Monoclonal antibodies that recognize distinct epitopes of the macrophage type three complement receptor differ in their ability to inhibit binding of Leishmania promastigotes harvested at different phases of their growth cycle.

The macrophage receptor CR3 has been shown by several investigators to be involved in the binding of Leishmania promastigotes to host macrophages. This receptor is known to recognize iC3b and to mediate direct lectin-like attachment of particles such as yeast zymosan. In the present study, two anti-CR3 monoclonal antibodies, M1/70 and 5C6, which ligate different epitopes of murine CR3, have been used in conjunction with sodium salicyl hydroxamate (Saha; inhibits covalent ester linkages of C3 to an activator surface) to block binding of L. donovani and L. major promastigotes harvested at different phases of their growth cycle. M1/70 inhibited all promastigote binding. 5C6 and Saha blocked in parallel only the binding of peanut agglutinin (PNA)-positive late log and early stationary phase parasites. These results suggest that the binding PNA-positive parasites to CR3 is iC3b-mediated, while entry of the more infective PNA-negative late stationary phase promastigotes into host macrophages may involve direct lectin-like binding to CR3.     

Monoclonal antibodies recognizing determinants specific for the promastigote stage of Leishmania mexicana

Two monoclonal antibodies (IX-IF9-D8 and IX-5H9-CI) produced to a membrane enriched fraction of Leishmania mexicana amazonensis promastigotes have been demonstrated to be specific for the promastigote (insect) form and not the amastigote (mammalian host) form of the parasite. The antigens recognized by these monoclonal antibodies are not found on amastigotes isolated from infected animals or on amastigotes isolated from a macrophage cell line J774 infected initially with promastigotes. The antigens are not re-expressed by amastigotes cultured at 34°C; however, amastigotes cultured at 24°C that have begun transformation into promastigotes do express these antigens. The level of expression of these antigens in cultures of amastigotes undergoing transformation into promastigotes, increases with time from 16 to 36 h and appears to correlate with the percentage of promastigotes. Two protein molecules with apparent molecular weights of 40 000 and 92 000 have been identified by radioimmune precipitation as associated with L. mexicana promastigote stage specific determinants.     

Transbilayer translocation of membrane phosphatidylserine and its role in macrophage invasion in Leishmania promastigotes

Infectivity of Leishmania promastigotes has been shown to be growth cycle-dependent and restricted to the stationary phase. By using annexin V-FITC binding and procoagulant activity measurement assays, we show here that the promastigotes in the stationary phase contain significantly higher amounts of phosphatidylserine (PS) on their surface as compared to the log phase promastigotes. We also demonstrate that the infectivity of the promastigotes is determined by the presence of PS on their surface. In addition, by using NBD-labelled phospholipids, we show that the promastigote plasma membrane contains ATP-dependent out-to-in and ATP-independent in-to-out PS translocases which regulate the PS localisation in two-halves of the membrane bilayer, and that the greater amounts of external PS observed in the stationary phase promastigotes is perhaps due to the slower ATP-dependent out-to-in PS movements in these cells, as compared to the log phase promastigotes.     

Differential microbicidal effects of human histone proteins H2A and H2B on Leishmania promastigotes and amastigotes

Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense against extracellular bacteria, fungi, and Leishmania promastigotes. In this study, we used human recombinant histone proteins to further study their leishmaniacidal effects and the underlying mechanisms. We found that the histones H2A and H2B (but not H1(0)) could directly and efficiently kill promastigotes of Leishmania amazonensis, L. major, L. braziliensis, and L. mexicana in a treatment dose-dependent manner. Scanning electron microscopy revealed surface disruption of histone-treated promastigotes. More importantly, the preexposure of promastigotes to histone proteins markedly decreased the infectivity of promastigotes to murine macrophages (Mφs) in vitro. However, axenic and lesion-derived amastigotes of L. amazonensis and L. mexicana were relatively resistant to histone treatment, which correlated with the low levels of intracellular H2A in treated amastigotes. To understand the mechanisms underlying these differential responses, we investigated the role of promastigote surface molecules in histone-mediated killing. Compared with the corresponding controls, transgenic L. amazonensis promastigotes expressing lower levels of surface gp63 proteins were more susceptible to histone H2A, while L. major and L. mexicana promastigotes with targeted deletion of the lipophosphoglycan 2 (lpg2) gene (but not the lpg1 gene) were more resistant to histone H2A. We discuss the influence of promastigote major surface molecules in the leishmaniacidal effect of histone proteins. This study provides new information on host innate immunity to different developmental stages of Leishmania parasites.     

