Induction of cellular and humoral responses by autoclaved and heat-killed antigen of Leishmania donovani in experimental visceral leishmaniasis

The potential of autoclaved and heat-killed antigen of Leishmania donovani to induce cell-mediated and humoral response has been evaluated in the present study. The vaccines were delivered thrice subcutaneously at an interval of 2 weeks. Two weeks after second booster, BALB/c mice were challenged with 10⁷ stationary phase promastigotes of L. donovani. Significant protection was achieved in immunized mice against L. donovani challenge with 69% to 76% and 59% to 64% reduction in parasite load in the liver and spleen respectively. Immunization induced significantly higher level of delayed type hypersensitivity (DTH) response in mice immunized with heat-killed antigen followed by autoclaved antigen. The immune response was assessed by quantifying Leishmania-specific antibodies and cytokine production. The antibody response was predominantly of IgG type with increased IgG2a production and lesser amount of IgM. The immunization preferentially stimulates the production of IFN-γ and IL-2 in splenocytes which suggests a Th1 type response with a concomitant down-regulation of IL-10 and IL-4. These results indicate a potential for the heat-killed and autoclaved antigen as a vaccine which could trigger cell-mediated immune response.     

Redundancy of interleukin-6 in the differentiation of T cell and monocyte subsets during cutaneous leishmaniasis

Leishmania (L.) major is a protozoan parasite that infects mammalian hosts and causes a spectrum of disease manifestations that is strongly associated with the genetic background of the host. Interleukin (IL)-6 is an acute phase proinflammatory cytokine, known in vitro to be involved in the inhibition of the generation of regulatory T cells. IL-6-deficient mice were infected with L. major, and T cell and monocyte subsets were analyzed with flow cytometry. Our data show that at the site of infection in the footpad and in the draining popliteal lymph node, numbers of regulatory T cells remain unchanged between WT and IL-6-deficient mice. However, the spleens of IL-6^−/− mice contained fewer regulatory T cells after infection with L. major. The development of cutaneous lesions is similar between WT and IL-6-deficient mice, while parasite burden in IL-6^−/− mice is reduced compared to WT. The development of IFN-γ or IL-10 producing T cells is similar in IL-6^−/− mice. Despite a comparable adaptive T cell response, IL-6-deficient mice develop an earlier peak of some inflammatory cytokines than WT mice. This data indicate that the role of IL-6 in the differentiation of regulatory T cells is complex in vivo, and the effect of an absence of this cytokine can be counter-intuitive.     

Expression of a tomato sugar transporter is increased in leaves of mycorrhizal or Phytophthora parasitica-infected plants

A full-length cDNA clone (LeST3), encoding a putative tomato sugar transporter, was isolated from mycorrhizal roots by using a PCR-based approach. Based on sequence similarity, conserved motifs and predicted membrane topology, LeST3 was classified as a putative monosaccharide transporter of the sugar transporter subgroup of the major facilitator superfamily. Southern blot analysis showed that LeST3 represents a single-copy gene in tomato. To investigate its function, LeST3 was expressed in a hexose transport-deficient mutant of Saccharomyces cerevisiae. Although LeST3 was correctly transcribed in yeast, it did not restore growth on hexoses of the S. cerevisiae mutant. LeST3 gene expression was increased in the leaves of plants colonised by the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices and in those of plants infected with the root pathogen Phytophthora parasitica. These data suggest that LeST3 plays a role in the transport of sugars into the sink tissues and responds to the increased demand for carbohydrates exerted by two AM fungi and by a root pathogen to cope with the increased metabolic activity of the colonised/infected tissues or to supply carbohydrates to the AM fungus.     

Molecular cloning and biochemical characterization of Leishmania donovani serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) catalyzes the inter conversion of serine and tetrahydrofolate (H(4)-folate) to form glycine and 5,10-methylene H(4)-folate and generates one-carbon fragments for the synthesis of nucleotides, methionine, thymidylate, choline, etc. In spite of being an indispensable enzyme of the thymidylate cycle, SHMT in Leishmania donovani remains uncharacterized. The study of L. donovani SHMT (ldSHMT) becomes important as this gene is preferentially expressed in the amastigote stage of parasite, which resides in human macrophages. Here we report cloning, expression and purification of a catalytically active ldSHMT. The homogeneity of recombinant protein was analyzed by denaturing gel electrophoresis and protein was found to be 95% pure having yield of 1mg/l. The recombinant protein is a tetramer of 216kDa as evidenced by gel filtration chromatography and uses serine and tetrahydrofolate as substrates with Km of 1.6 and 2.4mM, respectively. Further biochemical studies revealed that pH optimum of ldSHMT is 7.8 and enzyme is thermally stable up to 45 degrees C. ldSHMT was found sensitive towards denaturants as manifested by loss of enzyme activity at the concentration of 1M urea or 0.25M guanidine hydrochloride. This is the first report of purification and characterization of recombinant SHMT from any protozoan source. Studies on recombinant ldSHMT will help in evaluating this enzyme as potential drug target.     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Vaccination with a novel recombinant Leishmania antigen plus MPL provides partial protection against L. donovani challenge in experimental model of visceral leishmaniasis

