Early response gene expression during differentiation of cultured Leishmania donovani

The promastigote form of the unicellular parasite, Leishmania donovani, must differentiate into the amastigote form to establish an infection in a mammalian host. Identification of genes whose expression changes during differentiation could help reveal mechanisms of Leishmania gene regulation and identify targets for controlling the diseases caused by this human pathogen. Two genomic clones were isolated, P9 that is more highly expressed in promastigotes than in axenic amastigotes and A14 that is preferentially expressed in axenic amastigotes. Analysis of the DNA sequences revealed open reading frames that would encode 55.5 kDa and 100 kDa proteins, respectively, with no homology to known proteins. The mRNA level for these genes during 24 h time courses of parasite differentiation in culture was compared to two genes known to be differentially expressed, c-lpk2 and mkk. Changes in RNA level occurred within 2 h for each gene and continued in advance of morphological changes. The expression levels of these four genes in axenic amastigotes correlated with results from animal-derived parasites.     

An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans

Glycoconjugates have been shown to play important roles in Leishmania development. However, the ability to study these molecules and other processes would benefit greatly from improved methods for genetic manipulation and analysis of the amastigote stage. This is especially challenging for L. donovani, the agent of the most severe form of leishmaniasis, which can rapidly lose virulence during in vitro culture. Here we report on a clonal subline of an L. donovani 1S2D (LdBob or LdB), which differentiates readily from promastigotes to amastigotes in axenic culture, and maintains this ability during extended parasite cultivation in vitro. This derivative can be plated and transfected efficiently while grown as promastigotes or amastigotes. Importantly, LdB maintains the ability to differentiate while undergoing genetic alterations required for creation of gene knockouts and complemented lines. Like virulent L. donovani, LdB exhibits down-regulation of lipophosphoglycan (LPG) synthesis and up-regulation of A2 protein synthesis in amastigotes. We showed that knockouts of LPG2, encoding a Golgi GDP-mannose transporter, eliminated phosphoglycan synthesis in LdB axenic amastigotes. These and other data suggest that LdB axenic amastigotes will be generally useful as a differentiation model in studies of gene expression, virulence, glycoconjugate function and drug susceptibility in L. donovani.     

A simple and reproducible method to obtain large numbers of axenic amastigotes of different<Emphasis Type="Italic"> Leishmania</Emphasis> species

This work describes a simple method to yield large amounts of Leishmania amastigote-like forms in axenic cultures using promastigotes as the starting population. The method described induced extracellular amastigote transformation of Leishmania amazonensis (97%), Leishmania braziliensis (98%) and Leishmania chagasi (90%). The rounded parasites obtained in axenic cultures were morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as measured by their ability to revert back to promastigotes and to infect BALB/c mice. L. amazonensis and L. braziliensis promastigotes and axenic amastigotes differed in terms of their Western blot profiles. A 46 kDa protein was recognized by specific antibodies only in axenic and lesion-derived L. amazonensis amastigotes and not in promastigotes.     

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.     

Megasome biogenesis in Leishmania amazonensis : a morphometric and cytochemical study

Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process. Received: 18 May 2000 / Accepted: 31 August 2000     

Comparative analysis of megasomes in members of the Leishmania mexicana complex

Megasomes are large lysosome-like structures, previously described in amastigote forms of Leishmania belonging to the mexicana complex, whose major constituents are the cysteine proteinases. Routine observation of thin sections of amastigotes obtained from species of the mexicana complex revealed variations in size and number of megasomes according to the species, and also between amastigotes obtained from axenic cultures and from infected animals. Three-dimensional reconstruction of amastigotes, stereology and immunocytochemical localization of cysteine proteinase revealed significant differences between the three Leishmania species examined, L. amazonensis, L. mexicana and L. pifanoi. The relative volume of megasomes in lesion-derived amastigotes was higher than in axenic amastigotes of L. amazonensis and L. mexicana. The relative volume of megasomes from lesion-derived amastigotes of L. mexicana was 2-3 times higher than in L. amazonensis. Axenic amastigotes of L. pifanoi showed a small relative volume of megasomes and low cysteine proteinase activity, and were not able to produce lesions in the animals, whereas axenic amastigotes of L. mexicana and L. amazonensis did. There were significant differences in the structural organization, distribution within the cell, size and number of megasomes, and in the characteristics of cysteine proteinases found in the amastigotes of the three Leishmania species. These results suggest that these organelles and their constituents may be involved in the infectivity and virulence of Leishmania species.     

Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: the Leishmania genome is constitutively expressed

Leishmania are protozoan parasites that cause a wide spectrum of clinical diseases in humans and are a major public health risk in several countries. Leishmania life cycle consists of an extracellular flagellated promastigote stage within the midgut of a sandfly vector, and a morphological distinct intracellular amastigote stage within macrophages of a mammalian host. This study reports the use of DNA oligonucleotide genome microarrays representing 8160 genes to analyze the mRNA expression profiles of L. major promastigotes and lesion derived amastigotes. Over 94% of the genes were expressed in both life stages. Advanced statistical analysis identified a surprisingly low degree of differential mRNA expression: 1.4% of the total genes in amastigotes and 1.5% in promastigotes. These microarray results demonstrate that the L. major genome is essentially constitutively expressed in both life stages and suggest that Leishmania is constitutively adapted for survival and replication in either the sandfly vector or macrophage host utilizing an appropriate set of genes for each vastly different environment. Quantitative proteomics, using the isotope coded affinity tag (ICAT) technology and mass spectrometry, was used to identify L. infantum promastigote and axenic amastigote differentially expressed proteins. Of the 91 distinct proteins identified, 8% were differentially expressed in the amastigote stage, 20% were differentially expressed in the promastigote stage, and the remaining 72% were considered constitutively expressed. The differential expression was validated by the identification of previously reported stage specific proteins and identified several amastigote and promastigote novel stage specific proteins.     

Roles of free GPIs in amastigotes of Leishmania

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.     

Cell surface characterization of amastigotes of Trypanosoma cruzi obtained from different sources

The present study analyses the morphology and the exposition of surface carbohydrates and the Ssp4 antigen of amastigote forms of Trypanosoma cruzi (Y strain) obtained from three different sources: (a) intracellular, isolated from infected Vero cells 3 days after infection, (b) extracellular, isolated from the supernatant of Vero cells 15 days after infection, and (c) axenic, obtained by incubation of tissue culture trypomastigotes in LIT medium, at 37 °C for 4 days. No morphological differences were observed by light microscopy among these amastigotes. Transmission electron microscopy of thin sections showed a thick cell coat easily observed on the plasma membrane of axenic amastigotes. Carbohydrate-containing sites on the surface of the three different amastigotes were analysed using lectins, agglutination assays and flow cytometry. Mannose and/or glucose residues were found on the surface of all populations, but intracellular amastigotes showed the highest number. A small group of cells from the different populations expressed galactose and N-acetyl-glucosamine residues. The presence and distribution of the Ssp4 antigen in the different amastigote populations were evaluated using FITC and gold-labelled antibodies, and observed with an electronic programmable individual cell sorter and transmission electron microscopy. Ssp4 antigen was present on the membrane lining the flagellar pocket and on the cell surface, as well as inside the cytoplasmic vesicles of the host cell. Flow cytometry analysis of different amastigote populations showed that intracellular amastigotes presented the highest percentage of Ssp4-expressing cells. Received: 6 April 1997 / Accepted: 23 September 1997     

A protein of the leucine-rich repeats (LRRs) superfamily is implicated in antimony resistance in Leishmania infantum amastigotes.

Pentavalent antimonial containing drugs (SbV) are the mainstay for the control of the protozoan parasite Leishmania but resistance to this class of drug is now prevalent in several endemic areas. We describe here the use of functional cloning where an expression cosmid bank derived from Leishmania infantum was transfected in L. infantum axenic amastigotes and selected for potassium antimonyl tartrate (SbIII) resistance. This strategy allowed the isolation of a cosmid encoding for a novel resistance protein, LinJ34.0570, which belongs to the superfamily of leucine-rich repeat (LRR) proteins. Parasites overexpressing this LRR protein, which is part of the LRR_CC subfamily, were resistant to SbIII as axenic amastigotes and to SbV as intracellular parasites. This work pinpoints a novel protein that can contribute to antimonial resistance in Leishmania.     

