Receptor-mediated phagocytosis of Leishmania: implications for intracellular survival

The extracellular promastigote stage of Leishmania spp. is transmitted to mammals by a sand fly vector. Leishmania promastigotes ligate host macrophage receptors, triggering phagocytosis and subsequent internalization, a crucial step for survival. Parasites transform intracellularly to the amastigote stage. Many studies document different receptors detecting promastigotes and amastigotes, but the relative importance of each interaction is ill-defined. Recent studies suggest that the macrophage receptors utilized during phagocytosis impact the intracellular fate of the parasite. This review summarizes the receptors implicated in Leishmania phagocytosis over the past 30 years. It then proceeds to weigh the evidence for or against their potential roles in intracellular parasite trafficking.     

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.     

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.     

Role of the RGD sequence in parasite adhesion to host cells

For many protozoan parasites, one of the first events in the process of infection is attachment to the surface of host cells. This adhesion phase usually involves ligand-receptor interactions, and has stimulated interest in the biochemical characterization of those host cell and parasite surface components involved. In this article, Ali Ouaissi discusses the strategy employed by pathogens such as Trypanosoma cruzi, Trichomonas, Leishmania and Treponema pallidum, in binding to their host cells' fibronectin receptors. Two systems appear available - to bind to the dimeric cell surface fibronectin through the Arginine-Glycine-Aspartic acid (RGD) sequence that is not occupied by the host cell surface receptors, or to present a surface antigen representing a 'fibronectin-like' molecule containing the RGD sequence directly to the host cell fibronectin receptors.     

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.     

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.     

Role of intracellular cAMP in differentiation-coupled induction of resistance against oxidative damage in Leishmania donovani

Even though the human parasite Leishmania donovani encounters tremendous oxidative burst during macrophage invasion, a set of parasites survives and proliferates intracellularly, leading to transformation from promastigote to amastigote form and disease manifestation. The striking shifts in temperature (from 22 degrees C in the insect gut to 37 degrees C in the mammalian host) and pH (7.2 in the insect gut to 5.5 in the parasitophorous vacuole of macrophages) are the key environmental triggers for differentiation as these cause an arrest in the G1 stage of the cell cycle and initiate transformation. Using an established in vitro culture and differentiation system our study demonstrates that the differentiation-triggering environment induces resistance to oxidative damage and consequently enhances infectivity. Differentiation conditions caused a three- to fourfold elevation in cAMP level as well as cAMP-dependent protein kinase activity. Similar to stress exposure, positive modulation of intracellular cAMP resulted in blockage of cell cycle progression and induction of resistance against oxidative damage. Resistance against pro-oxidants from either stress or cAMP may be associated with upregulation of the expression of three major antioxidant genes, peroxidoxin 1, trypanothione reductase, and superoxide dismutase A. Positive modulation of the intracellular cAMP response enables cells to resist the cytotoxic effects of pro-oxidants. In contrast, downregulation of intracellular cAMP by overexpression of cAMP phosphodiesterase A resulted in a decrease in resistance against oxidative damage and reduced infectivity toward activated macrophages. This study for the first time reveals the importance of cAMP response in the life cycle and infectivity of the Leishmania parasite.     

A leucine-rich repeat motif of Leishmania parasite surface antigen 2 binds to macrophages through the complement receptor 3

Membrane glycoconjugates on the Leishmania parasites, notably leishmanolysin and lipophosphoglycan, have been implicated in attachment and invasion of host macrophages. However, the function of parasite surface Ag 2 (PSA-2) and membrane proteophosphoglycan (PPG) has not been elucidated. In this study we demonstrate that native and recombinant Leishmania infantum PSA-2, which consists predominantly of 15 leucine-rich repeats (LRR) and a recombinant LRR domain derived from L. major PPG, bind to macrophages. The interaction is restricted to macrophages and appears to be calcium independent. We have investigated the PSA-2-macrophage interaction to identify the host receptor involved in binding and we show that binding of PSA-2 to macrophages can be blocked by Abs to the complement receptor 3 (CR3, Mac-1). Data derived from mouse macrophage studies were further confirmed using cell lines expressing human CR3, and showed that PSA-2 also binds to the human receptor. This is the first demonstration of a functional role for PSA-2. Our data indicate that in addition to leishmanolysin and lipophosphoglycan, parasite attachment and invasion of macrophages involve a third ligand comprising the LRRs shared by PSA-2 and PPG and that these interactions occur via the CR3.     

