Molecular determinants of Leishmania virulence.

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Annu. Rev. Microbiol. 1990. 44:499---529 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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MOLECULAR DETERMINANTS OF LEISIlMANIA VIRULENCE Kwang-Poo Chang and Gautam Chaudhuri Department of Microbiology/Immunology, University of Health Sciences/Chicago Medical School, North Chicago, Illinois 60064

Dunne Fong Department of Biological Sciences, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855 KEY WORDS:

protozoan, parasites, pathogenicity, ectoenzymes, glycoconjugates

INTRODUCIION .... . . ........ . . . . .......... . . ............... . . . . . .... . .... . . . . . . . . . . . ...... . . . . . . . . Leishmania and L eishmanias is . ....... . . ... ........... . . . . . . . . ......... . .. . . . . . . . . . . .... . . . . . . . Parasite, Vector, and Animal Models with Special Reference to Virulence . .... .. . . .

499

HYPOTHESES AND IMPLICATIONS . . . . . . . ... . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . ....

501

Re�eptor-�,fedi.ated E�?cytosis of Leishmania. . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . LeIshmanIa DifferentiatIOn................ ............... ..... ........... . ..... .. . . . ....... . . .. . Intracellular Survival of Leishmania..........................................................

502

.

500 500

INTRACELLULAR PARASITISM AND LEISHMANIA VIRULENCE: FACTS,

MOLECULES OF POTENTIAL IMPORTANCE IN LEISHMANIAL VIRULENCE .. ........ . ...... . . . . . ... ........... . . . .... . .. ...... . . . ........... . .. . . .. . . ..

Surface Lipophosphoglycan (LPG) . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. .. . ... . . . . . . . . . . . .... . . . . . Surface Glycoproteins-Ectoenzymes .... . . . . ................ . . . . . . ....... . . . . . .... . . .. . . . . . . . . Cysteine Proteinases and Megasomes . ....... ..... . ... . .. ............... .. . . ......... ... ..... Heat-Shock Proteins.............................................................................. Other Stage-Specific Molecules . . . . .. ...... ...... .... . ...... . . .. .... . . ....... . . . . . . . ...... . . . . . .

.

504 506 507 507 509 515 516 517

REGULATION OF LEISHMANIA VIRULENCE.. . . . . . ......... . .. . ....... . . . .. . . . .... . .. . . . . .

517

CONCLUSION

519

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .

INTRODUCTION Host-parasite interactions determine the outcome of all microbial infections. Crucial are the genotypes and phenotypes of the hosts as well as the parasites. The latter elaborate multifarious virulence factors to break down the innate barrier of the host and to compromise or mislead its subsequent immune 499

0066-4227/90/1001-0499$02.00

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CHANG ET AL


response. This tenet of infectious diseases is true for all pathogens including parasitic protozoa such as Leishmania. This review introduces the unique features of these parasites and highlights recent advances at the cellular and molecular level. Basic research of Leishmania in the fields of biochemistry (48), cell biology (2, 27, 1 77), immunology (89, 90, 1 5 1 ), and molecular biology (7) was recently reviewed. Leishmania

and Leishmaniasis

Members of the genus Leishmania are a biologically diverse group of trypano somatid flagellates. Some species are nonpathogenic to man and parasitize lower vertebrates, e.g. lizards (49). Most species pathogenic to both humans and other animals can be differentiated genetically, biochemically, and im munologically ( 1 1,54, 83, 1 74). All species are transmitted by the phleboto mine sandflies, wherein they live extracellularly in the alimentary tract and assume different morphologic forms , usually as motile promastigotes ( 1 06). When delivered into the mammalian host by the vector, the pathogenic species infect macrophages, differentiate into nonmotile amastigotes and multiply intracellularly as such (27, 122). This intracellular parasitism con tinues in a chronic course locally or spreads to mucocutaneous tissues or visceral organs. Thus, leishmaniasis can be cutaneous, mucocutaneous, or visceral, often depending on the species involved. These diseases are mainly zoonotic. Their distribution is world-wide , limited by the distribution of the vector in warm climates. Incidences of leishmaniases have increased, es pecially in the developing world because of the lack of vaccines, difficulty of vector control, and reliance on chemotherapy with antiquated drugs such as antimonials.

Parasite, Vector, and Animal Models with Special Reference to Virulence The last decade or so has been marked by an increasing interest in Leishmania species and steady progress toward a better understanding of these parasites. These advances have been facilitated through the work of many investigators to refine the parasite systems for laboratory studies. Most species can now be adapted to grow readily as promastigotes in defined or semidefined com mercially available liquid media and on solid agar plates as colonies; animal models are available for infection experiments and for the isolation of amasti gotes (29). The latter can also be grown in vitro for short-term assays or long-term culture in macrophages and macrophage lines, respectively (30). The cutaneous species of South American origin become amastigote-like at elevated temperature (see 29) and can be grown continuously as "axenic amastigotes" in macrophage-free conditions ( 1 20). In addition, several spe cies of sandflies have been successfully reared as the vectors for experimental


LEISHMANIA VIRULENCE

501

transmission of leishmaniases ( 106, 168). The availability of these parasite cultures, animal models, and vector systems has made possible the study of all aspects of Leishmania in natural infections under laboratory conditions. The question of Leishmania virulence has been addressed by using spon taneously generated or specifically prepared variants of different infectivity. Leishmania virulence varies among clones (58, 77). Also well known is that amastigotes are usually more infective than promastigotes, and that the latter at the stationary phase of in vitro growth are more infective than those at early growth phases. Regardless of the in vitro growth phases, promastigotes tend to lose infec:tivity after prolonged cultivation in vitro. The change of virulence seen with growth phases in vitro parallels the development of promatigotes into infective forms in the sandfly for at least one Leishmania species ( 1 46). These infective and noninfective cells have been compared in attempts to identify vindence factors. In addition, variants of altered virulence have been selected, with or without prior mutagenization, by using animal infection (50, 95) or for rl�sistance to specific agents, e.g. ricin (79) and tunicamycin (80). The specifkity of the selective agents allows the prediction of target mole cules for biochemical analysis relevant to Leishmania virulence. INTRACELLULAR PARASITISM AS A BASIS OF VIRULENCE: FACTS, HYPOTHESES, AND IMPLICATIONS

LEISHMANIA

The most striking feature of leishmaniasis is the ability of all the pathogenic species to exclusively parasitize the macrophage phagolysosomes, where the parasites not only survive but continue to differentiate and multiply (2, 27, 1 22). Few microorganisms choose to live in such an inhospitable environment laiden with hydrolytic enzymes. Leishmania virulence must be closely related to this intralysosomal parasitism and hence to the sequential events of host parasite cellular interactions leading to its establishment. On the basis of information from the earlier work of many investigators, the sequential events of intracellular parasitism in leishmaniasis were summarized and the mechanisms speculated as follows (27, 28): (a) Promastigotes may enter macrophages via receptor-mediated endocytosis. Pre-existing receptors of macrophages may be exploited by these parasites for binding to multiple surface ligands of the macrophages. This binding may involve serum factors of the mammalian host, i.e. complement and natural antibodies, as well as digestive fluids of the sandfly vector, such as secretory products of the salivary gland and gut regurgitants. However, the possibility of nonreceptor mechanisms(s) for the uptake of Leishmania species by macrophages should be left open until pending conclusive identification of the putative receptor(s) and ligand(s) involved. (b) Leishmania species neutralize the lysosomal •

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CHANG ET AL

microbicidal factors via the parasites' surface. resistant molecules or excreted

by relying on the endogenous system of the macrophages and in part by using the parasite's own unusual pathway(s). (c) Stage-specific genes are expressed during Leishmania differ entiation in response to environmental signal(s) (d) Amastigotes thus adapt themselves to grow at acid pH in the parasitophorous vacuoles. Several points emerge from the foregoing summary of previous work. Leishmania species represent a unique entity in microbiology when one considers the evolution of intracellular symbiosis and parasitism ( 1 1 3, 1 64). The interactions of Leishmania with macrophages offer additional models to address many questions of interest in cell biology. From practical viewpoints , factors and detoxify the oxidative metabolites


.

these infection steps may be manipulated by chemical or immunological means for better prevention,

therapy, and control of leishmaniasis.

