Characterization of the receptor for insulin-like growth factor on Leishmania promastigotes

Insulin-like growth factor (IGF)-I constitutively present in the skin is one of the first growth factors that Leishmania parasites encounter after transmission to the vertebrate host. We have previously shown that IGF-I is a potent growth-promoting factor for Leishmania parasites. IGF-I binds specifically to a single-site putative receptor at the parasite membrane, triggering a cascade of phosphorylation reactions. In the present article we characterize the receptor for IGF-I on Leishmania (Leishmania) mexicana promastigotes. The receptor is a monomeric glycoprotein with a molecular mass of 65 kDa and is antigenically related to the alpha chain of human type 1 IGF-I receptor. Upon IGF-I stimulation the receptor undergoes autophosphorylation on tyrosine residues with activation of its signaling pathway. Activation of the IGF-I receptor also leads to phosphorylation of an 185-kDa molecule that is homologous to the substrate of the insulin receptor present in human cells, the insulin receptor substrate 1 (IRS-1).     

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.     

Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.     

Extracellular cultivation and morphological characterization of amastigote-like forms of Leishmania panamensis and L. braziliensis

Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32 degrees C for L. panamensis (WR442; L. braziliensis panamensis) and at 28 degrees C for L. braziliensis (M5052; L. braziliensis braziliensis). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

I. Extracellular Cultivation and Morphological Characterization of Amastigote-Like Forms of Leishmania panamensis and L. braziliensis

. Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32°C for L. panamensis (WR442; L. braziliensis panamensis ) and at 28°C for L. braziliensis (M5052; L. braziliensis braziliensis ). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

Temperature-induced expression of proteins in Leishmania mexicana amazonensis. A 22-kDa protein is possibly localized in the mitochondrion

Temperature increase is an integral part of Leishmania life cycle, and plays a major role in stage transformation. Analysis of the temperature-dependent pattern of protein synthesis on two-dimensional gel electrophoresis shows that, in addition to the conserved heat-shock type of response in which expression of the major 70-kDa and 83-kDa heat-shock proteins is observed, a group of low-molecular-mass (17-40 kDa) proteins is induced in promastigotes of Leishmania mexicana amazonensis at elevated temperatures. Immuno-gold labelling with antibodies raised against the heat-induced 22-kDa proteins was localized mainly in the mitochondrion of Leishmania parasites, though labelling was observed also in the nucleus. The correlation of this finding with various reports on induction of mitochondrial enzymes in response to temperature stress in other organisms is discussed.     

Metacyclogenesis of Leishmania spp: species-specific in vitro transformation, complement resistance, and cell surface carbohydrate and protein profiles

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.     

Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular Amastigote-Like Forms of Leishmania panamensis and L. braziliensis. II. Stage- and Species-Specific Monoclonal Antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania . Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to >200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Leishmania: overexpression and comparative structural analysis of the stage-regulated meta 1 gene.

The meta 1 gene of Leishmania major is upregulated in metacyclic promastigotes and encodes an 11.5-kDa protein with no significant similarities to other proteins in the existing databases. In this paper, we characterize the homologous meta 1 genes in L. amazonensis and L. donovani. Proteins encoded by this gene in all three species present a high degree of identity. The meta 1 gene cannot be replaced by gene targeting in L. major, suggesting an essential role for the protein, at least in promastigotes. Overexpression of the meta 1 protein in L. amazonensis generates parasites that are more virulent than wild-type organisms in vivo.     

Identification and isolation of the Leishmania transferrin receptor.

In a previous report, we have presented several lines of evidence, derived from widely different methodologies, suggesting that Leishmania has specific receptors for transferrin with a Kd similar to the mammalian transferrin receptor. This paper describes the identification, purification, and biochemical characterization of Leishmania transferrin receptor. The Leishmania transferrin receptor, detected on intact parasites by immunoperoxidase staining, was first identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using 125I-transferrin, as a 70-kDa protein. It has been isolated initially from Leishmania infantum promastigotes using affinity chromatography on a transferrin-Sepharose column and, subsequently, from Leishmania major promastigotes. The use of polyclonal antisera to the purified 70-kDa Leishmania transferrin receptor and to the purified rat transferrin receptor showed that the two receptors are antigenically distinct. The 70-kDa Leishmania transferrin receptor was subsequently characterized as an integral membrane glycoprotein. The monomeric state of the Leishmania transferrin receptor was demonstrated by gel filtration of purified receptor complexed with 125I-transferrin. Thus, the Leishmania transferrin receptor, unlike the mammalian receptor, is not a disulfide-linked dimer but a single 70-kDa polypeptide.     

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.     

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses.

To investigate human immune responses to defined leishmania Ag we have begun to characterize biochemically and immunologically, an abundant 42-kDa surface Ag of Leishmania amazonensis, a causative agent of human leishmaniasis. We have shown that this Ag, La gp42, is expressed on the surface of L. amazonensis promastigotes, being anchored to the membrane by a glycosyl-phosphatidylinositol moiety. As demonstrated by lectin blotting studies, La gp42 is glycosylated, binding both Con A and wheat germ agglutinin. Immunologically, La gp42 is strongly recognized by sera from patients with different forms of leishmaniasis as well as by patients with Chagas' disease. In addition, we show that purified La gp42 stimulates the proliferation of human T lymphocytes obtained from several leishmaniasis patients. Finally, the N-terminal sequence of La gp42 was obtained and a serologically cross-reactive 42-kDa protein with a homologous sequence was identified in Leishmania major.     

Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of (125)I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.     

Characterisation of a new Leishmania META gene and genomic analysis of the META cluster

The META1 gene of Leishmania is upregulated in metacyclic promastigotes and encodes a 12 kDa virulence-related protein, conserved in all Leishmania species analysed. In this study, the genomic region adjacent to the Leishmania amazonensis META1 gene was characterised and compared to the Leishmania major META1 locus as well as to syntenic loci identified in Trypanosoma brucei and Trypanosoma cruzi. Three new genes expressed with increased abundance of steady state mRNA in L. amazonensis promastigotes were identified, two of which are upregulated in stationary phase promastigotes, sharing the pattern of expression previously described for the META1 mRNA. One of these new genes, named META2, encodes a polypeptide of 444 amino acid residues with a repetitive structure showing three repeats of the META domain (defined as a small domain family found in the Leishmania META1 protein and in bacterial proteins hypothetically secreted and/or implicated in motility) and a carboxyl-terminal region similar to several putative calpain-like proteins of Trypanosoma and Leishmania.