Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is an infection encountered in tropical and subtropical regions of the world. Leishmania parasites are propagated by different species of the sandfly vector (genus Phlebotomus or Lutzomya), depending on the region. Old World species of Leishmania, such as L. donovani and L. major, cause pathology from southern Europe to Africa, the Middle East, and throughout southern Asia, whereas New World species (e.g., L. mexicana, L. amazonensis, and L. chagasi) are found throughout South and Central America and as far north as the southern states of the United States. Clinical manifestations differ widely, depending on the Leishmania species. The majority of mortality results from the visceral form of leishmaniasis, caused by L. donovani or L. chagasi; 90% of annual cases are reported in Bangladesh, Brazil, India, Nepal, and Sudan. The most common manifestations are cutaneous lesions; 90% of new cases of cutaneous leishmaniasis occur in Afghanistan, Brazil, Iran, Peru, Saudi Arabia, and Syria, and they are caused principally by L. major and the L. mexicana subgenus (64, 164). The Viannia subgenus is encountered solely in the Americas and is responsible for the clinical form named mucocutaneous leishmaniasis, which is characterized by facial disfigurement. In 2000, it was estimated that more than 12 million individuals were infected by the various Leishmania species in 88 countries, with an estimated 1.5 to 2 million clinical cases (164).
Two distinct developmental stages of Leishmania are recognized. Promastigotes are found within the sandfly and have an elongated shape and long flagellum. Promastigotes can be further classified as procyclic promastigotes, which multiply in the gut of the sandfly, or as the infective metacyclic promastigotes, which are found in the mouth parts and anterior gut and do not divide. These differentiate into round or oval amastigotes, which lack flagella, once in the host.
In its mammalian host, the lifestyle of Leishmania is that of an obligate intracellular pathogen infecting the hematopoietic cells of the monocyte/macrophage lineage, which it enters by phagocytosis. Since this cell type is specialized for the destruction of invading pathogens and priming of the host immune response, Leishmania has had to evolve a range of sophisticated mechanisms to subvert normal macrophage function. These include preventing the activation of deadly antimicrobial agents such as nitric oxide (NO) and also inhibition of many of the cytokine-inducible macrophage functions necessary for the development of an effective immune response. This enables the parasite to evade the innate immune response and to divide within the phagolysosome of the infected macrophage, from where it can spread and propagate the disease within the host. In this review, we focus on the molecular mechanisms whereby Leishmania can subvert host surveillance by altering the macrophage signal transduction machinery, thereby modulating the macrophage environment in its favor. It should be noted that studies generally use only one, or sometimes two, species of Leishmania at a time and a single developmental stage, and this gives the impression that the mechanisms revealed apply to all species. However, given the diversity of pathologies caused by Leishmania spp., it is inevitable that significant differences exist in the mechanisms of host cell manipulation. These differences may account for some of the conflicting results described in this review; our understanding is now at a stage where more studies directly comparing the effects of different species on macrophages would be extremely useful.
Parasite Surface and Secreted Molecules
In addition to being distinguished by morphology and location, the various developmental stages of Leishmania
parasites can be distinguished by their surface molecule composition. Procyclic promastigotes are covered by a 7-nm-thick glycocalyx. The glycocalyx of metacyclic promastigotes is even thicker, at least 17 nm, but it is almost completely absent from amastigotes (123
). This jacket comprises glycoproteins and other glycosylated species, which are anchored to the surface membrane by a distinctive glycosylphosphatidylinositol (GPI) linkage (reviewed in detail in reference 45
). The dominant surface molecule of promastigotes is lipophosphoglycan (LPG). Its structure varies between Leishmania
species, but it is composed principally of repetitive units consisting of a disaccharide and a phosphate, linked to the membrane by a GPI anchor. Leishmania
species differ markedly by the presence of glycan side chains, as well as by their composition and positioning on the LPG core structure. LPG of L. major
, for example, is highly branched, whereas that of L. donovani
is not (102
). Furthermore, the structure of LPG differs between procyclic and metacyclic promastigotes, being significantly longer in the latter, and is almost completely absent from amastigotes (103
). As discussed below, studies using purified LPG or mutant parasite strains defective in LPG production have shown that LPG plays many important roles in parasite survival and modulation of the immune response, and differences in LPG structure and distribution are important for the different properties of the different developmental stages of Leishmania
Another important surface molecule is the glycoprotein gp63 (promastigote surface protease). This is a zinc-dependent metalloprotease with a wide range of substrates, including casein, gelatin, albumin, hemoglobin, and fibrinogen (104). While around 10-fold less abundant than LPG, gp63 is still found throughout the promastigote surface (100, 123). However, its shorter length means that it is essentially buried under a sea of LPG. Like LPG, gp63 is down-regulated in the amastigote form (141). This reduced expression may be counteracted by the absence of LPG on the amastigote surface, meaning that gp63 is no longer masked and may therefore play an important role in amastigote survival and modulation of the host response (102, 123).
The most abundant promastigote surface molecule is glycosylinositol phospholipid (GIPL), a class of GPI-linked glycolipids. These molecules are 10 times more abundant than LPG, but their small size keeps them close to the parasite membrane, so it is unclear what role they play in interaction with the host (45, 101). Unlike LPG, which is continually shed, GIPL has a long half-life and so is believed to play a protective role at the promastigote surface (127).
The completion of the L. major database has revealed putative 65 cysteine peptidases; some of these clearly have roles in promoting parasite survival and disease progression, particularly during L. mexicana infection (for a recent review, see reference 109). At least 50% of the cysteine peptidase activity is localized within the lysosomes of the parasite, so is unlikely to pay a direct role in modulating macrophage signaling (69). However, it is also clear that a significant number of cysteine peptidases are released following amastigote death and as a result of their unusual intracellular trafficking pathway (17, 69). As described below, these released proteases can modulate macrophage activity by acting directly on the host cell surface or following entry into the macrophage endoplasmic reticulum from the phagosome.
