Etiological Agent Of Visceral Leishmaniasis Research Articles

Genome-wide gene expression profile induced by exposure to cadmium acetate in Leishmania infantum promastigotes.

Leishmania infantum is the etiological agent of visceral leishmaniasis in Mediterranean areas. The life cycle of the protist is dimorphic and heteroxene, as promastigotes develop inside the gut of sand-fly vectors and amastigotes multiply inside mammalian phagocytic cells. In previous studies, we analyzed the expression profiles of these stages and the modulation of gene expression triggered by temperature increase and acidification, both of which are crucial in the differentiation of promastigotes to amastigotes. Differential expression profiles of translation initiation and elongation factors were detected. Here we report that the presence of 1 mM cadmium acetate in the culture medium leads to a shock response consisting of growth arrest, morphological changes, the absence of motility, and the up-regulation of genes that code for: a heavy metal transporter, trypanothione reductase, a haloacid-dehalogenase-like hydrolase, and a metalloexopeptidase from the M20 family, among others. This response is probably controlled by the differential expression of regulatory genes such as those encoding initiation factors 4E, eukaryotic translation initiation factor 3 subunits 8 and 2α, and elongation factor 1β. The initiation factor 2α gene is induced in anomalous environments, i.e., those outside of the protist's normal life-cycle progression, for example, in response to the presence of cadmium ions, acidification without temperature increase, and vice versa. Our results suggest that the regulation of gene expression is a key component of the shock response.

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.

Leishmania donovaniAmastigotes Impair Gamma Interferon-Induced STAT1α Nuclear Translocation by Blocking the Interaction between STAT1α and Importin-α5

The protozoan parasite Leishmania donovani, the etiological agent of visceral leishmaniasis, is renowned for its capacity to sabotage macrophage functions and signaling pathways stimulated by activators such as gamma interferon (IFN-gamma). Our knowledge of the strategies utilized by L. donovani to impair macrophage responsiveness to IFN-gamma remains fragmentary. In the present study, we investigated the impact of an infection by the amastigote stage of L. donovani on IFN-gamma responses and signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in mouse bone marrow-derived macrophages. The levels of IFN-gamma-induced expression of major histocompatibility complex class II and inducible nitric oxide synthase (iNOS) were strongly reduced in L. donovani amastigote-infected macrophages. As the expression of those genes is mediated by the transcription factors STAT1alpha and IFN regulatory factor 1 (IRF-1), we investigated their activation in amastigote-infected macrophages treated with IFN-gamma. We found that whereas STAT1alpha protein levels and the levels of phosphorylation on Tyr701 and Ser727 were normal, IRF-1 expression was inhibited in infected macrophages. This inhibition of IRF-1 expression correlated with a defective nuclear translocation of STAT1alpha, and further analyses revealed that the IFN-gamma-induced STAT1alpha association with the nuclear transport adaptor importin-alpha5 was compromised in L. donovani amastigote-infected macrophages. Taken together, our results provide evidence for a novel mechanism used by L. donovani amastigotes to interfere with IFN-gamma-activated macrophage functions and provide a better understanding of the strategies deployed by this parasite to ensure its intracellular survival.

Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host

Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Deltaodc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Deltaodc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Deltaodc knockout lines were decreased approximately 80% compared to their wild-type counterpart. Furthermore, alpha-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Deltaodc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Deltaodc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.

Comparison of molecular markers for strain typing of Leishmania infantum

The epidemiology of Leishmania infantum, the etiological agent of visceral leishmaniasis, is changing rapidly; hence powerful typing tools are required in order to monitor the parasite populations spreading and to adapt adequate control measures. We compared here the resolving power of four molecular methods at the zymodeme level: PCR-RFLP analysis of kDNA minicircles ( kDNAPCR-RFLP) and antigen genes (cysteine proteinase b and major surface protease, cpb- and gp63PCR-RFLP), multilocus microsatellite typing (MLMT) and random amplification of polymorphic DNA (RAPD) were applied to samples of 25 L. infantum MON-1 strains obtained from different hosts (HIV+ patients, HIV− patients and dogs) coming from three Spanish foci: Madrid, Mallorca and Ibiza. While RAPD was not sufficiently resolving, the other three methods allowed genotyping within the zymodeme. KDNAPCR-RFLP and MLMT were the most discriminatory and appeared the most adequate for strain fingerprinting. In an eco-geographical context, cpbPCR-RFLP, MLMT and kDNAPCR-RFLP were all informative: they showed here a similar picture, with the existence of cluster(s) of isolates from the islands and other one(s) of mixed composition (Madrid and the islands). None of the markers revealed an association with the host type or the clinical form. In general, there was a significant correlation between each pair of distances calculated from the cpb, microsatellite and kDNA data, respectively, but visual inspection of the trees revealed a better congruence between cpb and microsatellite trees. The methods used here are complementary and each adapted to answer specific epidemiological questions. Their choice should be the result of a compromise between the required resolving power, the genetic features of the respective markers and the technical aspects.

Interplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex

Kinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules. Peaking at 15 min, the parasite-induced leakage was drastically enhanced by captopril (Cap), an inhibitor of angiotensin-converting enzyme (ACE), a kinin-degrading metallopeptidase. The enhanced microvascular responses were cancelled by HOE-140, an antagonist of the B 2 bradykinin receptor (B 2R), or by pre-treatment of promastigotes with the irreversible cysteine proteinase inhibitor N-methylpiperazine-urea-Phe-homoPhe-vinylsulfone-benzene (N-Pip-hF-VSPh). In agreement with the above-mentioned data, the promastigotes vigorously induced edema in the paw of Cap-treated J129 mice, but not Cap-B 2R –/ – mice. Analysis of parasite-induced breakdown of high molecular weight kininogens (HK), combined with active site-affinity-labeling with biotin-N-Pip-hF-VSPh, identified 35–40 kDa proteins as kinin-releasing cysteine peptidases. We then checked if macrophage infectivity was influenced by interplay between these kinin-releasing parasite proteases, kininogens, and kinin-degrading peptidases (i.e. ACE). Our studies revealed that full-fledged B 2R engagement resulted in vigorous increase of L. chagasi uptake by resident macrophages. Evidence that inflammatory macrophages treated with HOE-140 became highly susceptible to amastigote outgrowth, assessed 72 h after initial macrophage interaction, further suggests that the kinin/B 2R activation pathway may critically modulate inflammation and innate immunity in visceral leishmaniasis.