Role of hexosamine biosynthesis in Leishmania growth and virulence

Leishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphosphatidylinositol (GPI) glycolipids and N-linked glycans. To investigate whether these glycoconjugates are required for infectivity of promastigote and intracellular amastigote stages, we generated a Leishmania major mutant lacking the gene encoding glutamine : fructose-6-phosphate amidotransferase (GFAT). The L. major Δ gfat mutant is unable to synthesize GlcN-6-phosphate de novo and is auxotrophic for GlcN or GlcNAc. GlcN starvation leads to the rapid depletion of dolichol-linked oligosaccharides and GPI precursors, hypersensitivity to elevated temperatures encountered in the mammalian host and eventual parasite death. Short-term tunicamycin treatment induces a similar hypersensitivity to temperature, indicating that N-linked glycans are required for thermotolerance and viability. L. major Δ gfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GlcN(Ac) levels in the phagolysosome are low. In contrast, Δ gfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GlcN(Ac) for viability but can apparently scavenge all of their glucosamine requirements from the macrophage phagolysosome. These results highlight significant differences in the nutrient requirements of promastigote and amastigote stages and suggest that enzymes involved in UDP-GlcNAc biosynthesis are essential for pathogenesis in the mammalian host.     

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.     

Developmentally regulated sphingolipid degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.     

Isolation of infective promastigotes of Leishmania major from long-term culture by cocultivation with macrophage cell line.

Research on Leishmania-macrophage interaction is mainly focused on the impact of the parasite on macrophages and several known virulent factors have been described. Furthermore, studies on macrophage revealed several defense mechanisms including various cytokines which are released by macrophages to defend against parasite. In the present study, a new aspect of this interaction was evaluated: parasite characteristics, which emerge when they were cocultivated with macrophage. Two promastigote characteristics, survival at high temperature (32 degrees C) and infectivity rate were the focus of this study. In this study, an in vitro coculture model for promastigotes with macrophage cell line, J774 A1, was introduced using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane. After 5-7 days of coculturing at 32 degrees C, a few promastigotes survived longer than control group. Once this population of parasite was cultured at optimal temperature (26 degrees C), the emerged new clone was much more infective for J774 A1 cell line in comparison with the original one. Having this system and using the new clone of promastigotes, parasite infectivity rate was raised from 1-2% of original clone to 35-45%. Using this new introduced technique, infective promastigotes were isolated from 9 month old frequently sub-cultured clone of Leishmania major. This coculturing system allows investigators to prepare infective promastigotes from the frequently cultured parasites. Molecular and biochemical mechanisms of this phenomenon need to be investigated.     

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.     

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     

A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.

At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.     

Leishmania cell surface prohibitin: role in host–parasite interaction

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95–100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host–parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild‐type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI‐link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti‐prohibitin antibodies during macrophage–Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania–host interaction.     

Evidence that intracellular beta1-2 mannan is a virulence factor in Leishmania parasites

The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.     

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 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 Leishmania-macrophage interaction: a metabolic perspective

Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophages although they have the capacity to infect a variety of other phagocytic and non-phagocytic mammalian cells. Unlike most other intramacrophage pathogens, the major proliferative stage of Leishmania resides in the mature phagolysosomes of these host cells. In this review we highlight some of the strategies utilized by the intracellular amastigote stage of Leishmania to survive in this compartment. Remarkably, and in contrast to many other intracellular pathogens, Leishmania amastigotes have a minimalist surface glycocalyx which may facilitate uptake of essential lipids and promote exposure of phospholipids required for phagocytosis via macrophage apoptotic cell receptors. Leishmania amastigotes also differ from many other intracellular pathogens in having complex nutritional requirements which must be scavenged from the host cell. Amino acids and polyamines appear to be important carbon sources and growth-limiting nutrients, respectively, and their availability to intracellular amastigotes may be regulated by the activation state of host macrophages. Metabolic processes in both the parasite and host cell may thus be crucial determinants of disease outcome.