Confronting parasites from Canada

Share Embed


Descrição do Produto

Research Update

TRENDS in Parasitology Vol.18 No.12 December 2002

519

Research News

Confronting parasites from Canada Terry W. Spithill, Kris Chadee, Armando P. Jardim, Roger K. Prichard and Paula Ribeiro The Institute of Parasitology at McGill University, Quebec, Canada, was founded in 1934 and is the major site for research in molecular parasitology in Canada. Two main research themes are studied: pathogenesis and host defense mechanisms; and the molecular basis of drug resistance and drug target discovery. This article highlights recent research at the Institute in immunology, glycosome biogenesis, biochemistry and genetics of drug resistance, and helminth neurochemistry.

Published online: 24 October 2002 Parasitology as a research discipline is now entering the post-genome era [1]. Genomic analysis of mammalian hosts and parasites will provide us with the core knowledge of host and parasite genes and their predicted products, giving us tools to study host–parasite interactions at a whole genome level, thus allowing us to integrate gene expression with functional outcomes. The Institute of Parasitology at McGill University, Quebec, Canada, is embracing these opportunities and is adopting a molecular approach to the study of host–parasite relationships. Immunology and vaccine studies

The research in Kris Chadee’s laboratory focuses on Entamoeba histolytica molecules that induce inflammatory responses to alter the mucosal epithelium, thus initiating parasite invasion in the gut. Intrinsic E. histolytica proteins, secretory components released by live trophozoites, or products released by parasites co-cultured with colonic cells stimulate the expression of interleukin (IL)-8 in colonic epithelial cells [2]. IL-8 might have a major role in amoeba pathogenesis by causing nonspecific tissue injury, which is induced by neutrophil chemotaxis and activation within the tissues. Adam Belley and Chadee observed that E. histolytica releases prostaglandin E2, a potent stimulator of IL-8 [3] and mucus secretion in the gut; thus, initiating and/or exacerbating mucosal inflammation. Current research uses a proteomic approach to identify http://parasites.trends.com

parasite molecules that modulate the functions of the epithelial barrier. In particular, cysteine proteinases are being characterized for their ability to degrade protective mucins and/or epithelial-cell-adhesion proteins. The Gal-lectin of E. histolytica is a major surface molecule that allows the parasite to adhere to colonic mucin for colonization and to target cells for invasion (Fig. 1a). Gal-lectin is immunogenic and represents a potential vaccine candidate against amoebiasis. The region(s) of Gal-lectin involved in amoebic adherence to target cells and in stimulating release of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α and IL-12 from macrophages have been mapped [4,5]. Cell-mediated immunity mediated by T helper cell (Th) 1 responses is central in the host defense against E. histolytica. Because Gal-lectin contains its own Th1-adjuvant, a codon-optimized DNA vaccine encoding the carbohydratebinding domain was constructed. The vaccine stimulated Th1-like immune responses, and anti-Gal-lectin antibodies markedly inhibited amoebic adherence to target cells [6]. Now, research is aimed at developing a DNA vaccine to block parasite colonization and/or invasion using mucosal adjuvants. Terry W. Spithill’s laboratory is investigating the immunosuppressive mechanisms used by Fasciola hepatica to modulate host responsiveness. Fasciola cathepsin L proteases suppress lymphocyte proliferation in vitro and downregulate the expression of CD4 on T cells, probably by cleavage of CD4 from the T-cell surface [7]. Removal of CD4 on T cells by cathepsin L might be a parasite strategy to modulate host immunity. Spithill’s laboratory is also focused on malaria proteomics, malaria and liver fluke vaccine discovery, and developing technology for delivering multivalent DNA-based malaria vaccines using bicistronic vectors in the Plasmodium chabaudi–mouse model. One aspect of the work in Marilyn E. Scott’s laboratory is the effects

of protein malnutrition on nematode parasite survival, which showed that malnutrition increases the survival of Heligmosomoides by decreasing gutassociated IL-4 responses and increasing interferon (IFN)-γ levels following infection, resulting in reduced intestinal and systemic Th2 effector responses [8]. Gaetan M. Faubert’s laboratory is aiming to elucidate the immunology of Giardia infection and to develop vaccines that target the cyst wall proteins of Giardia [9]. Glycosome assembly in Leishmania

