Leishmania species: mechanisms of complement activation by five strains of promastigotes

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.     

Binding of Leishmania promastigotes to macrophages

Leishmania tropica promastigotes are easily attached to and engulfed by C3H peritoneal macrophages in vitro at 37 degrees C. Different sugars at 0.3-0.5 M inhibited in vitro the attachment of L. tropica promastigotes to C3H peritoneal macrophages with lactose (Gal-beta [1 leads to 4]Glc) being the most efficient. Inhibition of attachment is also affected by pre-treatment of promastigotes with galactose oxidase. Oligosaccharides extending from promastigote and amastigote cell surfaces contain an important proportion of non-reducing galactose as does the carbohydrate-rich factor (EF) excreted by promastigotes of L. tropica and L. donovani. This study suggests that Leishmania, an obligatory intracellular parasite, uses as a means of entering the host cell a cellular mechanism similar to that used in the removal of damaged cells from blood circulation. This mechanism is assumed to take advantage of the exposed sugars, particularly the exposed non-reducing galactose, on the parasite surface during the stage of attachment. Once the parasite is inside the cell, the EF it produces might have a protective function, being inhibitory to some of the host cell lysosomal enzymes.     

Leishmania mexicana mexicana: attachment and uptake of promastigotes to and by macrophages in vitro

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Killing of Leishmania tropica amastigotes by factors in normal human serum

Amastigotes of Leishmania tropica and L. donovani were incubated with fresh or heat-inactivated normal human serum. Viability was estimated by amastigote conversion to promastigote forms and by the ability of serum-treated amastigotes to infect human monocytes. L. tropica, a parasite that causes local skin infection, was killed by fresh but not by heat-inactivated serum. The serum cytotoxic effect on L. tropica was inhibited by EDTA but not by Mg-EGTA. C2-deficient serum killed normally; C6-deficient serum was ineffective. These data indicate that L. tropica is killed by the complement membrane attack complex, in a sequence of reactions initiated by components of the alternate pathway. In contrast, L. donovani, a parasite that causes systemic visceral leishmaniasis, was 10-fold less susceptible to the cytotoxic effects of normal serum. Thus, a profound difference exists in the susceptibility of amastigotes of two species of Leishmania to a defense mechanism present in human serum. Serum complement factors may play an important role in limiting L. tropica to the skin. The resistance of L. donovani to such factors may be the primary reason for its ability to escape from the site of inoculation and cause catastrophic, disseminated disease.     

Leishmania mexicana mexicana: Attachment and Uptake of Promastigotes to and by Macrophages In Vitro1

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and glutaraldehyde sensitive. The promastigote ligand is proteolytic enzyme and glutaraldehyde insensitive. Uptake follows attachment and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages. The first sub-population to be taken up survived better in macrophages over 24 h than subsequently engulfed sub-populations.     

Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation.

Living Schistosoma mansoni schistosomula incubated with normal chicken, guinea pig, human, and monkey sera were killed after 4 hr contact at 37 degrees C. The following data indicate that this action is dependent on the activation of the alternative complement pathway (AP): a) the inactivity of RB, RD, and zymosan-treated serum against schistosomula; b) the partial activity of RD restored in FD; c) the full effect of the C4-deficient guinea pig, C2-deficient human, and the agammaglobulinemic human sera; d) the consumption of both the AP and FB after the incubation of NHS with schistosomula; e) the detection of C3d breakdown product during the contact of the C2-deficient human serum with these young parasites. Killing by serum was decreased as the immature schistosomes developed and was completely absent against 4-day-old lung schistosomula (LS). In other experiments, it was demonstrated that schistosomula, in the presence of IgG, were able to initiate complement activation also through the classical pathway (CP). However, the CP does not appear to play a role in the schistosomulicidal activity of complement. The in vivo relevance of these observations is considered.     

Early mechanisms of Leishmania infection in human blood

In human blood, promastigotes bind natural antibodies and activate the classical complement pathway. C3-opsonized promastigotes immune-adhere within seconds to erythrocytes. Promastigote lysis by complement parallels C3 deposition kinetics, and ~90% of promastigotes are killed after 2.5 min. During infection, complement thus exerts strong selective pressure on Leishmania. Paradoxically, promastigote adaptation to the host immune adherence mechanism may provide the parasite a key to invasion.     

Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and (31)P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.     