Considerable efforts have further elucidated the molecular mechanisms of the sequential cellular events as outlined.

These investigations have provided well as new insights not

evidence in support of the earlier proposals as

predicted previously. Some of these recent findings are summarized below.

Receptor-Mediated Endocytosis of Leishmania MULTIPLE RECEPTORS

mania

Leish ( 139) because this event is

The receptors of macrophages for binding

promastigotes have received much attention

the very first step in the process of intracellular parasitism. Also, the functions and structures of macrophage receptors are becoming better known, and monoclonal antibodies

specific to different

binding domains are becoming

available. Presumed ligands or monoclonals are used to coat substratum on which the macrophages

are

placed so that their specific receptors are im

The binding of promastigotes to these macrophages is then assessed. This approach and the conventional competition-inhibition experiments have shown that Leishmania of different species bind to the complement receptors (CRlICR3) ( 1 3 , 1 07, 1 08, 1 42, 1 73), mannose-fucose receptor (MFR) ( 1 3 , 1 70, 1 72), fibronectin receptor (FnR) ( 1 1 9 , 1 35, 1 78), and a receptor for nonenzymatic glycosylation end products ( 1 1 0). A mannose-N acetylglucosamine receptor has also been implicated in the clearance of Leishmania parasites from the blood stream and thus their sequestration by macrophages in vivo (22). In addition, the binding of promastigotes to macrophages can be modulated by altering the microviscosity of their plasma membranes with cholesterol ( 1 14). Amastigotes may also bind specifically to macrophages via similar (178) or different ( 1 4 1 , 1 76) receptor(s). mobilized and thus unavailable for binding on the exposed surface.

CRI AND CR3 Considerable efforts have focused on the complement recep tor-(CR) mediated endocytosis of promastigotes by macrophages because it is



503

hypothetically credible from an immunological point of view (see 73 for review). To assume that promastigotes delivered into the mammalian host are immediately exposed to the complement present in the body fluids is reason able. Evidence indicates that complement components fixed on the infective parasites fail to mediate cytolysis, but instead help them gain entry into macrophages ( 1 3 , 1 07 , 1 1 1 ). Leishmania species may even play an active role by changing their surface to fix complement appropriately (124, 1 25) for binding CR 1 , through which endocytosis is known to provoke less respiratory burst, thereby ensuring better intracellular survival of the parasites (36, 1 09). Noninfective promastigotes may enter macrophages via other receptors, e.g. CR3 (36), which activates respiratory burst to a high level, resulting presum ably in the destruction of the parasites. The scheme as presented is attractive. Uncertainties surround the detailed mechanisms of different species. The data presented from different laboratories do not clarify if the entry of infective Leishmania into macrophages is always mediated by CR1 and not by CR3 and/or other receptors . Evidence also indicates that macrophages produce C3, which may thus opsonize Leishmania parasites locally under serum-free conditions ( 1 3). This observation argues against the notion that the native surface molecules of Leishmania may bind directly to different CR domains of macrophages ( 1 39, 1 42) (see section on RGD-binding domains below for further discussion). The binding of promastigotes to macrophages has been shown in vitro to involve these receptors (FnR fibronectin receptor; MFR mannose:-fucose receptor; FcR Fc receptor). Their importance relative to CRlICR3 has been discussed (73, 139). Worth noting is the fact that mac rophages also synthesize and release Fn, which may thus opsonize Leish mania to enhance their uptake by the phagocytes ( 1 1 9). However, the FnR is thought to play a role of lesser importance because it is not specific to macrophages and promotes rather than actively mediates phagocytosis ( 1 39). Because MFR- and FcR-mediated phagocytoses often elicit a strong respira tory burst, the entry of Leishmania parasites into macrophages via these pathways is thought to be unfavorable to the parasites' survival.

FnR, MFR, AND FcR

=

=

=

The CR and FnR, both in the THE RGD (Arg-Gly-Asp) BINDING DOMAINS integrin superfamily, recognize different peptides sharing a common RGD sequence , the specificity presumably depending on its adjacent amino acid residues. Recent work by two different groups implicates a putative "RGD containing Leishmania surface molecule" for direct binding of promastigotes to these two receptors of macrophages ( 1 35, 1 42). In the first study, peptide sequences with RGD on Fn and antibodies specific to this molecule were found to inhibit the binding of promastigotes to macrophages ( 1 35). In


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CHANG ET AL

addition, the antibody recognized a Leishmania surface protein of 63 kd (gp63), which is thus suggested to be a fibronectinlike, macrophage-binding molecule (11 9, 1 35). In the second study, a peptide of 21 amino acid residues containing RGD was synthesized presumably according to the gene sequence of the Leishmania surface glycoprotein (gp63) (19). This peptide was found to compete with promastigotes as well as gp63-coated beads for binding to macrophages (142). Moreover, a monospecific antiserum raised against this peptide was further shown to inhibit promastigote-macrophage binding and to recognize C3 (140). These findings appear to demonstrate direct binding of RGD sequence within a surface molecule of promastigotes to CR3. Recent work by others (104), however, indicates several mistakes in the gp63 gene sequence from the previous work (19), and the corrected sequence actually predicts no such peptide with RGD (104). The existing data thus suggest that Leishmania-macrophage binding either does not directly involve RGD se quence or involves this sequence in the intervening proteins of exogenous origin, i.e. Fn and C3 . Alternatively, the RGD sequence may be located in surface molecules of Leishmania other than gp63 . The attractive proposal is that promastigotes infect macrophages pre dominantly via their CRI for better survival of Leishmania parasites after their intracellular entry. Positive indentification of the receptors of concern for Leishmania by biochemical means will undoubtedly strengthen this very important concept. Such undertaking will also address the possibility that Leishmania-macrophage binding is based on a nonreceptor mechanism simply as a phenomenon of surface charges (149). The binding of Leishmania to macrophages in natural infection may well be more complicated than currently thought. The hypothesis of multiple ligand receptor cooperations proposed previously (27) likely reflects the true situa tion. Exposed on the surface of Leishmania species are multiple molecules. They may interact directly with a multitude of macrophage receptors with or without the intervention of soluble products from the host, as well as possibly from the sandfly vector [e.g. a vasodilatory peptide (1 34, 1 61 )]. It is not known whether all these membrane and soluble molecules of heterogeneous origin are involved, and how they interact among themselves, in Leishmania macrophage binding at the site of sandfly bites. The answers to these ques tions depend on further biochemical identification and biological characteriza tion of all factors involved. Leishmania

Differentiation

METACYCLOGENESIS The in vitro or in vivo generation of promastigotes from noninfective forms to infective or metacyclic forms has been reported for some species of Leishmania and referred to as metacyclogenesis (143).



