A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials (Sb(V)), but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory isolates, but our understanding of resistance in field isolates is lagging behind. We describe here a comparative analysis of a genetically related pair of Sb(V)-sensitive and -resistant Leishmania donovani strains isolated from kala-azar patients. The resistant isolate exhibited cross-resistance to other unrelated Leishmania drugs including miltefosine and amphotericin B. A comparative proteomics screen has highlighted a number of proteins differentially expressed suggesting that programmed cell death (PCD) is modified in the resistant parasite. Indeed drug-induced PCD progression was altered in the Sb(V)-resistant strain as determined using early and late markers of apoptosis. Two proteins, the heat shock protein HSP83 and the small kinetoplastid calpain-related protein (SKCRP14.1) were shown to be intimately implicated in the drug-induced PCD phenotype. HSP83 increased drug resistance and reduced drug-mediated PCD activation by interfering with the mitochondrial membrane potential, whereas SKCRP14.1 promoted antimonial-induced PCD but protected against miltefosine-induced PCD. This study highlights the important role of PCD in drug susceptibility/resistance in the protozoan parasite Leishmania.     

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.     

Possibility of membrane modification as a mechanism of antimony resistance in Leishmania donovani

Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resistance whose localization in the present study was confirmed on the pellicular plasma membrane of the parasite thereby indicating towards membrane modification as a mechanism of resistance in this resistant isolate.     

Transcriptomics throughout the life cycle of Leishmania infantum: High down-regulation rate in the amastigote stage

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in the Mediterranean Basin. The promastigote and amastigote stages alternate in the life cycle of the parasite, developing inside the sand-fly gut and inside mammalian phagocytic cells, respectively. High-throughput genomic and proteomic analyses have not focused their attention on promastigote development, although partial approaches have been made in Leishmania major and Leishmania braziliensis. For this reason we have studied the expression modulation of an etiological agent of visceral leishmaniasis throughout the life cycle, which has been performed by means of complete genomic microarrays. In the context of constitutive genome expression in Leishmania spp. described elsewhere and confirmed here (5.7%), we found a down-regulation rate of 68% in the amastigote stage, which has been contrasted by binomial tests and includes the down-regulation of genes involved in translation and ribosome biogenesis. These findings are consistent with the hypothesis of pre-adaptation of the parasite to intracellular survival at this stage.     

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.     

Antimony transport mechanisms in resistant leishmania parasites

Antimonial compounds have been used for more than a century in the treatment of the parasitic disease leishmaniasis. Although pentavalent antimonials are still first-line drugs in several developing countries, this class of drugs is no longer recommended in the Indian sub-continent because of the emergence of drug resistance. The precise mechanisms involved in the resistance of leishmania parasites to antimony are still subject to debate. It is now well documented that drug resistance in leishmania parasites is a multifactorial phenomenon involving multiple genes whose expression pattern synergistically leads to the resistance phenotype. The reduction of intracellular antimony accumulation is a frequent change observed in resistant leishmania cells; however, no comprehensive transport model has been presented so far to explain this change and its contribution to Leishmania resistance. The present review firstly covers the actual knowledge on the metabolism of antimonial drugs, the mechanisms of their transmembrane transport and intracellular processing in Leishmania. It further describes both the functional and molecular changes associated with Sb resistance in this organism. Possible transport models based on the actual knowledge are then presented, as well as their functional implications. Biophysical and pharmacological strategies are finally proposed for the precise identification of the transport pathways.     

Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.     

Heterologous expression of plasmodial proteins for structural studies and functional annotation

Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem^®. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.     

