. Treatment with MTC-305 was effective at reducing parasite burdens and improving the animals' clinical picture. Dogs treated with this compound did not present significant toxicity signs. These results were superior to those obtained using the reference drug, meglumine antimoniate, in monotherapy. These results would support a broader clinical trial, optimised dosage, and an expanded follow-up stage to confirm the efficacy of this drug.
ABSTRACT
Transmission of leishmaniasis in endemic areas is characterized by microfocality related to the presence of the vector. Most entomological studies in southwestern Europe have focused on sylvatic areas and town outskirts, very few have sampled town or urban centres, and no survey has investigated inside households. The aim of this study was to determine the sand fly species diversity and vector density in the surroundings of human leishmaniasis cases compared with environments in which there was no association. Sand flies were captured in 26 households associated with recently treated leishmaniasis patients, 15 neighbouring houses without associated cases, and in others environments. Overall 7495 sand flies belonging to six species were captured. The highest sand fly density was found in farmhouses where there is a great availability of blood sources and breeding sites. In the environment of human leishmaniasis cases, Sergentomyia minuta was the most prevalent species followed by Phlebotomus perniciosus. Nevertheless, lower Leishmania infantum infection rates and lower intensity of infection were detected in S. minuta sand flies than in P. perniciosus. The density of P. perniciosus in households with recently treated leishmaniasis patients varies between 0 and 108 sand flies per light trap/night, with the maximum values corresponding to farmhouses. This species appears to be adapted to both indoors and outdoors domestic biotopes, including urban households.
Subject(s)
Leishmania infantum , Leishmaniasis , Phlebotomus , Psychodidae , Humans , Animals , Spain/epidemiology , Leishmaniasis/epidemiology , Leishmaniasis/veterinaryABSTRACT
Dog are the main reservoir of Leishmania infantum, causing canine leishmaniasis, an incurable multisystemic disease that leads to death in symptomatic dogs, when not treated. This parasite causes visceral, cutaneous, and mucosal leishmaniasis in people in the Mediterranean Basin, North Africa, South America, and West Asia. This disease is mostly unknown by veterinarians outside the endemic areas, but the disease is expanding in the Northern Hemisphere due to travel and climate change. New methodologies to study the epidemiology of the disease have found new hosts of leishmaniasis and drawn a completely new picture of the parasite biological cycle. Canine leishmaniasis diagnosis has evolved over the years through the analysis of new samples using novel molecular techniques. Given the neglected nature of leishmaniasis, progress in drug discovery is slow, and the few drugs that reach clinical stages in humans are unlikely to be commercialised for dogs, but several approaches have been developed to support chemotherapy. New-generation vaccines developed during the last decade are now widely used, along with novel prevention strategies. The implications of the epidemiology, diagnosis, treatment, and prevention of canine leishmaniasis are fundamental to public health.
ABSTRACT
There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.
Subject(s)
Antimalarials , Lysine-tRNA Ligase , Malaria , Antimalarials/chemistry , Antimalarials/pharmacology , Drug Discovery , Humans , Lysine-tRNA Ligase/chemistry , Lysine-tRNA Ligase/genetics , Lysine-tRNA Ligase/metabolism , Plasmodium falciparum/metabolismABSTRACT
African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.
Subject(s)
Trypanosoma congolense , Trypanosomiasis, African , Animals , Cattle , Cyclin-Dependent Kinases , Drug Repositioning , Trypanosoma vivax , Trypanosomiasis, African/drug therapy , Trypanosomiasis, African/parasitology , Trypanosomiasis, African/veterinaryABSTRACT
Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between ß4 and ß5 subunits that catalyze chymotrypsin-like activity. A mutation in the ß5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the ß4/ß5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.
Subject(s)
Chagas Disease , Trypanosoma cruzi , Chagas Disease/drug therapy , Drug Discovery , Humans , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Trypanosoma cruzi/chemistryABSTRACT
Anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica is spreading to new areas in Morocco. Exposure to the vector, Phlebotomus sergenti, is the only proven risk factor. Our objective was to compare the densities and genetic characteristics of P. sergenti populations in two nearby localities in Morocco, one in an ACL endemic area (El Borouj) and another in a nonendemic area (Sidi Hajjaj). P. sergenti density was significantly higher in the endemic area than in the nonendemic town (p = 0.032). A different predominant P. sergenti mitochondrial lineage was evidenced in each one of the two localities, and for the first time, the P. sergenti lineage acting as a vector of L. tropica has been identified. Bioclimatic differences were detected between both localities. In conclusion we found differences in both the density and the mitochondrial lineage of P. sergenti populations that may explain the different epidemiological situation. Given that the density of P. sergenti in the locality without ACL cases seems sufficient to allow transmission, the main factor that would justify its nonendemic character could be the absence of P. sergenti Lineage IV, which seems to prefer warmer and drier climates.