Biological activity of Morita-Baylis-Hillman adduct homodimers in L. infantum and L. amazonensis: anti-Leishmania activity and cytotoxicity

Parasitology Research - This study is a report on the anti-Leishmania activity of Morita-Baylis-Hillman (MBH) homodimers adducts against the promastigote and axenic amastigote forms of Leishmania...     

Involvement of a Leishmania thymidine kinase in flagellum formation, promastigote shape and growth as well as virulence

Leishmania promastigote cells transmitted by their insect vector get phagocytosed by macrophages and convert into the amastigote form. In a recently performed proteomic study, a thymidine kinase (TK) was found to be preferentially expressed in amastigotes. Western blot analysis showing a marked increase in TK protein synthesis during stage differentiation from promastigotes to amastigotes confirmed this result. After comparison of the amino acid sequence of Leishmania donovani and Leishmania major thymidine kinases with thymidine kinases of other organisms the Leishmania protein has to be classified as a type II TK. Therefore, in accordance with the nomenclature of other thymidine kinases we named the Leishmania enzymes LdTK1 and LmTK1, respectively. The LdTK1 is localised within the cytoplasm of promastigotes. In amastigotes, increased expression and a clustered distribution of the protein can be observed. Lmtk1 single allele gene replacement mutants have significantly elongated flagellum. In contrast, lmtk1 double allele gene replacement mutants show a remarkably reduced flagellar length, diminished overall size and a deformed body shape. In addition, they have a 12-fold reduced growth rate. For both mutant strains, macrophage infectivity is clearly reduced compared to a L. major wildtype infection.     

Expression of Leishmania antigen on the surface membrane of infected human macrophages in vitro.

Resolution of leishmaniasis is associated with host immunological responsiveness to parasite antigens. In clinical disease, leishmania are found as amastigotes contained with macrophages. We investigated the possibility that Leishmania antigens are expressed on the infected macrophage surface by reacting infected macrophages with antibody to Leishmania. In vitro-infected human monocyte-derived macrophages were labelled with antibody to amastigotes when examined with immunofluorescent or immunoelectron microscopic techniques. Infected macrophages were poorly labelled by antibody to promastigotes (insect forms of Leishmania). Certain antisera that reacted with the surface membranes of amastigotes did not label the infected macrophage surface. These results indicate that human macrophages infected in vitro express Leishmania amastigote antigen(s) on their surface membranes, that such antigen(s) may not be present in large quantities in promastigotes, and that certain antigen(s) on the amastigote surface are not expressed on the surface membranes of infected macrophages.     

An assay to quantitate the binding of leishmania amastigotes to macrophages

A leishmania amastigote radiobinding assay has been developed using organisms labeled with tritiated uracil. These labeled amastigotes resemble freshly isolated unlabeled amastigotes in metabolic activity, bouyant density, morphology, viability and their ability to transform into promastigotes. Organisms routinely incorporate between 5 × 10⁻³ and 3 × 10⁻² cpm per amastigote, which allows the detection of as little as 1 × 10⁴ amastigotes per assay well. This radiolabeling technique has been used to quantitate the attachment of amastigotes to macrophages adherent to either 13 mm coverslips or to 96 well plates. It can also be used to screen monoclonal antibodies to macrophage surface proteins involved in amastigote binding. Once incorporated, the label remains amastigote associated, even after intact organisms have been internalized by macrophages. It remains parasite associated until the organisms have been degraded by macrophages, at which time label is released into the supernatant. Thus, a small adaptation of the binding assay can be used to compare the intracellular survival of amastigotes in macrophages following various experimental manipulations. This amastigote radiolabeling assay, therefore, represents an important step toward determining the receptors on macrophages involved in amastigote recognition and can also be used to study the degradation of intracellular pathogens by macrophages.     

Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania

Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg(-1) h(-1)) compared with wild-type cells (4.7 pmol mg(-1) h(-1)). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology.     

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.