Leishmania and Trypanosoma have several vital metabolic functions, including glycolysis, compartmentalized in a microbody organelle called a glycosome [10]. Enzymes involved in these pathways are nuclear-encoded, and imported post-translationally into the glycosome. Mistargeting of some glycosomal enzymes to the cytosolic compartment impairs parasite viability [11], making the glycosome biogenesis machinery an attractive target for chemotherapy. Targeting of proteins into the glycosome is primarily dependent on two motifs: PTS-1, a COOH-terminal tripeptide signal; and PTS-2, a more degenerate NH2-terminal nonapeptide signal sequence [10]. However, the mechanisms involved in translocating proteins across the glycosome membrane are unknown. The Leishmania peroxin (LdPEX) 5, a cytosolic receptor protein that selectively binds newly synthesized glycosomal matrix proteins bearing a PTS-1 signal, has been characterized [12]. LdPEX14, the second component of the glycosome assembly machinery, is a glycosomal membrane-associated protein that acts as a docking receptor, permitting PTS-1-laden LdPEX5 to bind to the cytosolic surface of the glycosome (Fig. 1b). To elucidate the molecular events and components connected with the import of proteins into the glycosome, Armando P. Jardim’s laboratory is using a functional proteomics approach with LdPEX5 and LdPEX14 as bait molecules to isolate novel proteins involved in

1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)02369-3

520

Research Update

TRENDS in Parasitology Vol.18 No.12 December 2002

functional genomics and heterologous expression in Caenorhabditis elegans to elucidate the role of ABC transporters, tubulins and ligand-gated ion channels in anthelmintic action, and to develop diagnostic molecular tools for resistance (Fig. 1c). Studies in the Elias Georges’ laboratory are based on defining proteins, such as the multidrug resistance protein MRP, involved in multidrug resistance in human cancer cells [18], with comparative studies in Plasmodium. Neurochemistry and gene expression

Fig. 1. (a) An Entamoeba histolytica trophozoite in contact with gerbil colonic epithelial cells before invasion. This host–parasite interaction involves the Entamoeba surface protein Gal-lectin, which is being targeted as a vaccine candidate to control Entamoeba invasion [6]. The cells were treated with periodic-acid Schiff reagent and stained with Alcian blue. (b) Confocal immunofluorescence image of Leishmania donovani promastigotes, double-stained for: (1) Leishmania peroxin (LdPEX) 5, a cytosolic receptor for the PTS-1 topogenic signal (red); and (2) LdPEX14, a glycosome membrane-associated protein (green). (c) The micro-injection of constructs encoding green fluorescent protein (GFP) into Caenorhabditis elegans ovaries is often used to study the expression of heterologous parasitic nematode genes. In (c), GFP expression is targeted to the somatic muscles of C. elegans. (d) Three-dimensional computer model of a novel serotonin receptor from C. elegans. A lateral view of the seven transmembrane helices is seen with the extracellular surface oriented towards the top. The seven helices (different colours) are arranged in a counterclockwise orientation from helix 1 (purple) to helix 7 (red). A predicted serotonin-binding site is formed from conserved residues of helix 3 (light blue) and helix 6 (orange) [19]. Scale bar = 5 µm (a), 2 µm (b) and 75 µm (c).

glycosome biogenesis. Identification of these novel proteins by mass spectrometry has been greatly facilitated by the rapidly growing Leishmania genome database [1]. Drug action and molecular genetics of drug resistance

Roger K. Prichard and Robin Beech’s labs are examining the basis of selective toxicity of anthelmintic drugs. Their work has shown that the macrocyclic lactone (ML) anthelmintics, such as ivermectin (IVM), act on glutamate-gated chloride channel (GluCl) [13] and aminobutyrategated chloride channel (GABACl) subunits [14] in Haemonchus contortus and other parasitic nematodes. These ligand-gated chloride channels are also implicated in the mechanism of ML resistance in H. contortus [14,15], in which specific GluCl and GABACl alleles are selected. Resistance alleles have been sequenced and compared with the wild-type (susceptible) alleles, and similar amino acid changes in the ligand-binding domain have been found in both the GluCl http://parasites.trends.com

