The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages

The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.     

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.     

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.     

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.     

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.     

Surface acid proteinase (gp63) of Leishmania mexicana. A metalloenzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages

Acid proteinase activity is associated with the major surface glycoprotein (gp63) of both extracellular promastigotes and intracellular amastigotes of the parasitic protozoan, Leishmania mexicana. The enzyme purified by monoclonal affinity chromatography from promastigotes is strongly inhibited by metal ion chelators, which is reversible by the addition of Zn(II). This proteinase loses its activity after dialysis against 1,10-phenanthroline. The apoenzyme thus prepared is reactivated substantially by Zn(II) and partially by Cu(II), Cd(II), Co(II), or Ni(II). From the recently published structure of the gene encoding gp63, we identify hitherto unrecognized sequences, which can be aligned to the consensus zinc-binding sites of other known metalloproteinases. Anti-gp63 polyclonal antibodies, but not the monoclonals, precipitate similar molecules from amastigotes. These molecules differ slightly from gp63 in electrophoretic mobility but have similar endopeptidase activity. Phagolysosomal degradation by macrophages of proteins entrapped in liposomes is prevented by coating them with native gp63. This protection is lost with heat denaturation of gp63 to kill its enzymatic activity. The proteolytic activity of the metalloenzyme on the surface of these parasites may thus protect their membrane from cytolytic damages during their survival, differentiation, and multiplication in the phagolysosomes of macrophages.     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.     

Involvement of<Emphasis Type="Italic">Leishmania donovani</Emphasis> major surface glycoprotein gp63 in promastigote multiplication

The major surface glycoprotein gp63 of the kinetoplastid protozoal parasiteLeishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector inL. donovani promastigotes, gp63-deficient transfectants were obtained. Reduction of the gp63 level resulted in increased generation times, altered cell morphology, accumulation of cells in the G2/M phase of the cell cycle, and increased numbers of binucleate cells with one or two kinetoplasts. Growth was stimulated, and the number of binucleate cells reduced, by addition to the culture of a bacterially expressed fusion protein containing the fibronectin-like SRYD motif and the zinc-binding (metalloprotease) domain of gp63. These observations support an additional role of gp63 in promastigote multiplication; the fibronectin-like properties of gp63 may be important in this process     

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.     

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.     

Host-specific evasion of the alternative complement pathway by schistosomes correlates with the presence of a phospholipase C-sensitive surface molecule resembling human decay accelerating factor.

Schistosoma mansoni parasites recovered from the blood stream were found to be nonactivators of the alternative complement pathway (ACP) when exposed to sera of homologous but not heterologous host species. Schistosomes could be converted into activators of the homologous ACP by treatment with phospholipase C. Antibodies to either human or guinea pig decay accelerating factor (DAF), a 70-kDa glycosylphosphatidylinositol anchored membrane glycoprotein which controls ACP activation on the mammalian cell plasma membrane, bound to the surface of immature schistosomes and immunoprecipitated a molecule of similar molecular mass from detergent extracts of surface iodinated parasites. Phospholipase C treatment drastically reduced the reactivity of the worms with the anti-DAF antibodies. These data suggest that schistosomes evade the ACP by inserting functional host DAF into their surfaces, possibly through adsorption of the molecule's lipophilic diacyglycerol membrane anchor moiety into the outer lipid bilayer of the parasite.     

Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form.

Leishmania sp. protozoa contain an abundant surface protease (gp63) that is important for the virulence of the parasite. We found that the average amount of gp63 expressed by Leishmania donovani chagasi promastigotes increases 6-11-fold as they develop from a less infectious form in logarithmic phase to a highly infectious form during stationary phase of cultivation in vitro. The predominant gp63 RNA switches from a 2.7 to a 3.0 kilobase (kb) RNA during the transition from log to stationary phase. Sequence analysis of gp63 cDNAs reveals that three different classes of gp63 RNAs, containing unique 3'-untranslated regions (3' UTRs), are expressed during growth to stationary phase. The predominant 2.7-(log) and 3.0-kb (stationary) class gp63 RNAs possess nearly identical coding regions, but they diverge in their 3' UTRs. A third class, consisting of 3.1- and 2.6-kb (constitutive) gp63 RNAs, is expressed at low levels throughout cultivation. This latter class encodes a gp63 with an additional 41 amino acids at its C terminus, replacing a potential signal for attachment of a glycolipid membrane anchor with a sequence that could be a transmembrane region. These findings are consistent with the regulated expression of different gp63 genes, resulting in different amounts of gp63 protein, during the promastigote's in vitro development to an infectious form.     

Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice

The Mr 63,000 membrane polypeptide (gp63) is one of the Leishmania receptors for host macrophages and has been shown to protect mice from infection. The gene encoding gp63, the major Mr 63,000 surface glycoprotein of L. major promastigotes, has been expressed as a fusion protein with the enzyme glutathione S- transferase encoded by the parasitic helminth Schistosoma japonicum. This fusion protein was recognized by polyclonal antibodies to the native Leishmania gp63 polypeptide. The insoluble gp63 fusion protein was purified by SDS-PAGE and electroelution and was used to raise antibodies in rabbits. These rabbit anti-gp63 antibodies recognized the fusion protein and the denatured parasite gp63 on immunoblots and by immunofluorescence on fixed promastigotes, but did not recognize the native molecule on live organisms. However, antibodies raised against native promastigote glycoproteins, affinity purified on solid-phase gp63 fusion protein, recognized both native and denatured gp63, suggesting the presence of native determinants in the recombinant protein. The gp63 fusion protein did not protect mice of either healer or nonhealer phenotype from challenge infection with live promatigotes. The implications of these results for the engineering of recombinant DNA-produced molecular vaccines are discussed.     

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement.     

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.     

Human pentraxin 3 binds to the complement regulator c4b-binding protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes

Using both hamster and mouse models of infection, we documented that the virulence of Leishmania donovani chagasi promastigotes decreases over time, when parasites are maintained in long term culture after isolation from an infected animal. Concomitant with this loss of virulence is a marked decrease in amount of the major promastigote surface glycoprotein, gp63, present in promastigotes. The latter was shown by a decrease in binding of polyclonal anti-gp63 serum to attenuated (cultivated long term) as compared to virulent (recently isolated) promastigotes, using immunofluorescence and Western blot assays. Binding of Con A to promastigote glycoproteins, separated by SDS-PAGE, documented a similar decrease. An alteration in the mechanism of promastigote attachment to macrophages was also noted: purified gp63 inhibited attachment of virulent promastigotes to human monocyte-derived macrophages, but it did not affect the attachment of attenuated promastigotes. Northern blot analysis showed that, despite marked differences in the amount of gp63 protein, the quantity of gp63 RNA was comparable in attenuated and virulent promastigotes. However, virulent promastigotes contained two major gp63 RNA species of 3.0 and 2.7 kb, whereas attenuated promastigotes had one predominant gp63 RNA of 2.7 kb and only minor amounts of 3.0 kb RNA. Thus, the decrease in gp63 expression in attenuated, contrasted to virulent, promastigotes is associated with qualitative, but not quantitative, differences in the gp63 messenger RNA.