The mouse macrophage receptor for C3bi (CR3) is a major mechanism in the phagocytosis of Leishmania promastigotes

We examined the role of the macrophage receptor for C3bi, the CR3, in the phagocytosis of Leishmania major promastigotes and report that M1/70, a monoclonal antibody to the CR3, inhibited the binding of leishmania to macrophages both when the assays were performed in the presence of normal serum and in its absence. In serum, leishmania activate complement and fix C3. Fixation and subsequent cleavage to C3bi occurs rapidly, and by as early as 5 min both forms of the molecule can be identified on the parasites' surface. Complement fixation results in an enhanced phagocytosis of leishmania promastigotes by mouse macrophages. In the case of L. major, 63% of this serum-enhanced binding is inhibitable by M1/70. Binding assays were also performed in the absence of serum with the use of thoroughly washed promastigotes. The addition of M1/70 inhibited binding under these conditions by 54%. Two other rat monoclonal antibodies directed against different antigens on the macrophage plasma membrane did not inhibit binding. M1/70 did not inhibit the binding of promastigotes to rat bone marrow cells, nor did it inhibit IgG-SRBC binding to mouse peritoneal macrophages. These data indicate that the inhibition observed in the presence of M1/70 was specific for the CR3 and that the macrophage receptor for C3bi plays a major role in the phagocytosis of Leishmania major promastigotes, even in the absence of serum.     

Mechanisms of acquired immunity in leishmaniasis

Self-curing cutaneous leishmaniasis depends on T cell-mediated immune activation of infected macrophages. Failure of immune control in inbred mouse models of metastasizing mucocutaneous and visceralizing forms of the disease involves, respectively, insusceptibility of the parasite and the generation of T cells that suppress a potentially curative response. Prophylactic immunization in man has so far been restricted to cutaneous leishmaniasis and based on inducing infection under controlled conditions with virulent Leishmania tropica major promastigotes. The feasibility of immunization against visceral leishmaniasis merits reconsideration. BALB/c mice are genetically vulnerable to L. tropica major, which produces a fatal visceralizing type of disease involving specific suppression of cell-mediated immunity. Potent and lasting protection can be induced by repeated intravenous immunization with irradiated promastigotes. The efficacy of this 'vaccine' is relatively heat-stable (1 h at 56 degrees C). Immunity is not attributable to antibody but to the generation of Lyt-1+2- T cells which, although possessing helper and macrophage-activating functions, do not express classical delayed-type hypersensitivity. The immunological features of this system and its relevance to the possibility of protection against human Leishmania donovani infection are considered.     

The macrophage-attachment glycoprotein gp63 is the predominant C3-acceptor site on Leishmania mexicana promastigotes

The interaction between Leishmania promastigotes and their vertebrate host's complement system results not only in parasite lysis but also, due to surface-bound complement components, in increased macrophage binding potential. In this study we demonstrate, with the use of isolated complement components, that activation is via the alternative complement pathway, initiated by direct deposition of C3 onto the parasite surface. The predominant C3 acceptor site on the promastigotes was initially identified as the glycoprotein gp63 by anti-C3 antibody immunoprecipitation of radioiodinated promastigotes following incubation in the alternative pathway initiators C3, and factors B and D. The C3-binding properties of gp63 were confirmed and quantified, in relation to other surface antigens, by incubating parasites in iodinated C3 and immunoprecipitating bound C3 with antibodies directed against different promastigote surface antigens. The other abundant surface antigen, the glycolipid 'excreted factor', did not show any C3-binding activity. Further demonstration was provided by incubating liposomes containing either gp63 or excreted factor in iodinated C3 and factors B and D. Only gp63-containing liposomes bound C3. Considering that both gp63 and the excreted factor have recently been implicated in attachment and uptake by macrophage, these findings may have considerable bearing in the determination of which of the macrophage surface receptors identify which parasite ligand.     

Complement-mediated serum cytotoxicity for Leishmania major amastigotes: killing by serum deficient in early components of the membrane attack complex

Leishmania major, the agent of Oriental sore, is an obligate intracellular parasite of macrophages in mammalian hosts. Man's immune defense against this organism requires participation of specifically sensitized lymphocytes and activated macrophages. Recent studies, however, have demonstrated that as little as 1/120 concentration of normal human serum is highly cytotoxic for the amastigote form of L. major. Initiation of the lethal process occurs rapidly, requiring only 30 sec of parasite exposure to serum, and is mediated by antibody-independent activation of the alternate complement pathway. The molecular mechanism of cytotoxicity is not known, but may require participation of the membrane attack complex, C5b-9. We investigated this possibility by treating amastigotes with human sera genetically deficient in complement components C5, 6, 7, 8, or 9. We then measured viability of treated parasites by amastigote-promastigote conversion. Our results were quite unexpected: not only did C9-deficient serum kill organisms, but sera singly deficient in each of the preceding components C6 to C8 were also cytotoxic. The degree of cytotoxicity was related both to serum concentration and to the point in the complement cascade at which deficiency occurred. Sera lacking C6 or C7 were less cytotoxic than those deficient in C8, which were less toxic than those deficient in C9. Cytoxicity of deficient sera was abolished by heating serum to 56 degrees C for 30 min. These findings indicate that an incomplete membrane attack complex may mediate cytotoxicity for L. major amastigotes. Moreover, our results raise important questions regarding the mechanism by which the complex is assembled on the surface of a living, unicellular eukaryotic organism.     