Identification of the promastigote surface protease in seven species of Leishmania

Twelve different strains of Leishmania, including L. major, L. donovani, L. infantum, L. tropica, L. mexicana, L. amazonensis, L. braziliensis, and L. enriettii were examined for the presence of an ectoenzyme structurally and functionally related to the promastigote surface protease found in L. major LEM 513. All strains examined possess a protease that is labelled by surface iodination of living promastigotes. The electrophoretic migrations of the labelled proteases are similar in all species showing distinct ectoprotease activity. In addition, proteases that cross-react immunologically with the polypeptide moiety of the surface protease of L. major LEM 513 were found in 10 strains. These proteases were in all cases labelled by surface radioiodination. Two of the strains, L. amazonensis and L. braziliensis, do not show a strict correlation between protease activity, surface iodination, and immunological cross-reactivity with the promastigote surface protease of L. major LEM 513, although both strains possess distinct neutral proteases with electrophoretic behavior similar to that of the enzyme of L. major. The amount of proteolytic activity detected at the surface of living cells depends on the strain tested, and correlates qualitatively with the amount of promastigote surface protease detected on zymograms. We conclude that the proteolytic activity found at the surface of Leishmania promastigotes is a common feature of the species infective for humans and that the promastigote surface protease described in this article is structurally and functionally conserved in Old and New World Leishmania.     

Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis

Significant differences were found in the ability of resident mouse peritoneal macrophages to ingest amastigote and promastigote forms of Leishmania mexicana amazonensis. Differences in the association index of the parasites to the macrophages were also found between infective and non-infective promastigotes. Evidence was obtained suggesting that the macrophage receptor, which recognizes mannose-6-phosphate-containing units found in lysosomal enzymes, is involved in the association with the macrophage of promastigotes, but not of amastigotes. Addition of mannose-6-phosphate, its structural analogue fructose-1-phosphate, Hansenula holstii phosphomannan or the mannose-6-phosphate-containing lysosomal enzyme -D-mannosidase to the interaction medium, markedly inhibits the association of the parasites with macrophages.     

Expression of a stage-specific lipophosphoglycan in Leishmania major amastigotes.

Amastigotes of Leishmania major were isolated from infected mice and radiolabeled for 2 h with [3H]galactose. An acidic [3H]glycoconjugate was extracted from a dilipidated residue fraction with the solvent water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactivity labeled glycoconjugate was found to possess the following characteristics that were similar to the lipophosphoglycan extractable from promastigotes: (i) migrated as a broad band upon electrophoresis on SDS polyacrylamide gels; (ii) deaminated with nitrous acid; and (iii) hydrolyzed with phosphatidylinositol-specific phospholipase C. Furthermore, analysis of the aqueous soluble material released by the latter enzyme revealed a negatively-charged [3H]polysaccharide intermediate in size compared to the analogous portions of LPG isolated from non-infective and metacyclic promastigotes. Most importantly, the [3H]polysaccharide was found to contain phosphate and was susceptible to mild acid hydrolysis, establishing that the intact molecule is a lipophosphoglycan. A structural difference, however, was found in the major, mild acid-generated fragment of the amastigote phosphoglycan, which was larger in size and not as anionic as the analogous fragment from the promastigote phosphoglycans. These results indicate that the amastigotes do express a lipophosphoglycan, but that it is structurally distinct from its promastigote counterparts.     

Changes in the proteome and infectivity of Leishmania infantum induced by in vitro exposure to a nitric oxide donor

Leishmania species are protozoan parasites that exhibit an intracellular amastigote form within mammalian macrophages and an extracellular promastigote form inside the sandfly vector. The generation of nitric oxide (NO) upon activation of macrophages is surely the principal killing effector of intracellular amastigotes but little is known about the potential action of NO against the promastigote phase during its multiplication inside the digestive tract of the sandfly vector. Therefore, we have approached this issue by using an in vitro model to study the effect of an NO donor, 3-morpholinosydnonimine (SIN-1), on the proteome and infectivity of promastigotes of Leishmania infantum. Exposure of promastigotes to SIN-1 during its logarithmic growth phase caused a dramatic effect on parasite protein expression and viability, consequently killing about 60-70% of the promastigotes. The significant changes in the proteome included the over-expression of enolase, peroxidoxin precursors, and heat-shock protein 70 (HSP70), under-expression of 20S proteasome alpha 5 unit, and phosphomannomutase and induced expression of 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) synthase and prostaglandine f2-alpha (PGD2) synthase. Interestingly, promastigotes that resisted treatment showed enhanced infectivity to J774 macrophages in comparison to the controls. This finding together with the appearance of the PGD2S and an over-expression of HSP70 isoforms in treated promastigotes led us to speculate the existence of NO-mediated programmed cell death (PCD) events as a potential mechanism of population regulation and selection of properly infecting forms that predominantly operate on the promastigote stage.     

Internal and surface subpopulations of the major surface protease (MSP) of Leishmania chagasi

Major surface protease (MSP) facilitates Leishmania promastigote evasion of complement-mediated lysis in the mammalian host and enhances host macrophage phagocytosis of the promastigotes. We previously showed that the steady-state abundance of MSP protein increases 14-fold during in vitro cultivation of L. chagasi promastigotes from logarithmic to stationary phase, despite the fact that the total amount of MSP mRNA does not increase. Furthermore, 10 major MSP isoforms are differentially expressed in different promastigote growth phases, and attenuation of parasites by long-term in vitro cultivation influences MSP isoform expression. Herein, we report that although about two-thirds of newly synthesized MSP becomes surface localized, the rest of the MSP does not reach the promastigote surface. This internal MSP is stable without detectable decrease in abundance up to 6 days after biosynthesis. Furthermore, surface-localized MSP is released at different rates from logarithmic and stationary phase virulent Leishmania promastigotes. These data are consistent with the hypothesis that the major mechanism regulating MSP abundance is the rate of loss of surface-localized MSP from the promastigote surface, and that internally localized MSP is very stable.     

Strain-specific recognition of live Leishmania donovani promastigotes by homologous antiserum raised against a crude membrane fraction of infected macrophages

Surface antigens on Leishmania promastigotes and infected macrophages are obvious targets in immunoprophylaxis for leishmanial infection. We have recently demonstrated that the polyclonal antiserum and monoclonal antibodies generated by homologous immunizations with the crude membranes of parasite-infected cells react effectively with the `neo-antigenic' determinants on the infected cell surface. In the present study, we investigated the utility of such polyclonal antisera for identifying `minor' surface components of promastigotes. The reactivity of anti-Leishmania donovani-(strain RMRI68) infected macrophage membrane (anti-IMm) antiserum was compared with that of anti-promastigote (anti-Pr) antiserum towards the infected macrophage surface and promastigotes of three Indian strains of L. donovani, RMRI68, AG83 and DD8. While anti-Pr antiserum showed no reactivity with the infected macrophage surface but reacted strongly with air dried and live promastigotes of all three strains, anti-IMm antiserum reacted with the infected cell surface and, interestingly, specifically recognized live promastigotes of the strain used for infection, i.e., strain RMRI68. The reactivity patterns of the two antisera with the immunodominant components of the L. donovani promastigote surface, i.e., purified LPG-KMP11 complex and gp63 molecules, indicated that unlike anti-Pr antiserum, the specificities in anti-IMm antiserum were mainly directed towards molecules other than the LPG-KMP11 complex and gp63. Antiserum generated in a similar fashion against the macrophage membrane of cells infected in vitro with strain AG83 also contained antibodies specific to strain AG83 promastigotes. The present approach may therefore greatly help in identifying specific antigen(s) important in clinical and epidemiological control of leishmaniasis. Received: 17 April 1998 / Accepted: 15 June 1998     

Effect of immunoglobulin M from normal human serum on Leishmania donovani promastigote agglutination, complement-mediated killing, and phagocytosis by human monocytes.

Serum from healthy, nonimmune humans contained immunoglobulin M (IgM) antibodies that agglutinated Leishmania donovani promastigotes, activated complement, and enhanced promastigote ingestion by human monocytes. The findings indicate that IgM antibodies have the capacity to affect the initial interaction of L. donovani promastigotes with human host defenses.     

The influence of temperature and serum deprivation on the synthesis of heat-shock proteins and alpha and beta tubulin in promastigotes of Leishmania major

We have examined changes in the relative synthesis of individual proteins in promastigotes of Leishmania major subjected to decreasing serum levels in vitro. We observed increases in the relative synthesis of the putative heat-shock proteins of 82 and 70 kDa and of proteins of 79 and 41 kDa but decreases in the synthesis of proteins of 38 and 28 kDa. The relative synthesis of alpha-tubulin increased, whereas that of beta-tubulin decreased, in promastigotes subjected to decreased serum concentrations. This uncoordinated regulation of the synthesis of the tubulin proteins was not reflected as an alteration in the relative levels of the messenger RNA of the respective proteins. We have also studied changes in the synthesis of proteins in L. major promastigotes subjected to a temperature change from 26 degrees C to 34 degrees C. The results indicate that the synthesis of putative heat-shock proteins of 82, 70, 65, 41, 23 and 22 kDa increased when the parasites were incubated at the higher temperature, although these proteins were synthesised in detectable amounts at 26 degrees C. We could not detect differences between infective and non-infective promastigotes, separated by binding to peanut agglutinin, in the synthesis of individual proteins in response to increased temperature. These results were confirmed by densitometer analysis of autoradiographs of labelled promastigote proteins, and the relative changes in the synthesis of the two major heat-shock proteins, as well as alpha- and beta-tubulin, were estimated.     

Differences in expression and exposure of promastigote and amastigote membrane molecules in Leishmania tropica.

The accessibility of particular Leishmania tropica promastigote (extracellular) and amastigote (intracellular) membrane molecules might be related to the relative abilities of the two stages to induce host immune responses. To examine the exposure of membrane antigens on resident macrophage-susceptible promastigotes and resident macrophage-resistant amastigotes, both stages were analyzed by polyacrylamide gel electrophoresis and immunoblotting after specific labeling and extraction procedures. Protein compositional studies, using metabolic labeling of promastigotes and amastigotes, demonstrated that both forms possessed numerous endogenously synthesized proteins. In addition, a marked difference was revealed in the external exposure of promastigote and amastigote membrane constituents when analyzed by 125I surface labeling or Western blot analysis. Whereas nine promastigote proteins were intensely to moderately iodinated, only one amastigote membrane component was similarly labeled (9.5K band). Western blot analyses with serum from a rabbit immunized with a mixture of both L. tropica stages indicated that the majority of promastigote molecules accessible to 125I may also react with immune serum. However, Western blots of extracted amastigotes identified several bands not seen on radiographs and thus not accessible to 125I. The external exposure of these amastigote molecules was confirmed in that immune serum adsorbed with viable, intact amastigotes was no longer reactive with amastigote extracts. Further, by Western blot analyses of sodium dodecyl sulfate- but not Nonidet P-40-extracted amastigotes, three amastigote-specific membrane antigens not previously observed with nonionic detergent extraction methods were identified. The autofluorographic pattern of amastigotes intrinsically labeled with N-[3H]acetylglucosamine, an amino sugar which is incorporated into membrane carbohydrates, was in excellent agreement with the pattern of antigens reactive with antibody in Western blots. Thus, with these cell surface labeling and extraction methods, promastigote and amastigote membranes were shown to be significantly different. Amastigotes possessed several membrane-associated molecules, but few appeared to be either accessible or reactive with 125I. Moreover, the majority of molecules not reactive with 125I, but reactive with antibodies, may be glycosylated. These observations are discussed relative to the ability of amastigotes both to survive within the degradative milieu of macrophage phagolysosomes and to evade host immune reactivity.     

The immunochemical structure and surface arrangement of Leishmania donovani lipophosphoglycan determined using monoclonal antibodies

Using intact Leishmania donovani promastigotes or purified L. donovani lipophosphoglycan (LPG) as immunogens, we have derived four LPG-specific monoclonal antibodies (MAbs). Two of these MAbs recognize an epitope consisting of the repeating phosphorylated galactose beta-1,4-mannose disaccharide portion of the molecule and cross-reacted with LPG from Leishmania major. These MAbs bound to the surface of living promastigotes of both species. The two other MAbs bound to the phosphosaccharide core structure of LPG and did not bind to the surface of living parasites, presumably due to masking of the core region. Experiments using all four MAbs with an LPG-deficient promastigote mutant indicated that both the repeat epitope and phosphosaccharide core were present in these cells, suggesting that incomplete assembly was responsible for the absence of intact LPG.     