505

This is marked, in Leishmania major in vitro, by the following events: (a) change of long slender forms in the log phase into short, actively motile forms in the stationary phase (146); (b) biochemical and antigenic changes of the terminal saccharide moiety of lipophosphoglycan (LPG) (56, 144, 145), resulting in the acquisition of resistance to complement-mediated cytolysis and the loss of agglutinability by galactose-specific lectin. Other Leishmania species of the Old World, e.g. Leishmania donovani, undergo similar changes (35, 67, 175). These changes are either less dramatic or inapparent in most New World! species (94, 148). In spite of these variabilities among different species, promastigotes appear to increase the expression of certain genes, presumably preadapting them to live subsequently in the mammalian hosts (94). How these changes can be defined in term of cellular differentiation and its relation to the cell cycle of promastigotes is of interest for further investiga tion. HEAT-SHOCK RESPONSE

A sudden increase in the ambient temperature is the most obvious environmental change during the transit of Leishmania parasites from insect vectors to mammalian hosts. This event has led to the studies of promastigotes in response to heat-shock or other stress. Here, the New World and Old World Leishmania also differ in their responses. At 35-37°C, cutaneous species of South American origin often lose motility and become rounded or elliptical forms (158) . These forms remain viable for some time, and change further to become amastigotelike (see 29) and can even be grown continuously as such under appropriate conditions ( 120). These axenic amastigotes are similar to lesion-derived amastigotes morpho logically and antigenically ( 121). Both of these axenic amastigotes and the promastigoltes heat-shocked for a short duration are more infective than the untreated promastigotes to laboratory animals (88, 156). In contrast, pro mastigotes of the Old World species. e.g. L. donovani and L. major, upon heat-shock often do not undergo such changes and fail to survive under such conditions. In the presence of macrophages, these promastigotes remain flagellated and motile for a long period and differentiate into amastigotes only gradually. Thermally regulated expression of certain Leishmania genes possi bly related to virulence is discussed in the next section. The signal(s) and its transduction triggering leishmanial differentiation remain a subject of interest for further investigation. Heat shock and other stress conditions, such as nutritional depletion, are perceived to play a role in differentiatilon. Drugs, such as 3-methoxybenzamide, appear to divert the Leishmania pathway from differentiation to multiplication, suggesting that ADP-ribosylation of proteins may be required for the initiation of the differen tiation event ( 1 60). The plasma membrane of Leishmania species contains unusual protein kinase(s), which may mediate the transduction of sig-

506

CHANG ET AL

nal(s) for leishmanial differentiation endogenous substrate(s) (37).


Intracellular Survival of

by

phosphorylation

of certain

Leishmania

The previous finding that Leishmania amastigotes are intralysosomal parasites (reviewed in 2, 27 , 28) receives further support from additional evidence: they reside in the acidic compartment of macrophages and are metabolically most active at the lysosomal pH (47, 115). These findings reinforce the notion that Leishmania amastigotes are possibly acidophilic or at least acid-tolerant. Their biosynthetic capabilities as such are important in considering not only the growth of these intracellular parasites but also their resistance to the microbicidal factors of macrophages and the modulation of these cells by the parasites in host immunity. Information is limited on the metabolism of amastigotes and their environment in the parasitophorous vacuoles. In con trast, the literature contains many reports on lymphokine-mediated killing of Leishmania by macrophages. The processing and expression of Leishmania antigens on infected macrophages has also continued to receive attention ( 169). These important immunological issues are undoubtedly related to the anti lysosomal and anti-oxidant activites of Leishmania species, both of which remain unclear (see 27, 28). Aside from the finding of trypanothione and trypanothione peroxidase (43), few efforts have been made to further characterize the specific enzymes already identified or known to detoxify oxidative metabolites during Leishmania-macrophage interactions. Some other nonspecific enzymes or molecules thought to neutralize the anti microbial factors of macrophages are discussed in the next section. Overall, considerable progress has been made during the past five years toward a better understanding of the molecular basis of Leishmania macrophage cellular interactions. Receptor-mediated endocytosis of Leish mania species by macrophages has been extensively studied. In vitro ex perimental evidence strongly supports a predominant role of CRlICR3 in this event. Leishmanial differentiation has been better delineated to include the preadaptation of promastigotes before their exposure to the conditions in the mammalian host and subsequent confrontation with macrophages for in tralysosomal parasitism. This is conceptually important in spite of certain species-specific differences observed. The final differentiation of Leishmania into amastigotes within macrophages is true for all species and is thus the most crucial event, although its initiation and regulatory mechanisms remain largely unknown (see the conclusion for further discussion). Which molecules expressed during leishmanial preadaptation and differentiation are directly relevant to Leishmania survival and virulence have not been clearly identi fied, but some potential candidates are included for discussion in the follow ing section .


507


MOLECULES OF POTENTIAL IMPORTANCE IN LEISHMANIAL VIRULENCE

The molecules described below are mainly glycoconjugates present on the cell surface of Leishmania (Figure 1). Work done to elucidate their biochemical properties, immunogenicity, and interactions with macrophages suggests potential contributions of these molecules to Leishmania virulence. Some stage-specific molecules of possible importance are also included for discus sion.

Surface Lipophosphog/ycan (LPG) The structure and possible functions of the glycoconjugates unique to Leish mania species have been recently reviewed (60, 1 65) . They are heterogeneous molecules and constitute the most abundant surface component; each cell has

Protein

Lipophosphoglycan

iii to to lTII � mil JJJJJ��h��h��h�l�� '"

Figure 1

Schematic presentation of glycosylphosphatidylinositol-anchored glycoproteins and

Jipophosphogl!ycans of Leishmania. Abbreviations stand for I, inositol; G, carbohydrate core; P, phosphate; E, e thanolamine . Reproduced with permi ssion from 165.

