Leishmania donovani: Thionins, plant antimicrobial peptides with leishmanicidal activity

The leishmanicidal activity of plant antibiotic peptides (PAPs) from the principal families, such wheat thionins, a barley lipid transfer protein and potato defensins and snakins were tested in vitro against Leishmania donovani. Only thionins and defensins were active against this human pathogen at a low micromolar range of concentrations. Thionins resulted as the most active peptides tested until now. They collapsed ionic and pH gradients across the parasite plasma membrane together with a rapid depletion of intracellular ATP without affecting mitochondrial potential. Hence the lethal effect of thionins was mostly associated to permeabilization of the plasma membrane leading to an immediate death of the parasite. The present work is the first evidence for leishmanicidal activity in plant peptides. Future prospects for their development as new antiparasite agents on human diseases are considered.     

One-step generation of double-allele gene replacement mutants in Leishmania donovani

Due to the apparent lack of sexual recombination in Leishmania spp., gene replacement strategies in this diploid organism necessitate the subsequent targeting of two gene alleles by using two constructs, bearing different antibiotic resistance markers. This approach is time consuming and often gives rise to spontaneous amplification of the targeted gene, nullifying efforts to create functional null mutants. Here, we show that by using two homologous recombination constructs in a co-transfection of Leishmania donovani promastigotes, we can obtain double-allele gene replacement mutants. This approach reduces the time required for the generation of functional null mutants and the number of in vitro passage cycles, potentially limiting culture-associated artefacts.     

Proteomic analysis of wild type and arsenite-resistant Leishmania donovani

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.     

Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids

Trypanosoma cruzi exhibits remarkable genetic heterogeneity. This is evident at the nucleotide level but also structurally, in the form of karyotypic variation and DNA content differences between strains. Although natural populations of T. cruzi are predominantly clonal, hybrid lineages (TcIId and TcIIe) have been identified and hybridisation has been demonstrated in vitro, raising the possibility that genetic exchange may continue to shape the evolution of this pathogen. The mechanism of genetic exchange identified in the laboratory is unusual, apparently involving fusion of diploid parents followed by genome erosion. We investigated DNA content diversity in natural populations of T. cruzi in the context of its genetic subdivisions by using flow cytometric analysis and multilocus microsatellite genotyping to determine the relative DNA content and estimate the ploidy of 54 cloned isolates. The maximum difference observed was 47.5% between strain Tu18 cl2 (TcIIb) and strain C8 cl1 (TcI), which we estimated to be equivalent to 73 Mb of DNA. Large DNA content differences were identified within and between discrete typing units (DTUs). In particular, the mean DNA content of TcI strains was significantly less than that for TcII strains (P < 0.001). Comparisons of hybrid DTUs TcIId/IIe with corresponding parental DTUs TcIIb/IIc indicated that natural hybrids are predominantly diploid. We also measured the relative DNA content of six in vitro-generated TcI hybrid clones and their parents. In contrast to TcIId/IIe hybrid strains these experimental hybrids comprised populations of sub-tetraploid organisms with mean DNA contents 1.65–1.72 times higher than the parental organisms. The DNA contents of both parents and hybrids were shown to be relatively stable after passage through a mammalian host, heat shock or nutritional stress. The results are discussed in the context of hybridisation mechanisms in both natural and in vitro settings.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp.

In this work, we have found an antiproliferative effect on Leishmania sp. promastigotes and axenic amastigotes by the human immunodeficiency virus (HIV) aspartyl-proteinase inhibitors, Ac-Leu-Val-Phenylalaninal, Saquinavir mesylate and Nelfinavir, the latter two being used as part of antiretroviral therapy. This effect appears to be the result of cell division blockage. In addition, these drugs induced in culture a decrease in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage. The finding of a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by these drugs allows us to propose this activity as the drug parasite target. A direct action of these HIV aspartyl-proteinase inhibitors on the parasite, would be correlated with the effect that highly active antiretroviral therapy have had in the decrease of HIV/Leishmania coinfection, opening an interesting perspective for new drugs research development based on this novel parasite proteinase family.     