Subject(s)
Leishmania tropica , Leishmaniasis, Cutaneous , Phlebotomus , Animals , Leishmania tropica/genetics , Leishmaniasis, Cutaneous/epidemiology , Leishmaniasis, Cutaneous/veterinary , Morocco/epidemiologyABSTRACT
Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL.
Subject(s)
Antiprotozoal Agents/therapeutic use , Benzoxazoles/therapeutic use , Boron Compounds/therapeutic use , Leishmaniasis, Visceral/drug therapy , Pyridines/therapeutic use , Animals , Antiprotozoal Agents/chemistry , Benzoxazoles/chemistry , Boron Compounds/chemistry , Cricetinae , Disease Models, Animal , Dogs , Humans , Mice , Pyridines/chemistry , Structure-Activity RelationshipABSTRACT
Here, we detail our optimized protocol for the identification of drug targets in Leishmania donovani using thermal proteome profiling. This approach is based on the principle that binding of a drug to its protein target can significantly alter the thermal stability of that protein. By monitoring changes in the thermal stability of proteins within drug-treated and untreated cell lysates, using mass spectrometry combined with tandem mass tag labeling, putative targets of the drug can be identified in an unbiased manner. For further details on the use and application of this protocol, please refer to Paradela et al. (2021).
Subject(s)
Drug Development/methods , Protein Stability/drug effects , Proteomics/methods , Chromatography, Liquid/methods , Leishmania/drug effects , Leishmania donovani/drug effects , Protein Binding , Proteome/analysis , Tandem Mass Spectrometry/methods , TemperatureABSTRACT
There is limited information regarding the role of wild mammals in the transmission dynamics of Leishmania infantum. A potential human leishmaniasis hot spot was detected in southern Spain that could not be explained solely by canine leishmaniasis prevalence. The aim of this work was to analyse the involvement of wild rabbits as the main factor affecting this Mediterranean hot spot. A survey of wild rabbits, dogs and sand flies was conducted in the human cases environment. A nearby region without clinical leishmaniasis cases was used as reference control. 51 wild rabbits shot by hunters were analysed by molecular techniques. 1100 sand flies were captured and morphologically identified. Blood collected from patients' relatives/ neighbours (n = 9) and dogs (n = 66) was used for molecular analysis and serology. In Mediterranean leishmaniasis hot spots such as Montefrío municipality (average incidence of 16.8 human cases per 100,000 inhabitants/year), wild rabbits (n = 40) support high L. infantum infection rates (100%) and heavy parasite burdens (average value: 503 parasites/mg) in apparently normal ear skin directly accessible to sand flies, enabling the existence of heavily parasitized Phlebotomus perniciosus females (12.5% prevalence). The prevalence of infection and median parasite load were very low among rabbits captured in Huéscar (n = 11), a human clinical leishmaniasis-free area for the last 18 years. P. perniciosus was the most abundant Phlebotomus species in all the domestic/peridomestic microhabitats sampled, both indoors and outdoors. Accordingly, leishmaniasis is clustering in space and time at this local scale represented by Montefrío due to the proximity of two competent host reservoirs (dogs and heavily parasitized wild rabbits) associated with overlapping sylvatic and domestic transmission cycles through the main vector, P. perniciosus. We highlight the usefulness of determining the prevalence of infection and parasite burden in wild rabbits as a control leishmaniasis measure with the advantage that the use of the ear offers.