CHANG

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more than lO6 copies. LPG is present in both leishmanial stages and is


released as "excreted factors." The molecular structure of LPG consists of three portions: terminal repeats of phosphorylated saccharides, a phosphory lated heptasaccharide core, and lyso-alkyl-phosphatidylinositol (Figure 2) (60, 118, 166). The glycosidic linkage of the inositol to a nonacetylated glucosamine is a striking feature of LPG. The terminal saccharide repeating units are also structurally unique and vary with Leishmania species. L. donovani promastigotes have an average of 16 copies, each consisting of [P04-6-Gal-(beta- 1 4)-Man-alpha- l ] (166). L. major promastigotes have multiple repeats of tri- and tetra-saccharides, consisting of galactose, man nose, glucose, and arabinose (59, 98). The difference in this portion of the LPG distinguishes antigenically different Leishmania species. Antigenic heterogeneity is further contributed to, probably even more significantly, by another group of related surface inositol glycolipids (GIPLs), which are composed of truncated saccharide units of variable lengths similar to LPG, but are anchored to the membrane via either alkylacylglycerol or lyso alkylglycerol (97, 118, 137, 154). Although GIPLs are antigenically distinct, they are thought to be precursors of LPG and the glycosylphosphatidylinositol (GPI) of the lipid anchor for certain Leishmania membrane glycoproteins (e.g. gp63) (see below). The tissue tropism of different Leishmania species ,

may be related to the variations in their surface glycolipids (60). Functional studies parallel to detailed structural analysis of LPG and related glycolipids suggest that these molecules play important biological roles in Leishmania-macrophage interactions. Purified LPG and the Fab fragments of monoclonal antibodies specific to these molecules inhibit the binding of promastigotes to macrophages (57). The receptors of macrophages for LPG appear to be CR3 and p150,95 of the integrin family, specifically the bacterial lipopolysaccharide-binding site (D. G. Russell, personal communication). In addition, LPG enhances the survival of promastigotes in macrophages (61). Experimental evidence suggests that the mechanism of this protection may be

based on the action of LPG to scavenge oxygen free radicals (24) and/or to inhibit relevant enzymes, e.g. lysosomal glycosidases (23, 99) and protein kinase C (99). Other proposed biological actions of LPG include inhibition of lymphoproliferative response and activation of T suppressor cells (see 165).

Repeating ®- Dlsaccharide

Figure 2

- Heptas8ccharide Core

Lyso -Alkyl-Pnospnalidyllnosilol

Structure of lipophosphoglycan from Leishmania donovani. Reproduced with permis

sion from 1 65 .



509

Leishmanial virulence has been related to quantitative and qualitative changes of promastigote LPG. In at least one strain of L. major (145), LPG changes with the in vitro development of infective forms in antigenicity by acquiring new epitopes ( 1 44) and in ultrastructural feature by assuming filamentous structural units (123). Promastigotes made resistant to ricin (79) show altered LPG with reduced saccharide moiety. Changes of these variants in their in vivo and in vitro infectivity have been observed ( 1 65), suggesting that LPG and related glycolipids may indeed contribute to leishmanial viru lence. Leishmania LPG and related glycolipids are highly immunogenic (40, 97, 1 36, 1 62), owing to their unique structural features. They have long been exploited as excreted factors for serotyping of Leishmania species (59, 1 65). Pathogenic Leishmania species contain an immunogenic Gal-alpha-I-3-Gal epitope recognized by sera from patients with leishmaniasis (6). Whether this epitope originates from LPG or GIPLs is unclear. LPG with appropriate adjuvants (59) or in combination with gp63 (2) have been shown to protect mice against cutaneous leishmaniasis.

Surface Glycoproteins-Ectoenzymes Zn-PROTEINASE

The general properties of this ectoenzyme have been re viewed ( 1 4). This molecule was initially recognized as a major surface antigen of promastigotes by using monoclonal antibodies (44) and surface radioiodination of living cells (27, see 39) . Purification from a number of species has been accomplished using different methods (17, 25, 105, 141, 1 55). The molecule is sized in most studies using SDS-PAGE to be 63 kd and exists as a homodimer in samples from L. major ( 1 6). It is estimated to constitute about 1 % of the total cellular proteins or 500,000 copies per cell ( 1 4). By p1eptide mapping and/or immunological cross reactivity, gp63 has been found in all major pathogenic Leishmania species (18) and in both stages (32, 33, 46, 63, 65, 102). It is present on the surface of the promastigotes in the sandfly (55). The molecule is posttranslationally modified by N glycosylation with asparagine-linked oligossacharides ( 14, 25, 26, 1 02) and by glypiation with GPI (15, 42). The N-glycans of gp63 from Leishmania mexicana amazonensis are the biantennary high-mannose type with heteroge neous lengths (Figure 3). Unprecedented is the finding of a terminal glucose residue associated with the longest oligosaccharide chain. The diglyceride moiety of the GPI appears to anchor gp63 to the membrane via myristic acid (42). Although other portions of the GPI still need to be characterized biochemically, they are known to cross-react immunologically with those in the variant surface antigens of the African trypanosomes ( 1 5). A change of gp63 in electrophoretic mobility seen during leishmarlial differentiation (32, 46) is thought to result from an alteration of its GPI ( 1 02) . The genes encoding gp63 have been cloned and sequenced from two

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different species (19, 104), showing 90% similarity at the amino acid level (Figure 4). This similarity is substantiated directly by partial peptide sequenc ing (60% of the total amino acid residues) of gp63 from a third species (Figure 4) (G. Chaudhuri, M. Chaudhuri, S. P. Latshaw, L. Moberly, R. G. Kemp, & K.-P. Chang , submitted for publication). The nucleotide sequence from one species further predicts cleavages of 100 amino acid residues as the prepeptide and propeptide from the N-terminus of gp63 (19). There are three potential sites of N-glycosylation, two of which are conserved (19, 104). Gp63 genes in L. major exist in one chromosome as a cluster of six copies, each consisting of three kb gene and intergenic sequences, and a single copy independent of the cluster (20). Other species also contain these genes in multiple copies arranged similarly, if not identically. Evidence supports the polycistronic transcription of gp63 genes, which gives rise to individual transcripts of two to three kb (19, 20). Some differences in the processing of these transcripts are noted between virulent and avirulent cells (171). Proteolytic activity of gp63 was first described in L. major (41). This activity was subsequently confirmed with gp63 purified from L. mexicana amazonensis, L. mexicana pifanoi (31, 32), and L. mexicana mexicana (71). Similar findings for other species are based on the comigration of proteolytic


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I MA II ALG FSGPFFEDAR I VA NV PNVRGK !II F 0 VP 197 IM A II ALG F S V G F FEGAR I L E S I S N VR IIK DF DVP OItPYPTP SNAIIIA VINSSTAVAKAREQYGCDTLEYLEVEDQGGAG

VI tiS S T A V A K A R E Q Y G C D T L E Y L E I E DQG O A G NIXSSTAV BQYOCNTLBYLBNBDQO

230

AG 5 H I KMRNAQDEL MAP AA AAGYY TAL T !of A I FQ �3 AG 5 II I KMRNA QDEL MAP A AAAGYY SAL T !of A I F Q

NAQDBLNAPAASAOYY

AVPQ

DL 0 F Y QAD F 5 KAE VMP WG Q :oJ A GCAFL TNKC ME Q 296 DLGFYQADFSKAEVMP WGRNAGCAFLSEKCMER

DLO'YQAD'SItABANPWOa

LIlli Ld.

S V TOWPAMPC�ESE DAI RCPT SRLSLGACGVTRm �I 'r K WPAM F e N ENE V T 101 R CPT 5 RLSI. GKCG V T R

LnoJ

II P '[J LPPY W 0 Y F T DP S L A O V 5 A F M D y e P V V V P Y S 362 H P D LP PY WQY F T O P SLAG I SAF !of 0 CC P VV E PYG YPALPPYWQYPTDPPLAOSSAPNDYCPVVVPYA

LnoJ

DG :1 C T 0 It A SEA H AS L L P F N V F S D A A R C o G $ C A Q RA 5 E A GAP F K G F S VFS 0 A ARC DOSCAQSASBADAPP It APSVPSDAAa c

11.•• J.d. 11.... J.d. 11....

