Infectivity and virulence of Leishmania donovani promastigotes: a role for media,source, and strain of parasite

Transformation of promastigotes of Leishmania donovani strain AG83 from amastigotes derived from an infected animal was studied in three media, Schneider's Drosophila medium (SDM), Medium 199 (M199), and biphasic M199 (B-M199) with 10% fetal bovine serum. The media, SDM and B-M199, both supported a more efficient transformation of promastigotes in comparison with M199. Infectivity studies in hamsters and BALB/c mice showed that promastigotes isolated in B-M199 were several folds more infective than those obtained from M199. Comparison of the infectivity and virulence of promastigotes of AG83, with a recent isolate of kala-azar, SL94, harvested under similar conditions, revealed greater infectivity of SL94 for both macrophages and animal models. The present study demonstrates that the medium used for the conversion of amastigotes to promastigotes plays a major role in determining the infectivity of the freshly transformed L. donovani promastigotes in hamsters and BALB/c mice. The source and the strain of the parasite also influence the outcome of L. donovani infection.     

Correlation between in vitro and in vivo infectivity of Leishmania infantum clones

Eleven clones of a single strain of Leishmania infantum (MCAN/ES/88/ISS441, Doba) were analyzed for biological behavior in vivo and in vitro. Different clones showed differences in growth dependent upon the two culture media employed. All clones displayed only slight differences in H2O2 and NaNO2 sensitivity compared to the original strain, whereas in vitro infectivity for mouse peritoneal macrophages differed significantly among the clones. In vivo infections in hamsters correlated strongly with in vitro infectivity. The phenotypic differences found suggest a polyclonal structure for the Leishmania infantum strain studied.     

Exploratory algorithm to devise multi-epitope subunit vaccine by investigating Leishmania donovani membrane proteins

Visceral leishmaniasis (VL) is a deadly parasitic infection which affects poorest to poor population living in the endemic countries. Increasing resistant to existing drugs, disease burden and a significant number of deaths, necessitates the need for an effective vaccine to prevent the VL infection. This study employed a combinatorial approach to develop a multi-epitope subunit vaccine by exploiting Leishmania donovani membrane proteins. Cytotoxic T- and helper T-lymphocyte binding epitopes along with suitable adjuvant and linkers were joined together in a sequential manner to design the subunit vaccine. The occurrence of B-cell and IFN-γ inducing epitopes approves the ability of subunit vaccine to develop humoral and cell-mediated immune response. Physiochemical parameters of vaccine protein were also assessed followed by homology modeling, model refinement and validation. Moreover, disulfide engineering was performed for the increasing stability of the designed vaccine and molecular dynamics simulation was performed for the comparative stability purposes and to conform the geometric conformations. Further, molecular docking and molecular dynamics simulation study of a mutated and non-mutated subunit vaccine against TLR-4 immune receptor were performed and respective complex stability was determined. In silico cloning ensures the expression of designed vaccine in pET28a(+) expression vector. This study offers a cost-effective and time-saving way to design a novel immunogenic vaccine that could be used to prevent VL infection.

Communicated by Ramaswamy H. Sarma     

Structure-based virtual screening,molecular docking,ADMET and molecular simulations to develop benzoxaborole analogs as potential inhibitor against Leishmania donovani trypanothione reductase

Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis and it affects 70 countries worldwide. Increasing drug resistant for antileishmanial drugs such as miltefosine, sodium stibogluconate and pentamidine has been reported in the VL endemic region. Amphotericin B has shown potential antileishmanial activity in different formulations but its cost of treatment and associated nephrotoxicity have limited its use by affected people living in the endemic zone. To control the VL infection in the affected countries, it is necessary to develop new antileishmanial compounds with high efficacy and negligible toxicity. Computer aided programs such as binding free energy estimation; ADMET prediction and molecular dynamics simulation can be used to investigate novel antileishmanial molecules in shorter duration. To develop antileishmanial lead molecule, we performed standard precision (SP) docking for 1160 benzoxaborole analogs along with reference inhibitors against trypanothione reductase of Leishmania parasite. Furthermore, extra precision (XP) docking, ADMET prediction, prime MM-GBSA was conducted over 115 ligands, showing better docking score than reference inhibitors to get potential antileishmanial compounds. Simultaneously, area under the curve (AUC) was estimated using ROC plot to validate the SP and XP docking protocol. Later on, two benzoxaborole analogs with best MM-GBSA ΔG-bind were subjected to molecular simulation and docking confirmation to ensure the ligand interaction with TR. The presented drug discovery based on computational study confirms that BOB27 can be used as a potential drug candidate and warrants further experimental investigation to fight against VL in endemic areas.     