The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis. The characteristics and in vitro stimulating capability of the recombinant proteins expressed by previously identified clones on the basis of their capacity to stimulate an indigenously established Leishmania-specific cell line leading to high level of IFN-γ suggested these to be potential candidates for immunoprophylaxis against leishmaniasis. In this study, we investigated the protective efficacy of purified recombinant proteins from two of the identified cDNA clones along with the adjuvant MPL, in a hamster model of experimental leishmaniasis. We demonstrate here that the immunization of animals with one of the recombinant proteins (rF14) having 97% similarity to C1 clone of L. chagasi ribosomal protein gene P0 (rLiP0) along with MPL provided partial protection against the virulent challenge of L. donovani. The absence of antigen-specific lymphoproliferative responses in these immunized animals may be responsible for the lack of complete and long-lasting protection.     

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.     

Leishmania major CorA-like magnesium transporters play a critical role in parasite development and virulence

Establishment of infection by Leishmania depends on the transformation of the invading metacyclic promastigotes into the obligatory intracellular amastigotes, and their subsequent survival in the macrophage phagolysosome, which is low in magnesium. We show that two Leishmania major proteins designated MGT1 and MGT2, which play a critical role in these processes, belong to the two-transmembrane domain (2-TM-GxN) cation transporter family and share homology with the major bacterial magnesium transporter CorA. Although both are present in the endoplasmic reticulum throughout the life cycle of the parasite, MGT1 is more highly expressed in the infectious metacyclic parasites, while MGT2 is enriched in the immature procyclic stages. The two proteins, although predicted to be structurally similar, have features that suggest different regulatory or gating mechanisms. The two proteins may also be functionally distinct, since only MGT1 complements an Escherichia coliΔCorA mutant. In addition, deletion of one mgt1 allele from L. major led to increased virulence, while deletion of one allele of mgt2 resulted in slower growth and total loss of virulence in vitro and in vivo. This loss of virulence may be due to an impaired transformation of the parasites into amastigotes. Deletion of both mgt1 alleles in the hemizygous MGT2 knockdown parasites reversed the growth defect and partially restored virulence. Our data indicate that the MGTs play a critical role in parasite growth, development and virulence.     

Leishmania major: species specific delayed hypersensitivity reaction induced by exogenous secreted antigen in the guinea pig

The cellular response to Leishmania major (L. major) is usually evaluated in vivo by the delayed-type-hypersensitivity (DTH) test using leishmanin. Leishmanin can give false-positive reactions in areas where there is a background of leishmaniasis. In a previous study, it was shown that a 56 kDa antigen purified from promastigote and culture supernatant of L. major induce strong DTH reactions in sensitized guinea pigs. In this study, the species-specificity of this antigen was further investigated. Three groups of guinea pigs were sensitized with L. major, L. tropica, and L. infantum and both flanks of sensitized animal were injected intradermally with purified 56 kDa antigen or soluble leishmania antigen (SLA). The extent of indurations were measured after 24, 48, and 72 h. In animals which were sensitized with three species of leishmania, only those immunized with L. major showed skin reactions to purified antigen by an increase in skin thickness. Since complex antigen mixtures such as SLA and leishmanin show cross-reactivity and can be non-specific, the result obtained here suggest that 56 kDa antigen may be a useful diagnostic tool for species specific diagnosis in field studies of leishmaniasis.     

Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis

The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmaniadonovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund’s adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1 × 10⁷L.donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.     

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.     

Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.     

Expression and characterization of a parasite-specific antigen on macrophages after infection withLeishmania donovani

A rabbit polyclonal antibody to crude soluble antigen ofLeishmania donovani promastigotes recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages and human monocyte derived macrophages infectedin vitro. The determinant was recognized on infected macrophage surface only when F (ab)₂ fragments of anti-leishmanial antiserum was employed in immunofluorescence. F(ab)₂ fragments of human patient sera also could recognize the determinant. The expression of this antigen was not stage-specific for the parasite. Immunochemical analyses revealed this antigen to be of 51 kDa protein. Specific leaching of membrane proteins by trypsin showed three bands of expressed antigens of 26, 11 and 10 kDa, which in all likelihood might be arising from the 51 kDa antigen. The antigen was not expressed until 12 h of post infection, reached a maximum level at 24 h and thereafter attained a steady state level as studied upto 96 h of post infection. This typc of antigen might have a great potential in immunodiagnostics and site-specific drug targeting.     