LSVONCDVTP

P it .' T DGI V It 5 Y A GLCA :oJ V PK T SII GI I K SY AGLCAN V

Plt'fTBOLI

0 R

ItSYAALCANVIt

D G A F R 395 0 GA F R DOAPa

C D TAT RT Y S V 0 V I I G 421 C D TATR T Y S V Q V II G TYSVQvao

LIlli

SN 0 Y T H C T PGLRVEL S T V 5 NA f EGG G Y I T C P P Y �I G 5 GY A� C TPG L RVEL 5 TV 5 5 AFEEGG Y I TCP P Y

LIlli Ld.

VE .� CoGNV 0 AAKD GGNTAA GRR GP R AA A T AL I. VE Y C QGNVQ AAK DGG N AAA GRRGP RA AA T A I. l

Lilli Ld. a....

AA I. L AVA L � AA I. L A V A L

J.de 11....

11....

SSOYAXCTPOLRPDLSTVSDAPBItOOYVTCPPY

VB '�CQONAQAI It

Figure 4

V V

494

Amino acid sequences of Leishmania gp63. Lmj and Ldc are sequences predicted

from the genes cloned and sequenced from L. major (19) and L. donovani chagasi ( 104), respectively. Lma represents peptide sequences of gp63 purified from L. mexicana amazonensis

(G. Chaudhulri, M. Chaudhuri, S. P. Latshaw, L. Moberly, R. G. Kemp, & K. ·P. Chang,

submitted for publication). Potential amino acid residues important for Zn-binding or catalysis are

indicated by bold-faced letters. The N-terminus of Lmj gp63 is numbered 1 and the numbers at the right hand side indicate the number of the Lmj last amino acid residue in the row. Arrow

indicates the probable cleavage point of Lmj gp63 precursor protein during maturation (19). X

represents uncertain residues in the Lma sequences. The asparagine residues as the potential sites of N-glycosyllation

are

underlined.


512

CHANG ET AL

activity with electrophoretic ally and immunologically similar molecules in fibrinogen gel ( 1 8). Evidence indicates that gp63 is a metallo-proteinase ( 1 6, 32, 9 1 ) with a signature sequence for zinc binding conserved in these enzymes ( 1 6, 32, 74, 1 04). Gp63 is thus inhibited by metal chelators, e.g. 1 , 10phenanthroline. Alpha-2 macroglobulin also inhibits the proteolytic activity of gp63 in solution, but not the membrane-bound form on the intact cells (66). Chemical modifications of specific amino acids implicate histidine, glutamic acid, and arginine residues in the catalytic mechanism of gp63 (G. Chaudhuri, M . Chaudhuri, S. P. Latshaw, L. Moberly, R. G. Kemp, & K.-P. Chang, submitted for publication), as found for other metallo-proteinases. At pH 4, gp63 actively degrades native substrates of biological relevance, e.g. C3, albumin, hemoglobin, and IgG (3 1, 32), and at neutral to alkaline pHs, it degrades denatured substrates, e.g. azocasein (41). The pH optimum and Km of gp63 cannot be accurately determined until very small substrates, e.g. dipeptides, are found. Using insulin B chain and glucagon, each consisting of about 30 amino acid residues as substrates, gp63 is shown to be a nonspecific endoproteinase, which prefers larger substrates and cleaves different peptide bonds at different pHs (G. Chaudhuri, M. Chaudhuri, S. P. Latshaw, L. Moberly, R. G. Kemp, & K . -P. Chang, submitted for publication). Other studies using substrates of 1 0 amino acid residues show site-specific cleav ages of peptide bonds with hydrophobic amino acids optimally at neutral pH (7 1 ) . The secondary structure of gp63 resembles neither the VSG of African trypanosomes nor other metallo-proteinases in that it consists of 50% anti parallel beta strands and 20% alpha-helix, as shown using Raman spectros copy (72). Crystallography of gp63 and site-directed mutagenesis of its gene will provide further insight into its catalytic mechanism for the possible development of inhibitors specific to this proteinase. By virtue of its proteolytic activity, universal presence, abundance, and surface localization, gp63 is thought to be a Leishmania virulence factor. The abundance of gp63 is indeed often positively correlated with Leishmania infectivity (8 1 , 82, 84, 1 7 1 ) [although one exceptional case has been reported (59)]. Evidence supports the formation of a covalent bond between C3 and surface gp63 of promastigotes ( 138). In addition, gp63 has been shown to cleave C3 into C3b and other C3 products (31) and to protect lipid-protein substrates from intralysosomal degradation within macrophages (32). These findings suggest that, because of gp63's proteolytic activity, it may function in the CR-mediated endocytosis of promastigotes and protection of Leish mania from intralysosomal microbicidal factors . Gp63 appears to share epi topes with fibronectin ( 1 35) and FcR (53). The implication of these findings in receptor-mediated endocytosis of promastigotes by macrophages awaits further investigation . This subject has been discussed in a previous section with particular reference to the absence of a RGD sequence in gp63 . While



513

evidence points to direct or indirect participation of this molecule in the binding of promastigotes to macrophages, it is unknown if this is simply a phenomenon of enzyme-substrate interactions. Leishmania gp63 is immunogenic. Anti-gp63 antibodies have been re ported in sera from patients with leishmaniasis (33, 63, 65, 1 16, 1 3 1). Immunization of genetically susceptible mice with gp63 together with LPG encapsulated in liposomes protects them against challenges with homologous species (2). Gp63 are thus potentially useful antigens for immunodiagnosis and immunoprophylaxis of leishmaniasis. The biochemistry and possible functions of mem brane-bound and secretory acid phosphatases (ACP) have been reviewed (39, 48). The cell surface ACP was initially discovered on the promastigote stage using ultrastructural cytochemistry (see 39). The two forms of ACP are antigenicaUy distinct (8, 52) and each probably exists as multiple isozymes present in both stages of most Leishmania species (9, 48, 62). One form of the membrane··bound ACP ( 132) and the secretory ACP (92) have been purified from L. donovani to homogeneity and found to be homodimers of 1 20 kd and 1 34 kd, respectively. Both forms of ACP are glycosylated (52, 93, 130). All these different forms of ACP are nonspecific monoesterases capable of hydrolyzing a variety of phosphorylated substrates optimally at pH 5.0-5.5; the membrane-bound enzymes are tartrate-resistant in contrast to the soluble forms (48, 52). The tartrate-resistant ACP purified from promastigotes re duces the respiratory burst of neutrophils (133) and is itself resistant to oxidative metabolites (147). In addition, it has been shown to de phosphorylate certain phosp holipid s and phosphoproteins (132). Thus, this ectoenzyme is thought to protect Leishmania species by interfering with the regulatory mechanism of the macrophages that produces microbicidal free radicals (48). Consistent with this proposal is the finding of tartrate-resistant ACP activity higher in virulent clones than in avirulent clones of L. donovani (77). No such correlation is found, however, in studies with other species, e.g. L. major (48, 52, 62). The possible function of the secreted ACP is unknown. Its production in large quantity elicits humoral immune response of the host and may conceivably contribute to the pathobiology in leishmaniasis (48, 52); however some pathogenic species, e.g. L. major, do not release ACP (62). ACID PHOSPHATASES