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.

SLC11A1 (formerly NRAMP1) and susceptibility to visceral leishmaniasis in The Sudan.

Genetic susceptibility to visceral leishmaniasis (VL) is indicated by differences in incidence and clinical phenotypes between ethnic groups in Sudan. In mice, innate susceptibility to Leishmania donovani, the etiological agent of VL, is controlled by Slc11a1 (formerly Nramp1). We therefore examined polymorphisms at SLC11A1 in 59 multicase families of VL from the high-incidence Masalit tribe in Sudan. Multipoint nonparametric analysis in ALLEGRO shows a significant linkage across SLC11A1 (Zlr scores 2.38-2.55; 0.008< or =P< or =0.012; information content 0.88). The extended transmission disequilibrium test shows biased transmission of alleles at 5' polymorphisms in the promoter (P=0.0145), exon 3 (P=0.0037) and intron 4 (P=0.0049), and haplotypes formed by them (P=0.0089), but not for 3' polymorphisms at exon 15 or the 3'UTR. Stepwise logistic regression analysis using a case/pseudo-control data set derived from the 59 families was consistent with main effects contributed by the intron 4 469+14G/C polymorphism. Although the two alleles for 469+14G/C lie on haplotypes carrying different alleles for the functional promoter GTn polymorphism, the latter did not itself contribute separate main effects. Sequence analysis of 36 individuals failed to identify new putative functional polymorphisms in the coding region, intron 1, intron/exon boundaries, intron 4/exon 4a, or in the 3'UTR. One novel promoter polymorphism (-86G/A) was located within a putative nuclear factor kappa B binding site that could be functional. Further work will determine whether additional polymorphisms occur upstream in the promoter, which could be in linkage disequilibrium with the intron 4 polymorphism. These studies contribute to knowledge of the role of SLC11A1 in infectious disease.

Emergence of zoonotic canine leishmaniasis in the United States: isolation and immunohistochemical detection of Leishmania infantum from foxhounds from Virginia.

Previously considered an exotic disease, canine leishmaniasis caused by Leishmania infantum has recently been detected within the foxhound population in the United States and parts of Canada. Leishmania infantum is the etiologic agent of visceral leishmaniasis in many areas of the world and dogs are considered a major reservoir host for human Leishmania infections. Human visceral leishmaniasis has recently emerged as an opportunistic infection among individuals co-infected with HIV/AIDS and in persons taking immunosuppressive drugs. We report the isolation of L. infantum from 3 naturally infected foxhounds from Virginia by culture of popliteal lymph node and bone marrow, and the development of an immunohistochemical test to detect the parasite in tissues.

Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme.

Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.

Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean–Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.

Leishmania donovani-infected macrophages: characterization of the parasitophorous vacuole and potential role of this organelle in antigen presentation.

Leishmania donovani amastigotes, the etiological agents of visceral leishmaniasis, are obligate intracellular parasites residing in membrane-bound compartments of macrophages called parasitophorous vacuoles (PV). The study of these organelles is of paramount importance to understanding how these parasites resist the microbicidal mechanisms of macrophages and how they escape the immune response of their hosts. Confocal microscopy of mouse bone marrow-derived macrophages infected with L. donovani amastigotes and stained for various prelysosomal/lysosomal markers and for major histocompatibility complex (MHC) molecules was used to define PV with respect to the endocytic compartments of the host cells and to address the issue of their potential role in antigen processing and presentation. Forty-eight hours after infection, many PV contained cathepsins B, D, H and L and they were all surrounded by a membrane enriched for the lysosomal glycoprotein lgp120/lamp 1 but apparently devoid of the cation-independent mannose 6-phosphate receptor, a membrane protein generally absent from the lysosomes. These data suggested that PV acquire within 48 hours the characteristics of a lysosomal compartment. However, both macrosialin and the GTP-binding protein rab7p (specific markers of the prelysosomal compartment) were found to be highly expressed in/on PV membrane. Thus, at this stage, PV appear to exhibit both lysosomal and prelysosomal features. Infected macrophages activated with IFN-gamma before or after infection showed PV strongly stained for MHC class II molecules but not for MHC class I molecules. This suggests that, if infected macrophages can act as antigen-presenting cells for class I-restricted CD8+ T lymphocytes, Leishmania antigens must exit the PV. MHC class II molecules reached the PV progressively, indicating that they were not plasma membrane-bound molecules trapped during internalization of the parasites. The redistribution of class II observed in infected cells did not alter their quantitative expression on the plasma membrane at least during the first 48 hours following the phagocytosis of the parasites. The invariant chains, which are transiently associated with class II molecules during their intracellular transport and which mask their peptide-binding sites, did not reach PV or were rapidly degraded in these sites, suggesting that PV-associated class II are able to bind peptides. This last assumption is strengthened by the fact that class II located in PV could bind conformational antibodies that preferentially recognize class II with tightly associated peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetoplastid Flagellates: Surface-Reactive Carbohydrates Detected by Fluorescein-Conjugated Lectins

Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL.