Fibronectin shedding by Leishmania may influence the parasite-macrophage interaction.

Fibronectin (FN) is a large extracellular matrix protein involved in the endocytosis of several types of particles by different phagocytes. Here we investigated the role of FN in the entry and destruction of Leishmania amazonensis promastigotes (flagellated form) by murine resident peritoneal macrophages. We also studied the lateral mobility of this protein on the surface of the parasite cells using a immunogold technique. We compared the effects of addition and depletion of FN on infective and non-infective populations of Leishmania promastigotes. The invasion by the latter but not by the former, was increased by FN, and the uptake of these cells was more sensitive to FN depletion from the culture medium. We also observed enhanced killing of intracellular infective promastigotes upon FN addition to the macrophage cultures. Immunocytochemical localization of FN on the surface of the flagellates revealed that the parasite cells released bound FN by membrane shedding in a constitutive fashion. Therefore we conclude that FN removal by shedding may be part of a physiological mechanism by which the parasites evade intracellular destruction by host cells.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model

Homogenous metacyclic promastigotes of Leishmania chagasi were isolated by buoyant density from in vitro heterogeneous cultures and used for biochemical characterization of isoforms of the major surface protease (MSP). Compared to stationary phase promastigotes, metacyclic cells had three times more MSP, produced 3-fold higher parasite loads in a mouse model in vivo, and were more resistant to complement-mediated lysis in vitro. These metacyclic L. chagasi expressed both the virulence-associated 59-kDa, and the constitutively expressed 63-kDa, isoforms of MSP.     

The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania

Infection of dendritic cells by the human protozoal parasite Leishmania is part of its survival strategy. The dendritic cell receptors for Leishmania have not been established and might differ in their interactions among Leishmania species and infective stages. We present evidence that the surface C-type lectin DC-SIGN (CD 209) is a receptor for promastigote and amastigote infective stages from both visceral (Leishmania infantum) and New World cutaneous (Leishmania pifanoi) Leishmania species, but not for Leishmania major metacyclic promastigotes, an Old World species causing cutaneous leishmaniasis. Leishmania binding to DC-SIGN was found to be independent of lipophosphoglycan, the major glycoconjugate of the promastigote plasma membrane. Our findings emphasize the relevance of DC-SIGN in Leishmania-dendritic cell interactions, an essential link between innate and Leishmania-specific adaptive immune responses, and suggest that DC-SIGN might be a therapeutic target for both visceral and cutaneous leishmaniasis     

Binding and release of C3 from Leishmania donovani promastigotes during incubation in normal human serum

We have examined the nature and extent of C3 deposition on Leishmania donovani, strain 1S, clone 2D, promastigotes. Total molecules of C3 bound/parasite after 60 min was similar for parasites incubated in normal human serum, normal human serum adsorbed to remove natural antibody, or either serum source chelated with Mg-EGTA to limit activation to the alternative pathway. A comparison of parasites grown to early, mid, late-log or stationary phases revealed no difference in the extent and kinetics of C3 binding. C3 bound covalently to the parasite primarily through a hydroxylamine resistant (putatively amide) linkage. Of the bound C3, 75% was present as hemolytically inactive iC3b. Nearly 50% of the bound C3 was spontaneously released within 30 min at 37 degrees C. This spontaneous release was due to an unusual proteolytic cleavage event that released C3 from the C3 acceptor on the parasite surface. These results define and characterize the unusual features of C3 binding to L. donovani promastigotes during incubation in serum.     

Leishmania infection: surfaces and immunity

Infections with Leishmania parasites are initiated by bites from infected sandflies; the injected promastigotes are attacked by phagocytic cells but succeed in entering cells of the macrophage family and surviving in them. The secrets of the success of the extracellular form in penetrating the host cell and of the intracellular form in surviving in a potentially hostile environment are yet to be unraveled. The infectivity of the extracellular promastigote is related to the expression on its surface of molecules that interact with the surface of the host cell. One of these molecules is the promastigote surface protease, or gp63, which is also a dominant surface antigen; this enzyme is thought to be involved in binding to the macrophage via the cell receptors for mannose and fucose and for the third component of complement. Another important surface component is the lipophosphoglycan, consisting of a series of phosphorylated disaccharides linked to a novel lipid anchor in the membrane. This is also released from the parasite surface and was earlier identified as a highly immunogenic antigen excreted into culture medium. It can activate complement and may in this way promote attachment of the parasite to the macrophage. Other surface structures include the acid phosphatase, a glyco-inositol phospholipid, another glycolipid, and membrane proteins of 80 and 17 kilodaltons. All of these may play a role in attachment of the promastigote to the macrophage host cell, as well as in the survival of the amastigote within the macrophage, perhaps by inhibiting the activities of destructive enzymes. The roles in infectivity of these components of the Leishmania surfaces and their interactions with the various receptors on macrophages are discussed. The immune responses induced by these and other parasite antigens during infections in humans and experimental animals are also described briefly, especially those responses that may contribute to protection from infection, or to diagnosis and epidemiology.     

Leishmania (Viannia) braziliensis: human mast cell line activation induced by logarithmic and stationary promastigote derived-lysates

Herein we investigate the ability of live promastigotes and total lysate of Leishmania (Viannia) braziliensis, derived from parasites in the logarithmic (L-Lb) or stationary phase (S-Lb), to induce human mast cell line (HMC-1) activation. In comparison with medium-treated cells, a significant histamine release was observed in HMC-1 cultures stimulated with S-Lb. Lipophosphoglycan also induced histamine release by HMC-1 cells. In immunocytochemical assays, we found a marked staining for tryptase in medium-treated HMC-1 cells, however, stimulation with L-Lb or S-Lb caused a marked decrease in the color reaction as well as in the number of tryptase-positive cells. L-Lb and S-Lb induced an evident decrease in the intracellular expression of IL-4 but not IL-12. Live stationary promastigotes were able to induce high levels of IL-4 release in HMC-1 cultures. Furthermore, these cells released significant amounts of IL-12 when incubated with both types of live promastigotes. These results indicate that L. (V.) braziliensis promastigotes differ in their ability to induce direct human mast cells activation, according to the growth phase of the parasite. Furthermore, the release of pro-inflammatory mediators and cytokines could represent an important phenomenon that might favor the initial establishment of the infection.     

Production of nitric oxide by murine macrophages induced by lipophosphoglycan of Leishmania major

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is the surface glycoconjugate lipophosphoglycan (LPG). In addition, LPG is shown to be useful as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we made attempts to characterize functions of the culture supernatant, and membrane LPG isolated from metacyclic promastigotes of Leishmania major. The purification scheme included anion-exchange chromatography, hydrophobic interaction chromatography and cold methanol precipitation. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by SDS-PAGE and thin layer chromatography. The effect of mLPG and sLPG on nitric oxide (NO) production by murine macrophages cell line (J774.1A) was studied. Both sLPG and mLPG induced NO production in a dose dependent manner but sLPG induced significantly higher amount of NO than mLPG. Our results show that sLPG is able to promote NO production by murine macrophages.     

The major surface protein of Leishmania promastigotes is a protease

The major surface protein of Leishmania promastigotes is evolutionarily conserved and is found in isolates of L. donovani, L. major, L. tropica, L. mexicana, and L. braziliensis. The data provided in this communication demonstrate that in L. major this integral membrane protein is a protease, which we now designate promastigote surface protease. The enzyme has an alkaline pH optimum and is active both in its detergent-solubilized form and at the surface of living or fixed promastigotes. A water-soluble form of promastigote surface protease is obtained following digestion with the phospholipase C responsible for the release of the variant surface glycoprotein of Trypanosoma brucei. Possible biological functions of promastigote surface protease during the life cycle of Leishmania parasites are discussed.     

Immunochemical characterization of a glyco-inositol-phospholipid membrane antigen of Leishmania major

A low m.w. polymorphic glyco-inositol-phospholipid (GIPL) of Leishmania major was studied by using three different mAb. This molecule is shown to be distinct from the previously described lipophosphoglycan of L. major in its m.w., antigenic properties, expression during parasite growth, and kinetics of synthesis and catabolism. GIPL is shown to be released from the parasite surface in a water-soluble form, probably by an endogenous phospholipase. GIPL is also detectable on the surface of infected macrophages, although not all epitopes are detectable in this state. GIPL can be metabolically labeled with [3H]galactose, [3H]inositol, [32P]phosphate, and [3H]palmitic acid. GIPL can also be labeled on the surface of living promastigotes with galactose oxidase and [3H]sodium borohydride. The kinetics of synthesis and catabolism are much faster than those of lipophosphoglycan. GIPL is sensitive to degradation upon parasite lysis and becomes undetectable by mAb after 20 h at 37 degrees C. The expression of GIPL on the surface of promastigotes is more abundant during the logarithmic phase of growth, and declines in stationary phase.     