Leishmania donovani: effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Indian clinical isolates to sodium stibogluconate

Although pentavalent antimonials are the first-line drug for treatment of visceral leishmaniasis all over the world, yet, in India, increasing number of patients are being reported to be unresponsive to sodium stibogluconate. Verapamil, a calcium channel blocker, affects drug uptake by preventing its efflux and thereby accumulation within the cell. In the present study, effect of verapamil on in vitro susceptibility of both promastigote and amastigote stages of 15 clinical isolates and standard strain of Leishmania donovani to sodium stibogluconate was evaluated by detection of acid phosphatase. Amastigotes were found more susceptible to sodium stibogluconate than the promastigotes (p<0.05) and in the presence of verapamil, IC(50) value of sodium stibogluconate was reduced only for those isolates, which had a higher IC(50). Verapamil alone did not have any effect on the parasites. The results indicate that amastigotes are more susceptible to sodium stibogluconate than promastigotes and verapamil can reverse the in vitro drug resistance of L. donovani clinical isolates to sodium stibogluconate.     

Tolerance to drug-induced cell death favours the acquisition of multidrug resistance in Leishmania

The control of the protozoan parasite Leishmania relies on few drugs with unknown cellular targets and unclear mode of action. Several antileishmanials, however, were shown to induce apoptosis in Leishmania and this death mechanism was further studied in drug-sensitive and drug-resistant Leishmania infantum. In sensitive parasites, antimonials (SbIII), miltefosine (MF) and amphotericin B (AMB), but not paromomycin (PARO), triggered apoptotic cell death associated with reactive oxygen species (ROS). In contrast, Leishmania mutants resistant to SbIII, MF or AMB not only failed to undergo apoptosis following exposure to their respective drugs, but also were more tolerant towards apoptosis induced by other antileishmanials, provided that these killed Leishmania via ROS production. Such tolerance favored the rapid acquisition of multidrug resistance. PARO killed Leishmania in a non-apoptotic manner and failed to produce ROS. PARO resistance neither protected against drug-induced apoptosis nor provided an increased rate of acquisition of resistance to other antileishmanials. However, the PARO-resistant mutant, but not SbIII-, MF- or AMB-resistant mutants, became rapidly cross-resistant to methotrexate, a model drug also not producing ROS. Our results therefore link the mode of killing of drugs to tolerance to cell death and to a facilitated emergence of multidrug resistance. These findings may have fundamental implications in the field of chemotherapeutic interventions.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry

Gastrointestinal nematode infections of livestock animals are prevalent and costly problems worldwide. Currently, infections are controlled by anthelmintic chemicals but increasing drug resistance has prompted research interest to shift towards alternative methods of control such as vaccine development and selection of worm-resistant animals. The present study analyses proteins from Trichostrongylus colubriformis infective L3s that are recognised by IgG of immune sheep. Following protein separation via two-dimensional electrophoresis and Western blot probing with plasma from sheep resistant to T. colubriformis, mass spectrometry-based proteomic analyses were used to identify immuno-reactive protein spots. We were able to identify 28 immune targets, including aspartyl protease inhibitor, enolase, chaperone proteins, galectin, glycolytic enzymes, kinase, phosphatase and structural muscle proteins such as myosin, paramyosin, calponin and DIM-1. The data suggest that immune responses to T. colubriformis are dispersed over a relatively large number of parasite antigens, including several cytoplasmically expressed proteins. The results have new implications for understanding the molecular mechanisms that underpin host-parasite interaction during gastrointestinal nematode infections.     

Using a Label Free Quantitative Proteomics Approach to Identify Changes in Protein Abundance in Multidrug-Resistant Mycobacterium tuberculosis

Reports in recent years indicate that the increasing emergence of resistance to drugs be using to TB treatment. The resistance to them severely affects to options for effective treatment. The emergence of multidrug-resistant tuberculosis has increased interest in understanding the mechanism of drug resistance in M. tuberculosis and the development of new therapeutics, diagnostics and vaccines. In this study, a label-free quantitative proteomics approach has been used to analyze proteome of multidrug-resistant and susceptible clinical isolates of M. tuberculosis and identify differences in protein abundance between the two groups. With this approach, we were able to identify a total of 1,583 proteins. The majority of identified proteins have predicted roles in lipid metabolism, intermediary metabolism, cell wall and cell processes. Comparative analysis revealed that 68 proteins identified by at least two peptides showed significant differences of at least twofolds in relative abundance between two groups. In all protein differences, the increase of some considering proteins such as NADH dehydrogenase, probable aldehyde dehydrogenase, cyclopropane mycolic acid synthase 3, probable arabinosyltransferase A, putative lipoprotein, uncharacterized oxidoreductase and six membrane proteins in resistant isolates might be involved in the drug resistance and to be potential diagnostic protein targets. The decrease in abundance of proteins related to secretion system and immunogenicity (ESAT-6-like proteins, ESX-1 secretion system associated proteins, O-antigen export system and MPT63) in the multidrug-resistant strains can be a defensive mechanism undertaken by the resistant cell.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0511-2) contains supplementary material, which is available to authorized users.     