Resistance to macrophage-mediated killing as a factor influencing the pathogenesis of chronic cutaneous leishmaniasis

Cutaneous leishmaniasis can be either a spontaneously healing or chronic disease, depending upon the strain of parasite and the immunological status of the host. We have investigated parasite factors responsible for the variable pathogenesis observed in leishmanial infections by testing the sensitivity of several leishmanial strains to intracellular killing in lymphokine (LK) activated mouse macrophages. Significant microbicidal activity against Leishmania tropica, a strain which heals in C57BL/6 (B6) mice, was found. In contrast, a strain (Maria) which has previously been shown to induce chronic nonhealing cutaneous lesions in B6 mice was resistant to killing in activated macrophages. This resistance to killing was observed in macrophages activated by LK obtained from either Bacille Calmette-Guérin-, L. tropica, or the Maria strain infected mice. The inability of LK activated macrophages to kill the Maria strain was shown not to be due to parasite induced inhibition of killing mechanisms, since Maria strain infected, LK treated macrophages exhibited tumoricidial activity similar to uninfected macrophages. Furthermore, LK activated macrophages simultaneously infected with the Maria strain and another intracellular pathogen, Toxoplasma gondii, killed Toxoplasma, but not the Maria strain. Temperature was also found to significantly influence the multiplication and killing of Leishmania parasites. As would be expected from their cutaneous nature, L. tropica and Maria strain parasites multiplied better at 35 degrees C than at 37 degrees C. Also consistent with the failure of cutaneous strains to visceralize in immunocompetent mice was the observation that the killing of leishmanial parasites was enhanced at the higher temperature. Thus, the temperature dependent growth capacity and sensitivity to killing of a given leishmanial strain in macrophages may be important factors influencing the pathogenesis of cutaneous leishmaniasis.     

Leishmania major and Leishmania tropica: I the in vitro effects of an immunomodulator, S2-Complex

This study was undertaken to try to determine the possible anti-leishmanial activity of S2-Complex, an organic complex of copper chloride, ascorbic acid, and nicotinamide. The promastigotes, axenic amastigotes, and intracellular amastigotes of both Leishmania major and Leishmania tropica were incubated with different concentrations of S2-Complex. The EC50 for each form was calculated. Results show that all forms of the parasites were dose dependently inhibited by S2-Complex. The promastigotes of both parasites were the most resistant with highest EC50 followed by axenic amastigotes. While intracellular amastigotes were the most sensitive with the lowest EC50.These results indicate that S2-Complex has a direct anti-leishmanial effect. When mice were treated with S2-Complex or BCG for four days before harvesting the macrophages, and the macrophages infected with both L. major and L. tropica, they showed increased phagocytosis and increased parasite killing. The results of S2-Complex were not statistically different from the immunomodulating agent BCG. These results indicate that S2-Complex has an immunomodulating effect in addition to the direct anti-leishmanial effect.     

Leishmania major parasite stage-dependent host cell invasion and immune evasion

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.     

Leishmania: chemotaxic responses of promastigotes and macrophages in vitro

Promastigotes of Leishmania move progressively up a concentration gradient of: various sugars, specific sugars attracting individual species of Leishmania; serum albumin and another unidentified constituent of serum; hemoglobin; and a factor generated by promastigotes in NNN medium. The movement of promastigotes up a concentration gradient of serum is optimal at a pH of 6.4 to 6.8 and a temperature of 28 degrees C and above. Cholinergic and adrenergic agents did not affect the attraction of serum for promastigotes, and cyclic nucleotides, inflammatory mediators, and macrophage products were not chemotaxic. It is postulated that the sugar chemotaxins influence the movement of promastigotes from the sand fly midgut to the esophagus, and serum chemotaxins may play a part in the entry of promastigotes into the skin of a mammal from the proboscis. Macrophages, the host cell of the obligate intracellular Leishmania species, were not attracted to any product of promastigotes. When, however, promastigotes interact with serum, complement is activated to form C5a which is chemotaxic for macrophages. Activation of complement by promastigotes is, at least partially, by the alternate pathway. Other chemotaxins resulting from promastigote interaction with serum may also be present. Promastigotes may also produce inhibitors of C5a activity.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.     

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.     

The Abl and Arg kinases mediate distinct modes of phagocytosis and are required for maximal Leishmania infection

Leishmania, an obligate intracellular parasite, binds several receptors to trigger engulfment by phagocytes, leading to cutaneous or visceral disease. These receptors include complement receptor 3 (CR3), used by promastigotes, and the Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are not well understood. Here we show that Abl family kinases mediate both phagocytosis and the uptake of Leishmania amazonensis by macrophages (Ms). Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated beads is decreased in Arg-deficient Ms, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Ms, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Ms are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Ms are defective in IgG1-opsonized amastigote uptake. Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous leishmaniasis, and imatinib treatment results in smaller lesions with fewer parasites than in controls. Our studies are the first to demonstrate that efficient phagocytosis and maximal Leishmania infection require Abl family kinases. These results highlight Abl family kinase-mediated signaling pathways as potential therapeutic targets for leishmaniasis.