NUCLEOTIDASES 5' nucleotidase and 3' nucleotidase/nuclease have been reported to exist on the surface of some trypanosomatid protozoa including Leishmania species (51 , 52). The 5' nucleotidase has an electrophoretic mobility of about 70 kd on SDS-PAGE and is active in degrading both ribo and deoxy-ribonucleotides at pH 7-8; the 3' nucleotidase/nuclease of 43 kd is


514

CHANG ET AL

much more abundant. The latter enzyme is most active with 3' AMP and prefers RNA over DNA as the substrate. Its activity is optimal at pH 8.5 when assayed as 3' nucleotidase and at pH 6.0 when assayed as nuclease. Both enzymes are glycosylated and non-stage-specific; isozymes exist at least for the 3' nucleotidase. Of particular interest is the Leishmania 3' nucleotidase/nuclease, which has been reported in plants and fungi, but not in mammals. The activity of this enzyme in Leishmania becomes elevated in the logarithmic phase of cell growth and is induced to increase up to a WOO-fold in medium depleted of nutrients, especially purine nucleobases and nucleosides (51). Because all trypanosomatid protozoa are incapable of de novo biosynthesis for purines, the action of these nucleotidases is thought to provide these essential nutrients by converting nucleotides into transportable nucleosides and Pi (51, 52). The alkaline pH optima of both enzymes suggest that they probably serve such functions for promastigotes in the sandfly gut better than for amastigotes in the phagolysosomes of the macrophages. TRANSPORTERS The plasma membrane of Leishmania has been shown biochemically and genetically to possess the transport systems for folate, glucose, nucleosides, proline, and ribose (see 52). More attention has been devoted to a cation- or proton-transporting ATPase, which is apparently crucial for the pH homeostasis of Leishmania species and the transport of nutrients necessary for their adaptation to the changing environments in their life cycle. The metabolic activities of amastigotes, such as oxygen utilization, incorporation of precusors into macromolecules, and the transport of glucose and proline, are optimal at acidic pH, coinciding with the natural environment of phagolysosomes (47, 115). An intracellular pH close to neutrality is maintained in both promastigotes and amastigotes, although their ambient pHs may vary (from pH 7.4 to as low as pH 4.75). This is thought to result from the activity of a Leishmania membrane ATPase/proton pump. This enzyme is active at pH 6. 5 and localized to the inner side of the plasma membrane; it is Mg++-dependent, but Ca++- and orthovanadate-sensitive (179). The enzyme is encoded by a tandem pair of slighty different genes (ATPase la and lb) of 2,922 bp each, which predicts a protein of about 107 kd; the transcript of 5.2 kb from ATPase la is present in both Leishmania stages, while that of 5.75 kb from ATPase lb is much more abundant in amastigotes than in promastigotes (100, 101). The developmentally regulated expression of the ATPase lb may be crucial for the transition of Leishmania from the sandfly gut into the acidic environment of phagolysosomes. Another de velopmentally regulated gene contains an open reading frame of 567 amino acid residues for a protein of about 62 kd, which shows significant homology at the amino acid level to the red blood cell glucose transporter and several


5 15


bacterial hexose transport proteins (21). The genes are arranged in tandem repeats of at least seven copies, producing a transcript of about three kb more abundantly in the promastigote stage. It is not known if amastigotes possess other glucose transporters or utilize substrates other than glucose as their energy sources. Adenosine-pyrimidine nucleoside transporters of Leishmania have also been identified (5), but they have not yet been characterized at the molecular level.

Cysteine Proteinases and Megasomes The cysteine proteinase, a conserved and wide-spread enzyme, has been found in 1.. mexicana complexes and possibly other cutaneous species of South American origin (127). It is present in an unusual organelle, or mega some a modified lysosome, which is noticeable in the promastigotes grown -

to stationary phase and becomes fully developed as they differentiate into

amastigotes (91, 94). In the latter, megasomes constitute as much as 15% of the total cell volume (127). The cysteine proteinase of these megasomes, partially purified from the amastigotes of L. mexicana mexicana, has an electrophoretic mobility of 31 kd on SDS-PAGE and is sensitive to the conventional inhibitors of such enzymes, e.g. leupeptin (126). Its proteolytic activity is optimal at acidic pH against preferred peptide substrates, such as Pro-Phe-Arg and Phe-Arg ( 1 26). The N-terminal sequence of a similar en zyme from the axenic amastigotes of L. mexicana pifanoi is determined and found to be similar to that of other cysteine proteinases (D. McMahon-Pratt, personal communication). Molecular cloning of the gene for this enzyme is being pursued. The effects of inhibitors specific to this acid cysteine proteinase suggest that this enzyme is functionally important to the amastigotes. Antipain and leupeptin inhibit the growth of both promastigotes and amastigotes in L. mexicana mexicana (34). This enzyme is proposed to serve a degradative role, possibly for the nutritional benefits of the amastigotes and for releasing ammonia or other amines to modulate the host lysosomal activity for the parasites' intracellular survival (126). The amino acid esters, such as leucine methyl ester, were found to rapidly lyse and kill intracellular amastigotes without affecting their host cells (128, 129). Subsequent studies indicate that the target of these amino acid esters is the megasome wherein the cysteine proteinase cleaves these substrates into amino acids; their accumulation results in osmotic disruption of the mega somes and thus the released lysosomal enzymes lyse the amastigotes (4, 128) . The lysis of amastigotes resulting from swelling and rupture of the mega somes can be prevented by inhibiting the cysteine proteinase with specific inhibitors, i.e. antipain or chymostatin or benzyloxycarbonyl-Phe-Ala-

516

CHANG ET AL


diazomethane (3, 28). Application of the leucine methyl esters in vivo, however, produces no effect on the course of leishmaniasis (68). It is highly unusual to find a cysteine proteinase as a developmentally regulated molecule present in greater quantities in the amastigote stage of certain Leishmania species. The functions of this Leishmania enzyme as well as its evolution and regulatory mechanism for stage-specific expression among these organisms deserve further investigation.

Heat-Shock Proteins Exposure of promastigotes to elevated temperatures results in an over expression of the classic heat-shock genes and other genes. Multiple protein bands emerge shortly after heat-shock and their number varies with different species ( 1 , 69, 87, 1 53). Most of these protein bands appear to be con stitutively expressed in promastigotes but become over-expressed in heat shocked promastigotes and in amastigotes . Only one of these proteins has been positively identified immunologically as equivalent to Hsp70 protein ( 1 53). Exposure of promastigotes to sodium arsenite results in an over expression of stress proteins, which

arc

not entirely identical to those seen

with heat shock (87). Northern hybridization with Drosophila Hsp70 and Hsp83 gene probes indicates that the heat shock�induced expression of these proteins in promastigotes is transcriptionally regulated ( 1 52, 1 53, 1 67). The numbers and sizes of the transcripts, and the level of their increases upon heat shock vary with different species. Leishmania heat-shock genes equivalent to Drosophila Hsp70 and Hsp83 genes are localized as multiple copies to two different chromosomes in a number of species (88, 1 52). Hsp83 gene is linked to the tandem repeats of the beta tubulin genes ( 1 50a). The genome of L. major contains a cluster of four different Hsp70-related sequences, with a distant fifth gene in the same chromosome (88) and four other chromosome sites ( 1 52). One of these genes appears to code for an organelle protein, as it has a putative mitochondrial signal peptide at the amino terminal end (88) . Each of these gene sequences contains an open reading frame for 658 amino acids, with long repetitive 3 ' untranslated sequences and two palindromic heat-shock elements in the in tergenic region (88). Also examined during heat-shock of Leishmania promastigotes are alpha and beta tubulin genes , which exist as tandemly repeating units in separate chromosomes ( 1 57). Tubulin biosynthesis is, as expected, much more active in the motile flagellated promastigotes than in the nonmotile amastigotes with regressed flagella (see 45). The expression of tubulin genes is regulated during leishmanial differentiation transcriptionally or posttranscriptionally, depending on the species examined (see 45 , 86). A single transcript is produced from alpha tubulin genes in both leishmanial stages. Promastigotes