Subject(s)
Disease Reservoirs/parasitology , Leishmaniasis, Visceral , Phlebotomus , Rabbits/parasitology , Animals , Dogs , Female , Humans , Leishmania infantum , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/veterinary , Phlebotomus/parasitology , Spain/epidemiologyABSTRACT
Cutaneous leishmaniasis due to Leishmania tropica represents a major public health problem due to its ability to spread into non-endemic areas by means of its vectors, and the associated dramatic psychosocial impact. The objective of this work was to compare the intra and extradomiciliary density, sex ratio and gonotrophic stage of sand flies from a recent active focus in Morocco. This field study is based on the need to optimize the effectiveness of control programs. Two different capture methods, CDC light traps and sticky traps, were used at two different times of the year, corresponding with the peaks of sand fly abundance. 7,815 sand flies were captured and classified into 13 species belonging to genera Sergentomyia (50.8%) and Phlebotomus (49.2%). Phlebotomus sergenti was the most abundant and frequent species of the genus Phlebotomus both inside (49.3%) and outside houses (52.1%) and it showed the highest density in extradomiciliary captures in June. The proportion of blood-fed females was similar indoors and outdoors (21.5% and 26.3%, respectively). Females in the three gonotrophic stages were found in 26% houses and this was significantly associated with some factors related to housing conditions. Therefore, P. sergenti seems well adapted to both indoors and outdoors biotopes where these females coexist with males. These findings suggest that the adoption of additional measures could benefit the strategy of the Moroccan health authorities, currently consisting of indoor insecticide spraying, given that transmission may also occur outdoors.
Subject(s)
Leishmaniasis, Cutaneous , Phlebotomus , Animals , Female , Leishmaniasis, Cutaneous/epidemiology , Male , Morocco , Sex RatioABSTRACT
Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S)-1-treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S)-1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)-1 and the LdOSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed.
Subject(s)
Antiprotozoal Agents/pharmacology , Enzyme Inhibitors/pharmacology , Intramolecular Transferases/antagonists & inhibitors , Leishmania donovani/drug effects , Piperidines/pharmacology , Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Intramolecular Transferases/metabolism , Leishmania donovani/enzymology , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Piperidines/chemical synthesis , Piperidines/chemistryABSTRACT
Leishmaniasis is a vector-borne disease transmitted by sand flies. A dozen species have been involved in the transmission of Leishmania infantum in the Mediterranean region. Climate change may alter sand fly distribution at particular altitudes and latitudes. The objective of this study was to interrogate the existence of stable populations of sand flies in high-altitude ecosystems and evaluate if these populations are enough to support autochthonous transmission of leishmaniasis. These altitudinal conditions can be found in Sierra Nevada (southern Spain). Therefore, we have determined the sand fly population dynamics in different biotopes located at elevations above 1,300 m a.s.l. and searched for evidence of leishmaniasis transmission. Five collecting sites above 1,300 m a.s.l. containing large livestock concentrations were selected. Sand flies were caught using CDC light traps from May to November, annually from 2008 to 2013, and these were morphologically identified. Association between sand fly density or presence and temperature/humidity was estimated by linear and logistic regression, respectively. Leishmania infantum detection in female sand flies was performed by PCR. Diagnosis of canine leishmaniasis (CanL) was carried out by indirect immunofluorescence and PCR. A total of 2,973 specimens of 5 sand fly species were collected from June to October. Phlebotomus perniciosus was the most frequent (100%), abundant (80.1%) and densest species (9.8 sand flies/trap). The minimum temperature on the day of capture was the most important variable factor for sand fly presence and P. perniciosus density. An increase in altitude showed a negative effect over the sand fly diversity and activity period, driving changes in seasonal dynamics similar to those reported by latitudinal changes. CanL prevalence was 23%, a similar rate to previous surveys carried out on randomly selected dogs from towns in southern Spain. A successful host-vector-pathogen network was found at this altitude characterised by 9.9% L. infantum infection rate in non-blood fed P. perniciosus and Phlebotomus ariasi females and high CanL prevalence that entails an increase in the leishmaniasis risk area driven by sand fly colonization.
Subject(s)
Dog Diseases/epidemiology , Insect Vectors , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/veterinary , Phlebotomus , Psychodidae , Altitude , Animals , Dog Diseases/transmission , Dogs , Ecosystem , Female , Leishmaniasis/transmission , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/transmission , Male , Mediterranean Region , Phlebotomus/parasitology , Population Dynamics , Prevalence , Psychodidae/parasitology , Seasons , Spain/epidemiology , TemperatureABSTRACT
The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved ß-tubulin gene (ß-TUB). The two cryptic species differed at least in 7 bp in the ß-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and ß-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.