Febrifugine analogues as Leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking,ADMET and molecular dynamics simulation

Visceral leishmaniasis affects people from 70 countries worldwide, mostly from Indian, African and south American continent. The increasing resistance to antimonial, miltefosine and frequent toxicity of amphotericin B drives an urgent need to develop an antileishmanial drug with excellent efficacy and safety profile. In this study we have docked series of febrifugine analogues (n = 8813) against trypanothione reductase in three sequential docking modes. Extra precision docking resulted into 108 ligands showing better docking score as compared to two reference ligand. Furthermore, 108 febrifugine analogues and reference inhibitor clomipramine were subjected to ADMET, QikProp and molecular mechanics, the generalized born model and solvent accessibility study to ensure the toxicity caused by compounds and binding-free energy, respectively. Two best ligands (FFG7 and FFG2) qualifying above screening parameters were further subjected to molecular dynamics simulation. Conducting these studies, here we confirmed that 6-chloro-3-[3-(3-hydroxy-2-piperidyl)-2-oxo-propyl]-7-(4-pyridyl) quinazolin-4-one can be potential drug candidate to fight against Leishmania donovani parasites.     

The Absence of HIF-1α Increases Susceptibility to Leishmania donovani Infection via Activation of BNIP3/mTOR/SREBP-1c Axis

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Leishmania donovani: identification of novel vaccine candidates using human reactive sera and cell lines

Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to the genus Leishmania. The development of specific resistance against re-infection after cure suggests that a vaccine approach is feasible. Various studies in humans and experimental animals strongly suggest that Th1 type of cell-mediated immune response is important for protection against the disease. A defined antigen that could elicit a specific T-cell-mediated immune response in the host would be an ideal candidate for the vaccine against this parasite. In order to select a candidate antigen, we established a screening system to identify the recombinant clone, expressing antigen having T-cell epitopes from a cDNA library. We screened the library using an established Leishmania specific cell line (LSCL) from a naive healthy human subject. The cell line with predominantly CD4+ cells behaved in a Leishmania specific manner. Fifty-two immuno-reactive clones were screened against the LSCL in vitro and we identified three cDNA clones expressing recombinant antigens that could induce proliferation of these cells to produce INFgamma. The protective efficacy of one of these recombinant proteins was investigated in a hamster model of experimental visceral leishmaniasis and showed protection against a virulent challenge. The identified antigens might be potential candidates for vaccine against Leishmania.     

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania) infantum

The aim of the present study was to detect natural infection by Leishmania (Leishmania) infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA), the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA) gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.     

In vivo antileishmanial action of Ir-(COD)-pentamidine tetraphenylborate on Leishmania donovani and Leishmania major mouse models

Ir-(COD)-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same IC50 value on intracellular forms of Leishmania as pentamidine (15 microM). In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9/Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 mumoles/kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate. However, only 23% of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 mumoles/kg/day x 5 (54% of parasite suppression), the iridium complex exhibited 32% of parasite suppression after a treatment at 76 mumoles/kg/day x 5 administered subcutaneously. This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.     

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.     

Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL.     