and GABACl resistance alleles when compared with their respective wild-type alleles. The resistant and susceptible forms of the ligand-gated chloride channels have been expressed separately in Xenopus oocytes to form ion channels; using these oocytes, studies of the chloride conductance responses to GABA and glutamate, and to IVM, have revealed marked differences between the channels expressed from the two alleles. For example, with the susceptible GABACl alleles, IVM enhanced the chloride conductance response to GABA, whereas with the resistant alleles, this response was ablated by IVM [14]. It is apparent that the mode of action and mechanism of resistance to ML involves both GluCl and GABACl in parasitic nematodes. In addition to ligand-gated chloride channels, ML resistance and IVM tolerance also appears to be modulated by selection on P-glycoprotein and possibly other ATP-binding cassette (ABC) transporter genes [16,17]. A major axis of research at the Institute is to use

The helminth nervous system coordinates all major activities of the parasite in the host and also represents an attractive target for chemotherapy. Researchers at the Institute are exploring mechanisms of chemical neurotransmission in Haemonchus, Onchocerca and Schistosoma mansoni. Paula Ribeiro’s laboratory is focusing on the properties of biogenic amine neurotransmitters, such as serotonin, histamine and the catecholamines, which have important roles in the regulation of parasite motility, feeding and reproduction. A functional genomics approach is being used to clone and characterize novel biogenic amine cell-surface receptors [19] (Fig. 1d). A G-protein coupled receptor was isolated from S. mansoni (SmGPCR) and selectively activated by histamine [20,21]. A combination of sequence analyses and mutagenesis demonstrated that SmGPCR was a new structural class of the amine GPCR, which differed significantly from mammalian histamine receptors, and could be unique to flatworms [21]. SmGPCR will be investigated further as a potential target for anthelmintic development, and to elucidate its function in vivo. Little is known about helminth transcription factors and how their activities are regulated in the parasite. A recent study showed that a new S. mansoni proteasome subunit (SmPOH) stimulated AP1-mediated transcription when expressed transiently in a heterologous system [22]. Further studies revealed that the effect of SmPOH on transcription was associated with a stabilization of the transcription factor, c-Jun, and an apparent relocalization of c-Jun towards the nuclear periphery. Studies are continuing to explore the properties of the flatworm proteasome and its role in regulating transcription factor degradation and activity.

Research Update

TRENDS in Parasitology Vol.18 No.12 December 2002

Future perspectives

Using recent major grants to re-equip the Institute (see: http://www.mcgill.ca/ parasitology/), our research will increasingly use functional genomemining and genome analyses to identify key parasite and host proteins. We are committed to using proteomics to study protein–protein interactions, to identify novel proteins of functional importance, and to meet the challenge of assigning function to unidentified reading frames found in parasite genomes. An important ongoing responsibility for the Institute is the teaching and training of graduates in parasitology who will accept the exciting challenge to confront the continued threat of parasitic diseases.

3

4

5

6

7

Acknowledgements

Research at the Institute is supported by grants from several agencies: Fonds pour la Formation de Chercheurs et l’Aide a la Recherche; WHO; Canadian Institutes of Health Research; Natural Sciences and Engineering Research Council of Canada; Crohn’s and Colitis Research Foundation of Canada; Canadian Foundation for Innovation; Canada Research Chairs Program; Fort Dodge Animal Health; Cooperative Research Centre for Vaccine Technology, Australia; Australian Centre for International Agricultural Research; and McGill University. References 1 Ashton, P.D. et al. (2001) Linking proteome and genome: how to identify parasite proteins. Trends Parasitol. 17, 198–202 2 Yu, Y. and Chadee, K. (1997) Entamoeba histolytica stimulates interleukin-8 from human colonic