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.     

Comparative Fitness of a Parent Leishmania donovani Clinical Isolate and Its Experimentally Derived Paromomycin-Resistant Strain

Paromomycin has recently been introduced for the treatment of visceral leishmaniasis and emergence of drug resistance can only be appropriately judged upon its long term routine use in the field. Understanding alterations in parasite behavior linked to paromomycin-resistance may be essential to assess the propensity for emergence and spread of resistant strains. A standardized and integrated laboratory approach was adopted to define and assess parasite fitness of both promastigotes and amastigotes using an experimentally induced paromomycin-resistant Leishmania donovani strain and its paromomycin-susceptible parent wild-type clinical isolate. Primary focus was placed on parasite growth and virulence, two major components of parasite fitness. The combination of in vitro and in vivo approaches enabled detailed comparison of wild-type and resistant strains for which no differences could be demonstrated with regard to promastigote growth, metacyclogenesis, in vitro infectivity, multiplication in primary peritoneal mouse macrophages and infectivity for Balb/c mice upon infection with 2 x 10⁷ metacyclic promastigotes. Monitoring of in vitro intracellular amastigote multiplication revealed a consistent decrease in parasite burden over time for both wild-type and resistant parasites, an observation that was subsequently also confirmed in a larger set of L. donovani clinical isolates. Though the impact of these findings should be further explored, the study results suggest that the epidemiological implications of acquired paromomycin-resistance may remain minimal other than the loss of one of the last remaining drugs effective against visceral leishmaniasis.     

A proteomic analysis of penicillin resistance in Streptococcus pneumoniae reveals a novel role for PstS, a subunit of the phosphate ABC transporter

Resistance to penicillin is widespread in the Gram-positive bacterium Streptococcus pneumoniae, and while several mutations are known to be implicated in resistance other mechanisms are likely to occur. We used a proteomic screen of two independent mutants in which resistance was selected in vitro. We found a number of differentially expressed proteins including PstS, a subunit of the phosphate ABC transporter of S. pneumoniae. This protein was increased in both mutants, a phenotype correlated to increased RNA expression of the entire phosphate ABC transporter operon. Inactivation of the pstS gene led to increased susceptibility to penicillin in the wild-type strain. To further link the expression of the ABC phosphate transporter with penicillin resistance, we looked at pstS mRNA levels in 12 independent clinical isolates sensitive and resistant to penicillin and found an excellent correlation between resistance and increased expression of pstS. Inactivation of pstS in one of the clinical isolates significantly reduced penicillin resistance. Global approaches are ideally suited for the discovery of novel factors in the biology of resistance.     

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.     

In silico analysis of a therapeutic target in Leishmania infantum: the guanosine-diphospho-D-mannose pyrophosphorylase

Leishmaniases are tropical and sub-tropical diseases for which classical drugs (i.e. antimonials) exhibit toxicity and drug resistance. Such a situation requires to find new chemical series with antileishmanial activity. This work consists in analyzing the structure of a validated target in Leishmania: the GDP-mannose pyrophosphorylase (GDP-MP), an enzyme involved in glycosylation and essential for amastigote survival. By comparing both human and L. infantum GDP-MP 3D homology models, we identified (i) a common motif of amino acids that binds to the mannose moiety of the substrate and, interestingly, (ii) a motif that is specific to the catalytic site of the parasite enzyme. This motif could then be used to design compounds that specifically inhibit the leishmanial GDP-MP, without any effect on the human homolog.