Generation of ceramide in murine macrophages infected with Leishmania donovani alters macrophage signaling events and aids intracellular parasitic survival

In the present study, we examined the involvement of intracellular ceramide in host pathogen interaction of BALB/c mouse peritoneal macrophages infected with the obligate intracellular protozoan, Leishmania donovani. Our findings indicate that the level of intracellular ceramide was enhanced as a result of the in vitro infection. While the elevated ceramide was largely due to de novo synthesis, activation of the sphingomyelinases was also observed. The enhanced ceramide was responsible for the downregulation of classical PKC activity, upregulation of calcium independent atypical PKC- expression and activity of calcium independent PKC. Ceramide also impaired the phosphorylation of MAPK. Evidently, ceramide suppressed the generation of nitric oxide during leishmanial infection and also facilitated the survival of leishmanial parasites in the intramacrophageal milieu. These data present newer insight to the signaling events in leishmania-infected murine macrophages, which might offer ceramide as a new therapeutic target in the future.     

Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

Background

Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these ‘hostile’ environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent.

Methodology/Principal Findings

We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain.

Conclusions/Significance

The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.     

Leishmania donovani: In vitro culture and [1H] NMR characterization of amastigote-like forms

WhenLeishmania donovani promastigote forms, were cultured in TC-199 medium at 28°C and subsequently incubated at 38°C, they turned into aflagellate (amastogote-like) forms. A return of the incubation-culture temperature to 28°C these amastigote-like forms to revert to promastigotes. The amastigotes obtained by heat-shock, were viable and retained antigenic capacity being recognized by the sera of naturally infected patients. These forms, remained also capable of multiplying inside the J-774A. 1 macrophages. When the amastigote-like forms are kept in culture at 38°C retained their rounded appearance and their biological characteristics for more than 3 months subculturing every 6 days. These amastigote-like forms, when used for subcultures at 28°C, transformed into promastigotes capable of multiplying, as flagellate forms. The amastigotelike forms obtainedin vitro can be used in biochemical studies related to chemotherapy and immunology studies, as part of an effort to combat this parasite. The end-products of of glycolysis were studied in both the amastigote-like and promastigote forms ofL. donovani, by proton magnetic resonance analysis of the culture media. Alanine, succinate, and acetate, were predominant, and to a lesser extent pyruvate, glycine and D-lactate. Our results suggest that both forms ofLeishmania use different biochemical strategies to obtain their energy.     

Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg²⁺ ions were essential for enzyme activity; among other metal ions Mn²⁺ can replace Mg²⁺ to a certain extent whereas Ca²⁺, Co²⁺ and Zn²⁺ could not substitute for Mg²⁺. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF phenylmethylsulfonyl fluoride - DTT dithiothreitol - TCA trichloroacetic acid - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - EGTA Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid     

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.     

In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis

Background

Antitumor cyclopalladated complexes with low toxicity to laboratory animals have shown leishmanicidal effect. These findings stimulated us to test the leishmanicidal property of one palladacycle compound called DPPE 1.2 on Leishmania (Leishmania) amazonensis, an agent of simple and diffuse forms of cutaneous leishmaniasis in the Amazon region, Brazil.

Methodology/Principal Findings

Promastigotes of L. (L.) amazonensis and infected bone marrow-derived macrophages were treated with different concentrations of DPPE 1.2. In in vivo assays foot lesions of L. (L.) amazonensis-infected BALB/c mice were injected subcutaneously with DPPE 1.2 and control animals received either Glucantime or PBS. The effect of DPPE 1.2 on cathepsin B activity of L. (L.) amazonensis amastigotes was assayed spectrofluorometrically by use of fluorogenic substrates. The main findings were: 1) axenic L. (L.) amazonensis promastigotes were destroyed by nanomolar concentrations of DPPE 1.2 (IC50 = 2.13 nM); 2) intracellular parasites were killed by DPPE 1.2 (IC50 = 128.35 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 = 1,267 nM); 3) one month after intralesional injection of DPPE 1.2 infected BALB/c mice showed a significant decrease of foot lesion size and a reduction of 97% of parasite burdens when compared to controls that received PBS; 4) DPPE 1.2 inhibited the cysteine protease activity of L. (L.) amazonensis amastigotes and more significantly the cathepsin B activity.

Conclusions/Significance

The present results demonstrated that DPPE 1.2 can destroy L. (L.) amazonensis in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support the potential use of DPPE 1.2 as an alternative choice for the chemotherapy of leishmaniasis.