517

have several transcripts for beta tubulin genes, but only one transcript remains or becomes predominant as the parasites differentiate into amastigotes. The divergenc�� of the regulatory mechanisms for tubulin biosynthesis in different species is further verified by recent findings of stage-specific transcriptional (78, 1 52, 1 67) or posttranscriptional regulations ( 1 03, 163). Most intriguing is the increase in virulence seen with briefly heat-shocked promastigotes ( 1 56). What molecular changes in these promastigotes account for their increased virulence remain uncertain. If the over-expressed heat shock proteins are involved in this phenomenon, their precise role(s) remains equivocal.

Other Stage-Specific Molecules The lack of a de novo pathway for purine biosynthesis is a biochemical peculiarity of all trypanosomatid protozoa, including Leishmania. Their purine salvage pathway is thus very active. This pathway is very similar

between amastigotes and promastigotes of L. donovani, except for adenosine metabolism (96). The activity of adenosine kinase for phosphorylation of adenosine into AMP is 50-fold higher in amastigotes than in promastigotes; amastigotf�s also deaminate adenosine to inosine via adenosine deaminase, while promastigotes convert adenosine to adenine via adenase and then deaminate adenine to hypoxanthine via adenine deaminase. Adenine phos phoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase are thus preferentially used for the synthesis of nucleotides by amastigotes and promastigotes, respectively (70). Some of these stage-specific enzymes have been recently purified for further characterization (e.g. 1 2). Additional stage-specific antigens and genes have been identified using monoclonal antibodies (75) and differential cDNA hybridization (78, 150). Such efforts undoubtedly will be intensified and facilitated by new molecular approaches, such as polymerase chain reaction. Further characterization of these stage�-specific molecules is crucial for determining their possible signifi cance in Leishmania virulence. REGULATION OF

LEISHMANIA

VIRULENCE

Leishmania may lose virulence because their molecular machinery cannot produce factors crucial for infection. The regulation of virulence may be related to gene dosages, transcription, posttranscriptional processing, trans lation, andlor posttranslational modifications of the protein factors or the enzymes for the biosynthesis of other nonprotein molecules, e. g. LPG . The paucity of information about the regulatory sequences for Leishmania gene expression has only allowed the distinction between transcriptional


5 18

CHANG ET AL

and posttranscriptional regulation. Under such broad grouping, the expression of most protein molecules examined appears to be transcriptionally regulated. Evidence supports the regulation of leishmanial virulence by posttrans lational modifications of proteins, i .e. N-glycosylation, via DNA amplifica tion. The initial evidence for this is based on the finding that leishmanial promastigotes lose infectivity in vitro and in vivo when treated with tunicamy cin (TM) (64, 80, 117), an inhibitor of the N-acetylglucosamine- l -phosphate transferase (NAGT)-the first enzyme in the dolichol pathway of protein glycosylation. Subsequently, TM-resistant promastigotes of L . mexicana amazonensis are produced and found to maintain virulence upon prolonged in vitro cultivation in contrast to the wild-type (82). Significantly, the incorpora tion of 2-[3H]-mannose into proteins as a measure of N-glycosylation, the accumulation of a glycoprotein (gp63), and the NAGT activity are all higher in the variants and the virulent wild-type compared to the avirulent cells (81, 82). This observation has led to the proposal that the accumulation of gly coproteins as virulence factors is subjected to regulation by the activity of their N-glycosylation. The asparagine-linked oligosaccharides of the gly coproteins may be important for the expression of their enzyme activity, as shown in the case of Leishmania acid phosphatase (93, 1 30), and/or for the stability of the protein moiety against its denaturation or degradation. The precise mechanism, however, remains to be determined. Of particular interest is the finding in the TM-resistant variants of amplification of specific chromosomal DNA into extrachromosomal super coils (38), as found previously with methotrexate-resistant Leishmania (10) . Since the latter variants show no change in infectivity, the virulent phenotype of TM-resistant cells appears to result from the amplification of specific DNA sequences, but not DNA amplification per se . The extrachromosomal DNA of L. mexicana amazonensis originates from a large chromosome and is ampli fied up to a 1 00-fold as multiple copies of a 63-kb circle (38). A portion of the circular DNA hybridizes with Alg 7, a putative gene encoding yeast NAGT (80). TM-resistant variants produced from four additional Leishmania species contain extrachromosomal circles of 30-70 kb, but all have a consensus region of 20 kb (K. Katakura, Y. Peng, R. Pithwalla, & K . -P. Chang, submitted for publication). Mutiple transcripts produced from this consensus region are translationally active and the products in the variants are recog nized by a monospecific antibody raised against NAGT purified from bovine cells. Taken together, this evidence suggests that amplification of the NAGT gene in the variants is responsible for their TM-resistance and also for the increase in the overall activity of the dolichol pathway for N-glycosylation. While work is still underway to further characterize this microsomal enzyme and its gene, an apparent gene dosage effect increases the activity of an enzyme relevant to leishmanial virulence. Whether this occurs naturally as a regulatory mechanism for other virulence factors awaits further investigation.


519


Leishmanial proteins are subjected to other types of posttranslational mod ifications, e.g. glypiation and phosphorylation. Whether they also play cer tain roles in the regulation of Leishmania virulence factors has not been examined. Our preliminary evidence suggests that glypiation of gp63 may affect its protease activity (G. Chaudhuri & K.-P. Chang , unpublished data) . CONCLUSION