Overexpression and increased DNA topoisomerase II-like enzyme activity in arsenite resistant Leishmania donovani

Western immunoblot analyses of whole cell lysates probed with a human specific monoclonal anti-topoisomerase IIalpha antibody identified a 190 kDa protein over expressed in the arsenite resistant Leishmania donovani strain. The crude nuclear extract of the resistant strain showed higher topoisomerase II-like enzyme activity. suggesting a possible regulatory role of putative topoisomerase II in arsenite resistant Leishmania.     

Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran

Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran.     

Role of a differentially expressed cAMP phosphodiesterase in regulating the induction of resistance against oxidative damage in Leishmania donovani

Differentiation-coupled induction of resistance of Leishmania parasites to macrophage oxidative damage was shown to be associated with an increased cAMP response. This study explores the significance of the cAMP response in the parasite by identifying a differentially expressed cAMP phosphodiesterase (LdPDEA) and deciphering its role in regulating antioxidant machineries in the parasite. LdPDEA, a high K_M class I cytosolic cAMP phosphodiesterase, was expressed maximally in log-phase promastigotes, but was significantly reduced in stationary-phase promastigotes and amastigotes. Chemical inhibition or silencing of PDEA conferred enhanced resistance to pro-oxidants in these cells and this led to studies on trypanothione biosynthesis and utilization, as trypanothione is one of the major modulators of antioxidant defense in kinetoplastidae. Despite enhanced arginase and ornithine decarboxylase activity, trypanothione biosynthesis seemed to be unaffected by PDEA blockage, whereas significant elevations in the expression of tryparedoxin peroxidase, ascorbate peroxidase, and tryparedoxin were detected, suggesting a definite shift of trypanothione-pool utilization bias toward antioxidant defense. Moreover, parasites that overexpressed PDEA showed reduced resistance to oxidative damage and reduced infectivity toward activated macrophages. This study reveals the significance of a cAMP phosphodiesterase in the infectivity of Leishmania parasites.     

Leishmania donovani: assessment of leishmanicidal effects of herbal extracts obtained from plants in the visceral leishmaniasis endemic area of Bihar, India

One obstacle faced in the effective control of visceral leishmaniasis (VL) is the limited number of available treatment options. Furthermore, control efforts have been hindered further by the emergence of Leishmania resistance to many of the available drugs. In this study, we investigated the anti-leishmanial properties of 30 medicinally important plants from the VL endemic area of Bihar, India and compared them to two available anti-leishmanial drugs (sodium antimony gluconate and amphotericin B) and two plant lectins (phytohemagglutinin and concanavalin A) on Leishmania donovani promastigotes in vitro at 24 and 48 h after initiation of culture. We identified eight plant extracts in addition to phytohemagglutinin and amphotericin B that significantly inhibited the growth of promastigotes (p < 0.03). We further studied the minimum effective concentrations as well as the effect on axenic amastigotes viability and the cell cytotoxicity on human peripheral blood of four (Agave americana, Azadirachta indica, Eclipta alba and Piper longum) of the eight plant extracts that induced significant promastigotes killing (p = 0.00098). Effect-based dose finding analysis revealed that the threshold concentration of A. americana required to eliminate L. donovani after 24 h was 0.05 mg/ml. A. indica and P. longum plant extracts eliminated L. donovani promastigotes after 48 h at concentrations of 0.1 and 0.5 mg/ml, respectively. E. alba eliminated the promastigotes at a concentration of 0.5 mg/ml within 24 h. The axenic amastigote killing response was 1.90-, 2.52- and 1.3-fold higher than the promastigote killing response with A. indica, A. americana and E. alba plant extracts, respectively. A. americana and A. indica, respectively, led to approximate 2.5- and 1.3-fold declines in mitochondrial dehydrogenase activity compared with control. E. alba stimulation resulted in an up-regulation of dehydrogenase activity (p = 0.00329). The CSA from P. longum was found to be least cytotoxic; the observed difference in mitochondrial activity was insignificant (p = 0.16314). Further studies may reveal the pharmacological significance of many of the plants with anti-leishmanial properties identified in the present study.     

In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

The leishmanicidal effect of crude ethanolic extract of stem bark of Dysoxylum binectariferum and its fractions has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Ethanolic extract was lethal to promastigotes as well as amastigote forms in macrophage system at the concentration of 100 microg/ml. Chloroform fraction significantly inhibited promastigote multiplication and was also active against amastigotes in infected J774A.1 macrophages at 100 microg/ml. Hexane fraction was moderately active and the other fractions were inactive against both the forms. When tested in vivo in hamsters, ethanolic extract was toxic at 500 mg/kg whereas exhibited marginal activity (67.7+/-5.3%) at 250 mg/kg x 5, p.o. on day 7 post treatment (p.t.) which increases slightly (69+/-4.7) by day 30 p.t. Chloroform and n-hexane fractions exhibited 64.3+/-4% and 47.8+/-4.6% parasite inhibition at the dose of 100 mg/kg x 5 p.o., respectively. The pure compound, rohitukine, obtained from chloroform fraction showed weaker in vitro activity and was ineffective in infected hamsters. The lead potential of this plant need further investigations.