Down regulation of KMP-11 in Leishmania infantum axenic antimony resistant amastigotes as revealed by a proteomic screen

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials, but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory promastigote isolates, but the mechanism leading to drug resistance in amastigote isolates is lagging behind. Here we describe a comparative proteomic analysis of a genetically related pair of antimonial-sensitive and -resistant Leishmania infantum axenic amastigote strains. The proteomics screen has highlighted a number of proteins differentially expressed in the resistant parasite. The expression of the protein argininosuccinate synthetase (ARGG) was increased in the drug resistant mutant while a decrease in the expression of the kinetoplastid membrane protein (KMP-11) correlated with the drug resistance phenotype. This proteomic screen highlighted several novel proteins that are putatively involved in resistance to antimonials.     

Population structure of Tunisian Leishmania infantum and evidence for the existence of hybrids and gene flow between genetically different populations

Twenty-seven strains of Leishmania infantum from north and central Tunisia belonging to the three main MON zymodemes (the MON-typing system is based on multilocus enzyme electrophoresis (MLEE) of 15 enzymes) found in this country (MON-1, MON-24 and MON-80) and representing different pathologies (visceral, cutaneous and canine leishmaniasis) have been studied to understand the genetic polymorphism within this species. Intraspecific variation could be detected in L. infantum by the use of 14 hypervariable microsatellite markers. In addition to microsatellite repeat length variation, a high degree of allelic heterozygosity has been observed among the strains investigated, suggestive of sexual recombination within L. infantum groups. The two major clusters found by using Bayesian statistics as well as distance analysis are consistent with the classification based on isoenzymes, dividing Tunisian L. infantum into MON-1 and MON-24/MON-80. Moreover, the existence of hybrid strains between the MON-1 and the non-MON-1 populations has been shown and verified by analysis of clones of one of these strains. Substructure analysis discriminated four groups of L. infantum. The major MON-1 cluster split into two groups, one comprising only Tunisian strains and the second both Tunisian and European strains. The major MON-24 cluster was subdivided into two groups with geographical and clinical feature correlations: a dermotropic group of strains mainly from the north, and a viscerotropic group of strains from the centre of Tunisia. The four viscerotropic hybrid strains all originated from central Tunisia and were typed by MLEE as MON-24 or MON-80. To our knowledge, this is the first report describing relationships between clinical picture and population substructure of L. infantum MON-24 based on genotype data, as well as the existence of hybrids between zymodemes MON-1 and MON-24/MON-80, and proving one of these hybrid strains by molecular analysis of the parent strain and its clones.     

Multilocus sequence analysis of the genus Bradyrhizobium

The use of multilocus sequence analysis (MLSA) for the taxonomy of Bradyrhizobium was assessed. We compared partial sequences for atpD, recA, gyrB, rpoB and dnaK for a set of reference strains representing named species and genospecies, and a number of new isolates from Lupinus albus, Arachis hypogaea and Ornithopus compressus from Spain. The phylogenies of the individual genes were compared with previous DNA–DNA hybridization results. High hybridization values were well reflected, but intermediary hybridization values were less clearly apparent. However, the phylogeny of a concatenated dataset of the five genes did reflect all values and thus is more informative of overall genome similarity. Our results indicate that only for the genes gyrB, rpoB and dnaK there is a small gap between the interspecies sequence similarities and the intraspecies similarity, and therefore cut-off levels for species delineation cannot be set, although high sequence similarity (>99%) does permit identification. In a few instances, a reference strain did not group as expected for one of the five genes tested. This may be a result of horizontal gene transfer and recombination events occasionally involving housekeeping genes. This observation indicates it is best to consider more than one gene for taxonomic inferences. The majority of the new isolates from the three host species was identified as Bradyrhizobium canariense. Four strains from L. albus from León, Spain, formed a separate group close to Bradyrhizobium japonicum.     

