No abstract

A class of membrane molecules has been identified whose primary translation product includes a COOH-terminal protein sequence that signals attachment of a glycosyl-phosphatidylinositol anchor at a COOH-terminal residue that is newly formed by cleavage of the signaling sequence. This class includes a wide diversity of protein types from eukaryotes at many stages of evolution. The structures of the glycosyl-phosphatidylinositol anchors are being resolved, but their functions aside from membrane attachment and dynamics remain to be determined.

African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.

The WHO recognizes human African trypanosomiasis, Chagas disease and the leishmaniases as neglected tropical diseases. These diseases are caused by parasitic trypanosomatids and range in severity from mild and self-curing to near invariably fatal. Public health advances have substantially decreased the effect of these diseases in recent decades but alone will not eliminate them. In this Review, we discuss why new drugs against trypanosomatids are required, approaches that are under investigation to develop new drugs and why the drug discovery pipeline remains essentially unfilled. In addition, we consider the important challenges to drug discovery strategies and the new technologies that can address them. The combination of new drugs, new technologies and public health initiatives is essential for the management, and hopefully eventual elimination, of trypanosomatid diseases from the human population.

Two forms of protein-membrane anchor have been described for the externally disposed glycoproteins of eukaryotic plasma membranes; namely, the hydrophobic transmembrane polypeptide and the complex glycosylphosphatidylinositol (G-PI) moiety. The chemical structures of the major species of G-PI anchors found on a single variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei were determined by a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, chemical modification, and exoglycosidase digestions. The G-PI anchor was found to be heterogeneous with respect to monosaccharide sequence, and several novel glycosidic linkages were present. The results are pertinent to the mechanism of the biosynthesis of G-PI anchors.

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the ␣1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the ␣1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1⌬ null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.Candida albicans is a commensal organism carried by a significant proportion of healthy individuals. It is the most common opportunistic fungal pathogen of humans causing superficial infections of the mucosa and in the immunocompromised host life-threatening systemic infections (1-4). The cell wall is the immediate point of contact between fungus and host and plays an important role in adherence, antigenicity, and the modulation of the host immune response (5-9). The outer layer of the cell wall is enriched in highly glycosylated mannoproteins (10), and both the protein and carbohydrate components have been implicated in the host-fungal interaction (5, 6, 11). The study of glycosylation in C. albicans therefore has its own relevance in identifying the carbohydrate epitopes involved in pathogenesis.Cell surface mannoproteins contain both O-and N-linked oligosaccharides. The O-linked oligosaccharides, attached to serine or threonine, consist of a linear chain of one to five ␣1,2-linked mannose residues (12)(13)(14) and are known to be required for full virulence (14). The process of N-glycosylation has been studied extensively in Saccharomyces cerevisiae. N-Linked glycosylation is initiated in the endoplasmic reticulum with the transfer of the Glc 3 Man 9 GlcNAc 2 oligosaccharide precursor to the protein target (15, 16). The oligosaccharide precursor is then processed by endoplasmic reticulum-resident glucosidases and a mannosidase to yield the mature triantennary Man 8 GlcNAc 2 core (17). Outer chain ...

Sand flies are the exclusive vectors of the protozoan parasite Leishmania1, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel (PSG)2,3. Here, we analyse for the first time the inocula from Leishmania mexicana infected Lutzomyia longipalpis sand flies. This revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of PSG found to accompany the parasites during transmission. Collectively, these results have important implications for understanding the relationship between parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasise that to fully understand transmission of vector-borne diseases the interaction between all three participants must be considered.Leishmaniasis is a parasitic disease that currently infects some 12 million people worldwide, causing severe morbidity and mortality4. Infection is initiated by distinct life cycle stages, metacyclic promastigotes, that are introduced into the skin by fly bite along with sand fly saliva5-7. Leishmania are known to express various "virulence factors" in the sand fly, which may facilitate transmission to and infection of the mammalian host8-12. However, despite these discoveries our knowledge of parasite molecules that facilitate sand fly transmission is still limited. Furthermore, a number of key issues of transmission remain unresolved, such as the true infective dose, the mechanism of parasite delivery and the biological consequences of these upon infection. Significantly, in all Leishmania-vector combinations examined to date a gel-like plug, the parasite-derived PSG, blocks the anterior parts of the midgut coincident with the accumulation of metacyclic promastigotes2,3. An important structural component of PSG is fPPG, an unusual mucin-like glycoprotein unique to Leishmania13,14. Here we address these issues regarding transmission and reveal a novel contribution made by L. mexicana PSG to the infection process.Correspondence and requests for materials should be addressed to P. A.B. (pbates@liv.ac.uk To begin to understand the nature of the infective inoculum, the number and composition of L. mexicana parasites delivered during transmission was determined. A membrane feeding system was adapted to collect parasites egested by infected sand flies, revealing an average of 1086 parasites delivered per bite, highly enriched in metacyclic promastigotes (86-98%) ( Table 1). The only previous investigations quantitating egested parasites have been made using microcapillary forced feeding15,16. When this method was employed on L. mexicanainfected sand flies an average of 105 promastigotes were collected per...

Vespa et al., 1994;Biron and Gazzinelli, 1995 Aliberti et al., 1996;Fearon and Locksley, 1996; Trinchieri and Scott, 1996). In addition, the early Seder et al., 1993; Abbas et al., 1996; Fearon macrophage proinflammatory cytokines by Trypanoand Locksley, 1996). During infection with T.cruzi, the soma cruzi is considered to be important in controlling induction of parasite-specific CMI is likely to be involved the infection and the outcome of Chagas' disease. Here in at least two aspects of Chagas' disease pathophysiology we show that the potent tumour necrosis factor-α-, (Vespa et al., 1994;Fearon and Locksley, 1996; Brener interleukin-12-and nitric oxide-inducing activities of and Gazzinelli, 1997). The first is the control of parasite T.cruzi trypomastigote mucins were recovered quantireplication and its spread in the vertebrate host tissues. tatively in a highly purified and characterized glycosyl-The second is the inflammatory reaction observed in the phosphatidylinositol (GPI) anchor fraction of this infected host tissues, which is likely to be a major cause material. The bioactive trypomastigote GPI fraction of cardiac tissue damage during the acute and chronic was compared with a relatively inactive GPI fraction phases of the disease. prepared from T.cruzi epimastigote mucins. The trypoRelatively little is known about the protozoan parasite mastigote GPI structures were found to contain addimolecules that initiate the synthesis of pro-inflammatory tional galactose residues and unsaturated, instead of cytokines and nitric oxide (NO) by macrophages. Recent saturated, fatty acids in the sn-2 position of the alkylstudies have suggested a role for glycosylphosphatidylacylglycerolipid component. The latter feature is esseninositol (GPI) anchors from Plasmodium falciparum tial for the extreme potency of the trypomastigote (Schofield and Hackett, 1993;Schofield et al., 1996; GPI fraction, which is at least as active as bacterial Tachado et al., 1996Tachado et al., , 1997 and Trypanosoma brucei endotoxin and Mycoplasma lipopeptide and, therefore, (Magez et al., 1998) in this process. However, no evidence one of the most potent microbial proinflammatory of the biochemical purity of the P.falciparum GPIs was agents known.provided, making estimates of their concentrations dubiKeywords: Chagas' disease/cytokines/glycosylphosphaous. Furthermore, the possibility of Mycoplasma lipopeptidylinositol/inflammation/nitric oxide tide (Mühlradt et al., 1997) contamination (that has confounded other studies into proinflammatory factors) cannot be formally excluded. In the case of the T.brucei GPI work, highly purified and structurally characterized