8

9 10

11

12

13

epithelial cells without parasite–enterocyte contact. Gastroenterology 112, 1536–1547 Belley, A. and Chadee, K. (1999) Prostaglandin E2 stimulates rat and human colonic mucin exocytosis via the EP4 receptor. Gastroenterology 117,1352–1362 Seguin, R. et al. (1995) Identification of the galactose adherence epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages. Proc. Natl. Acad. Sci. U. S. A. 92, 12175–12179 Campbell, D. et al. (2000) A subunit vaccine candidate region of the Entamoeba histolytica galactose-adherence lectin promotes interleukin-12 gene transcription and protein production in human macrophages. Eur. J. Immunol. 30, 423–430 Gaucher, D. and Chadee, K. (2002) Construction and immunogenicity of a codon-optimized Entamoeba histolytica Gal-lectin based DNA vaccine. Vaccine 20, 3244–3253 Prowse, R.K. et al. (2002) Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cells. Parasite Immunol. 24, 57–66 Ing, R. et al. (2000) Suppressed T helper 2 immunity and prolonged survival of a nematode parasite in protein-malnourished mice. Proc. Natl. Acad. Sci U. S. A. 97, 7078–7083 Faubert, G. (2000) Immune response to Giardia duodenalis. Clin. Microbiol. Rev. 13, 35–54 Parsons, M. et al. (2001) Biogenesis and function of peroxisomes and glycosomes. Mol. Biochem. Parasitol. 115, 19–28 Blattner, J. et al. (1998) Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism. Proc. Natl. Acad. Sci. U. S. A. 95, 11596–11600 Jardim, A. et al. ( 2000) Peroxisomal targeting signal-1 receptor protein PEX5 from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization. J. Biol. Chem. 275, 13637–13644 Forrester, S.G. et al. (2002) A glutamate-gated chloride channel subunit from Haemonchus contortus: Expression in a mammalian cell line, ligand-binding and modulation of anthelmintic

521

14

15

16

17

18

19

20

21

22

binding by glutamate. Biochem. Pharmacol. 63, 1061–1068 Feng, X–P. et al. Study of the nematode putative GABA type A receptor subunits: Evidence for modulation by ivermectin. J. Neurochem. (in press) Blackhall, W.J. et al. (1998) Haemonchus contortus: Selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectinselected strains. Exp. Parasitol. 90, 42–48 Xu, M. et al. (1998) Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog. Mol. Biochem. Parasitol. 91, 327–335 Huang, Y-J. and Prichard, R.K. (1999) Identification and stage-specific expression of two putative P-glycoprotein coding genes in Onchocerca volvulus. Mol. Biochem. Parasitol. 102, 273–281 Daoud, R. et al. (2001) Major photoaffinity drug binding sites in MRP1 are within TM10-11 and TM16-17. J. Biol. Chem. 276, 12324–12330 Hamdan, F. et al. (1999) Characterization of a novel serotonin receptor from Caenorhabditis elegans: Cloning and functional expression of two splice variants. J. Neurochem. 72, 1372–1383 Hamdan, F. et al. (2001) A novel Schistosoma mansoni G protein – coupled receptor is responsive to histamine. Mol. Biochem. Parasitol. 119, 75–86 Hamdan, F. et al. (2002) Codon–optimization improves heterologous expression of a Schistosoma mansoni cDNA in HEK293 cells. Parasitol. Res. 88, 583–586 Nabhan, J. et al. (2001) A Schistosoma mansoni Pad1 homologue stabilizes c-Jun. Mol. Biochem. Parasitol. 121, 163–172

Terry W. Spithill* Kris Chadee Armando P. Jardim Roger K. Prichard Paula Ribeiro Institute of Parasitology, McGill University, 21,111 Lakeshore Rd, Ste-Anne de Bellevue, Quebec, Canada H9X 3V9. *e-mail: [email protected]

Meeting Report

Frontiers in research on parasitic protozoa Wendy Gibson and Michael Miles Frontiers in research on parasitic protozoa was the theme of the Autumn Symposium of the British Section of the Society of Protozoologists, held 2 September 2002, in London, UK.

from genomics to drug treatment and vaccination, with a representative cast of pathogens including Plasmodium, kinetoplastids, Entamoeba and Eimeria. Genomes and proteomes

Protozoologists fall into two camps, studying either free-living or parasitic protozoa. This year’s British Section of the Society of Protozoologists (BSSP) symposium focused on parasitic protozoa, http://parasites.trends.com

Few free-living protozoa have their own genome projects (Tetrahymena, Dictyostelium), whereas some genera of parasitic protozoa (Plasmodium, Trypanosoma) now boast several.

Many genome projects are justified because so much insight on parasite–host relationships, pathogenicity and recent evolution can be gained by comparative genomics of closely related organisms. Al Ivens (Sanger Institute, Cambridge, UK) and Adam Witney (St George’s Hospital, London, UK) described the latest technologies for genome sequencing and for microarray analysis of gene expression, including the complexities of meticulously

1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)02416-9

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.