Leishmania major lipophosphoglycan: Discrepancy in toll-like receptor signaling

Lipophosphoglycan (LPG) is structurally characterized by a series of phosphoglycan repeat units. Cellular LPG, isolated from promastigotes, has a very similar structure to culture supernatant LPG, but differs in the average number of phosphorylated oligosaccharide repeat units and in glycan composition. Comparison of these LPGs with capillary electrophoresis and immunoblotting indicate that these molecules are highly conserved structurally and composed of galactosylated Gal-Man repeats but their size and molecular weight are very different which is due to glycan portion. There are 30 and 20 repeat units in sLPG and mLPG, respectively. Both LPGs induced nitric oxide in macrophages cell line while sLPG had the higher stimulatory effect. In the presence of anti-TLR2 nitric oxide stimulated by LPG was reduced to control levels. In addition, in the presence of anti-TLR4, nitric oxide stimulated by LPGs was not affected. We propose that lipophosphoglycan induces nitric oxide production via TLR2 signaling pathway.     

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s > 60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.     

Immunolocalization and characterization of two novel proteases in Leishmania donovani: Putative roles in host invasion and parasite development

Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 °C and for MP-Ld is 37 °C showing their thermostability upto 60 °C. Broad substrate (both natural and synthetic) specificity and the effect of Ca²⁺ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development.     

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀ ∼ 20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀ ∼ 7.2 μM) as compared to diospyrin (IC₅₀ ∼ 12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀ ∼ 0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2 mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.     

Genetic structure of Phlebotomus (Larroussius) ariasi populations, the vector of Leishmania infantum in the western Mediterranean: Epidemiological implications

In recent years there has been growing interest in analyzing the geographical variations between populations of different Phlebotomus spp. by comparing the sequences of various genes. However, little is known about the genetic structure of Phlebotomus ariasi. In this study, we were able to sequence a fragment of the mitochondrial Cyt b gene in 133 sandflies morphologically identified as P. ariasi and proceeding from a wide geographical range covering 35 locations in 11 different regions from five countries. The intra-specific diversity of P. ariasi is high, with 45 haplotypes differing from each other by one to 26 bases and they are distributed in two mitochondrial lineages, one limited geographically to Algeria and the other widely dispersed across Mediterranean countries. The Algerian lineage is characterized by having 13 fixed polymorphisms and is made up of one sole haplotype. The European/Moroccan P. ariasi lineage is characterized by being made up of a great diversity of haplotypes (44) which display some geographical structuring. This could be one of the multiple factors involved in the epidemiological heterogeneity of the foci of leishmaniasis. Phlebotomus chadlii is the sister group of European/Moroccan P. ariasi. The separation of the Algerian haplotype, H45, from the rest of the specimens, European/Moroccan P. ariasi and P. chadlii, is well supported by the bootstrap analysis.     

Leishmania donovani: Genetic diversity of isolates from Sudan characterized by PCR-based RAPD

Drug unresponsiveness in patients with visceral leishmaniasis (VL) is a problem in many endemic areas. This study aimed to determine genetic diversity of Leishmania donovani isolates from a VL endemic area in Sudan as a possible explanation for drug unresponsiveness in some patients. Thirty clinically stibogluconate (SSG)-sensitive isolates were made SSG-unresponsive in vitro by gradually increasing SSG concentrations. The sensitive isolates and their SSG-unresponsive counterparts were typed using mini-circle kDNA and categorized using PCR-RAPD. All the isolates were typed as L. donovani, the resulting PCR-RAPD characterization of the SSG-sensitive isolates gave three distinct primary genotypes while, the SSG-unresponsive isolates showed only a single band. L. donovani isolates from eastern Sudan are diverse; this probably resulted from emergence of new L. donovani strains during epidemics due to the pressure of widespread use of antimonials.In this communication the possible role of isolates diversity in antimonial unresponsiveness and the in vitro changing PCR-RAPD band pattern in SSG-unresponsive strains were discussed.     

N-Myristoyltransferase from Leishmania donovani: Structural and Functional Characterisation of a Potential Drug Target for Visceral Leishmaniasis

N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovaniNMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 Å resolution. The structure has as its defining feature a 14-stranded twisted β-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis.     

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.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.