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

Effect of sand fly saliva on Leishmania uptake by murine macrophages

Leishmania promastigotes are introduced into the skin by blood-sucking phlebotomine sand flies. In the vertebrate host, promastigotes invade macrophages, transform into amastigotes and multiply intracellularly. Sand fly saliva was shown to enhance the development of cutaneous leishmaniasis lesions by inhibiting some immune functions of the host macrophages. This study demonstrates that sand fly saliva promotes parasite survival and proliferation. First, macrophages gravitated towards increasing concentrations of sand fly saliva in vitro. Secondly, saliva increased the percentage of macrophages that became infected with Leishmania promastigotes and exacerbated the parasite load in these cells. Thus, during natural transmission, saliva probably reduces the exposure of promastigotes to the immune system by attracting macrophages to the parasite inoculation site and by accelerating the entry of promastigotes into macrophages. Saliva may also enhance lesion development by shortening the generation time of dividing intracellular amastigotes.     

Differential effects of polyamine derivative compounds against Leishmania infantum promastigotes and axenic amastigotes

The natural polyamines are ubiquitous polycationic compounds that play important biological functions in cell growth and differentiation. In the case of protozoan species that are causative agents of important human diseases such as Leishmaniasis, an exogenous supply of polyamines supports parasite proliferation. In the present study, we have investigated the effect of three polyamine derivatives, (namely bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd) and spermine (BNIPSpm)), on the proliferative stages of Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean basin. A significant reduction of promastigotes and axenic amastigotes growth was observed in the presence of increasing concentrations of the drugs, although the mechanisms leading to the parasite growth arrest seems to be different. Indeed, by using a number of biochemical approaches to analyse the alterations that occurred during early stages of parasite-drug interaction (i.e. membrane phosphatidylserine exposure measured by annexin V binding, DNA fragmentation, deoxynucleotidyltranferase-mediated dUTP end labelin (TUNEL), mitochondrial transmembrane potential loss), we showed that the drugs had the capacity to induce the death of promastigotes by a mechanism that shares many features with metazoan apoptosis. Surprisingly, the amastigotes did not behave in a similar way to promastigotes. The drug inhibitory effect on amastigotes growth and the absence of propidium iodide labelling may suggest that the compounds are acting as cytostatic substances. Although, the mechanisms of action of these compounds have yet to be elucidated, the above data show for the first time that polyamine derivatives may act differentially on the Leishmania parasite stages. Further chemical modifications are needed to make the polyamine derivatives as well as other analogues able to target the amastigote stage of the parasite.     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies

Several (glyco)(sphingo)lipids from different human pathogens have been characterized, and frequently many of these molecules are participating in host-pathogen interaction. In Leishmania (Leishmania) amazonensis, for example, amastigotes present on their surface glycosphingolipids (GSLs) with the structure Galbeta1-3Galalpha, which is recognized by 30 kDa receptor of macrophages. Furthermore, other Leishmania species, such as Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis present glycosylinositolphospholipids (GIPLs) which are involved in Leishmania-macrophage interaction. It is worth to mention that these antigens are not expressed in mammalian cells. Leishmania promastigotes also present inositol phosphorylceramide (IPC), a unique sphingolipid characteristic of fungi and plants. It was observed that IPC synthesis is essential for parasite division, since Aureobasidin A, an inhibitor of IPC synthase, inhibited significantly promastigote and amastigote growths. Recently, it was also demonstrated that GIPLs, IPC and sterols are preferentially present in the parasite membrane microdomains resistant to Triton X-100 at 4 degrees C. The disruption of these microdomains by incubating parasites with methyl-beta-cyclodextrin inhibited significantly macrophage infectivity by Leishmania. Other pathogens, such as fungi, also present unique glycolipids which may have an important role for the fungal development and/or disease establishment. Taking together these results, this review will discuss different biological roles for (glyco)(sphingo)lipids of different pathogens.     

Molecular identification of a malaria merozoite surface sheddase

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface "sheddase," but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite.     

Plasma membrane and cytoskeletal constituents inLeishmania mexicana

The presence and the localization of actin, spectrin and ankyrin are studied by immunofluorescence and immunoblotting inLeishmania mexicana promastigotes growing in vitro.These proteins, amphitropic in nature, coexist both in soluble and insoluble forms. Our results demonstrate that the Triton insoluble form of these proteins constitutes beside tubulin the cytoskeletal scaffold of promastigotes in close association with the plasma membrane, the axoneme and the basal body of the parasite.     

Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages

Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. We have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study we have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here we show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, we show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.