There is no evidence of leishmanial toxigenicity , as found in some pathogenic bacteria. Available evidence points to multiple molecules , which appear to work in concert for Leishmania to infect macrophages, and to survive and multiply in their phagolysosomes. Most extensively studied are two catego ries of surface glycoconjugates: lipophosphoglycans (LPG) and related glyco lipids (GIPL) as well as gp63 as a Zn-proteinase and other ectoenzymes. The surface metallo-proteinase is of special interest because increasing evidence supports the functional significance of similar enzymes in biosystems includ ing microbial pathogenicity . Figure 5 depicts the possible functions of various virulent determinants proposed in leishmanial infection of macrophages. Evidence supports the development of virulent phenotype with leishmanial differentiation from noninfective to infective promastigotes and then to amastigotes (see section on Leishmania differentiation) . Presumably , Leish mania spedes undergo these changes in response to adverse conditions encountered sequentially in the following environments during part of their life cycle: (a) the nutritionally barren region in the digestive tract of the sandfly; (b) body fluids of the mammalian host with arrays of immune factors; and (c) the intralysosomal compartment of macrophages with microbicidal hydrolases. Perhaps, because they are exposed successively to these changing conditions , Leishmania species express specific genes that allow these para sites not only to survive in a particular adverse environment but also preadapt them to face the next wave of stresses. Preliminary evidence for this notion is described in the foregoing sections. Within the framework of our general understanding in developmental biology, available data also indicate that this leishmanial differentiation as described differs from other developmental systems in several aspects: (a) The signal(s) and the molecular changes seen during Leishmania differentia tion for thl� development of resistance are not well defined. Other microbial models for the development of resistant stages, e.g. sporulation or encysta tion, are often marked by a profound cellular remodeling triggered by a change of, for example, pH, osmotic pressure, and bile salt concentrations . As yet, no single factor is clearly identified that triggers a complete leishman ial differentiation to the amastigote stage. Although several developmentally regulated genes have been identified in Leishmania species, the relevance of

520

CHANG

ET AL

� /


Promasti

ole

2

Lysosome

Lytic enzymes--� Amastigole

Figure 5

Schematic depiction of the sequential events and the mechanisms involved in leish

manial infection of macrophages-the exclusive host cells in leishmaniasis.

(1) Upon delivery

into the mammalian host by the sandfly vector, infective promastigotes may be exposed to the host humoral soluble factors, e.g. complement, antibodies, oxidative metabolites. Respiratory burst of phagocytes may be prevented or reduced by the action of Leishmania surface molecules, e.g. acid phosphatase, other ectoenzymes, and/or LPG.

(2-4) Receptor-mediated endocytosis

of promastigotes by macrophages. Lei shmanial surface molecules, such as LPG and the Zo

proteinase (gp63), bind directly to the receptor(s) and/or indirectly through the intervention of host factors, e.g. C3 products , antibodies, fibronectin. mastigotes into phagosomes and their acidification.

(6)

(5) Internalization of bound pro Fusion of Leishmania-containing

phagosomes with lysosomes and protection of the parasites by their surface molecules, such as

LPG and the Zn-proteinase (gp63) .

(7-8) Differentiation of promastigotes into amastigotes and

multiplication of the latter in the phagolysosomes. The Leishmania parasites are protected by surface molecules, e.g. Zn-proteinase and/or by secreted factors.



521

their products to leishmanial resistance or survival and thus virulence remains to be established. (b) Leishmania differentiation toward the amastigote stage is not precisely programmed. It is very gradual and always heterogeneous among individual cells within the same species. There are also differences between different species in the ease of such differentiation. For example, heat-shock alone triggers promastigotes of the New World cutaneous Leish mania to differentiate partially toward the amastigote stage, but does not always do so for Old World species. Additional signals appear to be required for the latter to develop first into the metacyclic forms and then to the amastigotes. (c) Leishmania are not programmed to fully commit themselves to terminal! or one-way differentiation. Various stages of Leishmania species as described are reversible, specifically between the metacyclic and other promastigote forms in culture. This ability is different from, for example, the African trypanosomes. The procyclic trypanosomes differentiate into metacy elies, which do not usually revert, but instead further differentiate into a third stage in the mammalian host. Leishmania differentiation in the reverse direc tion from amastigotes to promastigotes is more rapid and more uniform in response to a simple signal of temperature shift-down. Leishmania differenti ation toward amastigotes thus may have evolved more recently and perhaps is still evolving for the adaptation of these parasites to live in the mammalian host. Developmentally regulated genes specific to the amastigote stage may yet prove to play important role(s) in leishmanial virulence. Interestingly, this is not the cas.e for molecules that are thought important in leishmanial infection of macrophages. For example, Leishmania gp63, LPG, and acid phosphatase have all been found in both stages of the parasites. Data indicate, however, that the expression of the two ectoenzymes as virulent determinants is related to their N-glycosylation. In vitro evidence suggests that this may be regulated by amplification of the gene encoding a glycosyltransferase of the dolichol pathway for the glycosylation. The effects of other posttranscriptional mod ifications on leishmanial virulence factors remain to be studied. The possible advantage of such regulatory mechanisms over direct transcriptional control of virulence genes is at present unclear. All potential determinants of leishmanial virulence so far inferred are based on correlative evidence or that obtained from protective or inhibitory types of biological experiments . Leishmania species can now be successfully trans fected with foreign genes by electroporation for expression (76, 85). Viruses have also Ibeen reported in Leishmania and may be exploited for transforma tion of these cells (159). In addition, antisense oligo-nucleotides can be synthesized on the basis of available nucleotide sequences to inhibit the expression of specific Leishmania genes. These genetic approaches may prove valuable for the study of relevant genes and their products to obtain

522

CHANG ET AL

direct evidence of their roles in Leishmania virulence. Intensive efforts to apply these new tools are expected during the next several years to gain significant insights in these and other areas of Leishmania biology.


ACKNOWLEDGMENTS

KPC and GC are supported by NIH-NIAID Grant No. AI-20468; DF is supported by NIH-NIAID Grant No. 2 1 364 and the Charles and Johanna Busch Memorial Fund. Literature Cited 1 . Alcina, A . , Fresno, M. 1988. Early and late heat-induced proteins during Leish mania transfonnation. mexicana Biochem. Biophys. Res. Commun. 156: 1 360-67 2. Alexander, J . , Russell , D. G. 1985. Par asite antigens, their role in protection, diagnosis and escape: The Leish maniases. Curr o Top. Microbial. Im munol. 120:43-67 3. Alfieri, S. C . , Ramazcillcs, C . , Zilbcr farb, V . , Galpin, I . , Nonnan, S. E., Rabinovitch, M . 19 88. Proteinase in hibitors protect Leishmania amazonensis amastigotes from destruction by amino acid ester. Mol. Biochem. Parasitol. 29: 1 9 1-201 4. Antoine, J. C . , Jouanne , C . , Ryter, A. 1989. Megasomes as the targets of leucine methyl ester in Leishmania ama zonensis amastigotes. Parasitology 99: 1- 10 5 . Aronow, B., Kaur, K ., McCartan, K. , Ullman, B. 1987. Two high affinity nu cleoside transporters in Leishmania donovani. Mol. Biochem. Parasitol. 22:29-37 6. Avila, J. L., Rojas, M . , Galili, U. 1989. Immunogenic Gal alphal-3Gal carbo hydrate epitopes are present on pathogenic American Trypanosoma and Leishmania. J. Immunol. 142:2828-34 7. Bard, E. 1989. Molecular biology of Leishmania. Biochem. Cell Bioi. 67: 5 1 6-24 8. Bates, P. A . , Dwyer, D. M. 1987. Biosynthesis and secretion of acid phos phatase by Leishmania donovani pro mastigotes. Mol. Biochem. Parasitol. 26:289-96 9. Bates, P. A . , Hennes, I . , Dwyer, D. M. 1989. Leishmania donovani : Im munochemical localization and secretory mechanism of soluble acid phosphatase. Exp. Parasitol. 68:335-46

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