Modulation of phagolysosome biogenesis by the lipophosphoglycan of Leishmania

Promastigotes of the protozoan parasite Leishmania are inoculated into the mammalian host by an infected sandfly and are phagocytosed by macrophages. There, they differentiate into amastigotes, which replicate in phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs), plays an important role in the ability of promastigotes to survive the potentially microbicidal consequences of phagocytosis. Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, has received considerable attention over the past several years. Of interest for this review, lipophosphoglycan confers upon Leishmania donovani promastigotes the ability to inhibit phagolysosome biogenesis. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-harboring phagosomes from interacting with late endosomes and lysosomes. Thus, similar to several other pathogens, Leishmania promastigotes hijack the host cell's cytoskeleton early during the infection process. Here, we review this phenomenon and discuss the potential underlying mechanisms.     

The major surface protein of Leishmania promastigotes is a fibronectin-like molecule

The major surface glycoprotein of Leishmania chagasi promastigotes showed crossreactivity with fibronectin (Fn), a large glycoprotein that is a major constituent of the extracellular matrix of most mononuclear cells. Polyclonal and monoclonal antibodies against Fn precipitated two molecules of 63-58 kDa from the lysates of both 125I and [35S]methionine-labeled promastigotes. In addition, a monoclonal antibody against a 15-kDa fragment of Fn containing the Arg-Gly-Asp-Ser (RGDS) sequence and several polyclonal monospecific mouse antibodies against a synthetic RGDS peptide also recognized the above two molecules. The attachment of Leishmania promastigotes to mouse peritoneal macrophages in vitro was partially inhibited when promastigotes were treated with F(ab')2 fragment of an anti-Fn IgG. Identical results were obtained by saturating the Fn receptors on macrophages using different peptides containing the RGDS sequence. Moreover, antigen preparations rich in glycoprotein 63 could efficiently promote the attachment and spreading of 3T3 mouse fibroblasts to surfaces coated with the antigen. These results clearly suggest that the gp63 of L. chagasi promastigotes is an Fn-like molecule that shares certain biological and molecular characteristics with Fn.     

Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by 125I labeling and immunoprecipitation.

Externally oriented surface membrane constituents of promastigotes from several Leishmania species were radiolabeled with 125I. Autoradiographs of cell surface-labeled and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the stocks revealed distinctive patterns of bands in the molecular weight range of 6,000 to 240,000. Immunoprecipitation of detergent extracts of the labeled promastigote stocks with anti-Leishmania donovani membrane serum demonstrated that each of the stocks contained some antigenically cross-reactive determinants. The electrophoretic patterns of these determinants serve both to distinguish the parasite stocks (by unique, species-specific patterns) and to indicate antigenic similarities in stocks thought to be different by other biochemical criteria. At least 12 cross-reactive cell surface antigens in two New World leishmanias are recognized by polyvalent anti-L. donovani serum, suggesting that these common leishmanial antigens may account for the documented serological cross-reactivities among various Leishmania species. In all stocks tested, an iodinated protein was identified which had a relative molecular weight of 65,000 under reducing conditions but which demonstrated an increase in relative mobility in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Distinctive patterns of the antigens common to the several stocks were also demonstrated with the use of monoclonal antibodies.     

Large-scale production of amastigotes by a human monoblastoid cell line

In this study, a human monoblastoid cell line (TPH-1) was tested in vitro for the production of Leishmania amastigotes. The number of TPH1 cells increased with time and 6 days after promastigote infection the percentage of infected cells was around 45%. Pre-treatment of TPH1 cells with retinoic acid induced the cells to differentiate into unreplicating macrophage-like cells. Ninety per cent was parasitized 6 days after promastigote infection; the number of amastigotes quintuplied during this period of time; this result was irrespective of the Leishmania species used for experiments. Viable and infective parasites were obtained from treated and nontreated cells. TPH1 cells merit further consideration for research concerning new molecules active against Leishmania.