This list is far from exhaustive, and it is likely that other surface molecules play important roles in modulating specific aspects of the host immune response. In particular, amastigotes express some poorly characterized, ß-mercaptoethanol-activated metalloproteases (141), and they are even able to incorporate membrane lipids from the host cell (100). Furthermore, other secreted molecules, such as proteophosphoglycans and acid phosphatases, have been directly linked to parasite survival and pathogenicity (89). This review will discuss the role of some of these molecules in subversion of the host defense response.
Initial Interaction and Phagocytosis
Having entered their mammalian host during the blood meal of a female sandfly, Leishmania
promastigotes must first evade complement-mediated lysis until they are engulfed by a macrophage. L. major
procyclic promastigotes cannot resist complement action, whereas the metacyclic form, which is specialized for transmission to the host, can fully avoid complement-driven lysis (128
). This difference in complement resistance has been shown to depend upon branched LPG on the parasite surface. LPG is longer on the surfaces of metacyclic promastigotes and seems to prevent the attachment of C5b-C9 subunits of the complement complex, which are for cellular lysis. L. donovani
promastigotes, however, prevent C5 convertase formation by fixing the inactive C3bi subunit on their surfaces (128
). The surface glycoprotein gp63, a protease, has been reported to protect L. amazonensis
and L. major
against cellular lysis by converting C3b into C3bi, thus favoring parasite opsonization and internalization (16
). It has also been proposed that a surface protein kinase of L. major
may phosphorylate members of the complement system and thereby inactivate the cascade (63
Parasite surface molecules also play an important role during attachment to the macrophage. In vivo opsonization of Leishmania metacyclic promastigotes by C3b and C3bi permits the interaction with the macrophage complement receptor 1 (CR1) and CR3, respectively. However, since C3b is rapidly converted to C3bi by gp63, it appears that CR3 is the more important receptor, and interaction with CR1 is only transient (73). Attachment via CR3 rather than CR1 is advantageous to the parasite, since it will not trigger the oxidative burst during phagocytosis (108). Promastigotes can also attach to the macrophage via the mannose-fucose receptor, which binds to mannan residues of LPG (10). LPG can also interact with C-reactive protein (CRP), an early inflammatory product, and thus triggers phagocytosis via the CRP receptor (31) without leading to the macrophage activation that is usually seen following CRP receptor-mediated phagocytosis (12). Furthermore, gp63 and LPG interact with fibronectin receptor and CR4, respectively (15, 155), although LPG appears to play only a minor role during attachment and internalization (reviewed in reference 38). More recently, a number of Leishmania surface molecules that play a role during the initial Leishmania-macrophage interaction have been identified, although the macrophage receptors for these molecules are not yet clear. For instance, Chiang and Sefton (27) have identified an ICAM-related molecule, ICAM-L, that may be necessary for the interaction between the parasite and the murine macrophage cell line J774. A recent study has reported that the greater infectiousness of metacyclic promastigotes is due in part to elevated surface phosphatidylserine (157), while blocking antibodies against another parasite surface molecule, GIPL, have been shown to inhibit attachment of L. major (154). Amastigotes can also be internalized in an Fc receptor-dependent fashion following opsonization with specific antibodies (57). The large number of receptors implicated suggests a degree of redundancy among parasite-macrophage interactions, although it appears that several interactions are necessary for internalization (15).
Following their attachment to the macrophage, Leishmania promastigotes are internalized to the relatively benign environment of the endosome, where they begin to differentiate into amastigotes. Unlike amastigotes, promastigotes are vulnerable to degradation by the acidic and hydrolytic environment of the phagolysosome. They must therefore retard endosome maturation and phagosome-endosome fusion, a process dependent on LPG (39). This retardation has been observed by the absence or delayed arrival of late endosomal markers such as rab7 and LAMP-1 (142) and may be related to the LPG-dependent accumulation of F-actin (67). The mechanism is not completely understood, but it has been shown to depend upon calcium presence (156) and protein kinase C (PKC) inhibition (66). Furthermore, LPG appears to change the shape of membranes, leading to steric repulsion between the phagosome and the endosome (107). The delay in phagolysosome maturation provides a window during which promastigotes can differentiate into the more resistant amastigotes. This is consistent with our earlier observation that parasite survival and infectiousness in different strains of mice correlate with macrophage phagocytic activity (120). Interestingly, while LPG is important for the survival of promastigotes in the early stages of uptake, it is not expressed by amastigotes, indicating that its role is transient and confined to only the early stages of infection.
Another survival strategy used by Leishmania parasites is the inhibition of the hydrolytic enzymes and other destructive molecules that are secreted into the phagolysosome. Two newly discovered Leishmania molecules, named peroxidoxins LcPxn1 and -2 (4), and a superoxide dismutase (54) are believed to deplete nitrite derivatives and reactive oxygen intermediates (ROI), which are the most important microbicidal small molecules. Furthermore, there is evidence that LPG itself can enhance promastigote survival by neutralizing vacuolar ROI (25). LPG may also protect against lysosomal enzymes, perhaps by its strong negative charge and galactose-mannose repeating units (41). The proteolytic activity of the surface molecule gp63 is optimal at the acidic pH found in phagolysosomes, supporting the suggestion that it targets lysosomal enzymes (143). However, its role is questionable, as parasites with mutations in the six gp63 genes are still capable of survival, differentiation, and replication within macrophages (70).