Tenth Annual Quebec
Molecular Parasitology Symposium
10ième Symposium Annuel
de Parasitologie Moléculaire du Québec
June 8 - 9, 2010
Trottier Building, 3630 University Street
McGill University
Department of Biology
Montréal, Québec, Canada
McGill Main Campus Map
Trottier Building, 3630 University Street
SSMU Ballroom, 3480 McTavish, 3rd floor
1
CHPI 2010 Program
Tuesday, June 8th (Trottier 0100)
9 :00-9 :15
Opening Remarks (Dr. Armando Jardim, Director of the CHPI)
9:15-10:15
Keynote Speaker: Dr. Greg Lanzaro
“Sand Fly Saliva and Leishmania Transmission”
10:15-10:30
COFFEE BREAK (Trottier Lobby)
Session I:
Immunity
(Trottier 0100)
(Chair: Mary Stevenson)
10:30-10:55 Louis-Philippe Leroux, Manami Nishi, and Florence Dzierszinski: Subversion of
the MHC-II pathway by Toxoplasma gondii: surprising discoordinate expression
of MHC-II and the Invariant chain (Ii).
10:55-11:20 Floriana Berretta, Piccirillo C.A., and Stevenson M.M.: Decreased numbers of
Foxp3+ regulatory T cells correlates with decreased IL-2 production and Th1
polarization of effector CD4+ T cells in P. chabaudi AS infected C57BL/6 mice.
11:20-11:45 Fikregabrail A. Kassa, Marina Tiemi Shio, Marie-Josée Bellemare, Momar
Ndao and Martin Olivier: Identification of inflammation-related biomarkers from
human serum adhering to the malaria pigment hemozoin.
11:45-12:10 Diane Tshikudi M., François Désautels, Abhay Satoskar and Tatiana Scorza:
MIF: a down-regulator of early T cell-dependent IFN responses in Plasmodium
chabaudi adami (DK) infected mice.
12:10-1:45
LUNCH
(Trottier Lobby)
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Session II: Biochemistry and Physiology
(Chair: Elias Georges)
(Trottier 0100)
2:00-2:25
Rona Strasser, A.V. Pilar, and A. Jardim: Leishmania donovani receptor-cargo
protein complexes form in the cytosol and dock at glycosomal protein Peroxin14,
modulating its conformation.
2:25-2:50
Laura-Isobel McCall, Greg Matlashewski: Involvement of the Leishmania
donovani virulence factor A2 in the parasite stress response.
2:50-3:15
Virginie Barrère, Roger Prichard, Luis Alvarez : Benzimidazole (BZ) resistance
in sheep nematode Haemonchus contortus, Single Nucleotide Polymorphism
(SNP) associations on beta tubulin isotype 1 gene.
3:15-3:30
COFFEE BREAK (Trottier Lobby)
3:30-3:55
Normand Cyr, Armando Jardim: Biophysical characterization of the Leishmania
donovani PEX14 and its role in the glycosomal translocation machinery.
3:55-4:20
Pranav Kumar, Éric Madore, Frédéric Raymond, Michel J. Tremblay : Gene
expression modulation in nelfinavir–resistant Leishmania donovani amastigotes is
associated with gene amplification, gene deletion, formation of supernumerary
chromosomes and chromosome loss.
4:20-4:45
Daniel Feingold, Joseph A. Dent and Laura Nilson: Three novel Cys loop ligandgated ion channels in Drosophila melanogaster: Potential pesticide targets?
4:45-7:00
POSTER SESSION & aperitifs
7:00-9:00
DINNER
(SSMU Ballroom)
(SSMU Ballroom)
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Wednesday, June 9th
(Trottier 0100)
9:15-10:15
Keynote Speaker: Dr. Derek McKay
“Immunomodulation by the tapeworm parasite, Hymenolepis diminuta"
10:15-10:30
COFFEE BREAK (Trottier Lobby)
Session III: Signalling
(Trottier 0100)
(Chair: Timothy Geary)
10:30-10:55 Claudia M. Wever, Patrick Janukavicius, Claire Bottomley, Danielle Farrington,
Igor Putrenko and Joseph A. Dent: Validating Acetylcholine-Gated Chloride
Channels as Novel Nematocide Targets.
10:55-11:20 Nicholas Patocka, Paula Ribeiro: Exploring Serotonin Signaling in Schistosoma
mansoni: Transporter and Receptor.
11:20- 11:45 Elizabeth Ruiz-Lancheros, Timothy Geary: Novel approach for receptor
deorphanization in the model nematode, Caenorhabditis elegans.
11:45-12:10 Kevin MacDonald and Paula Ribeiro: Silencing Acetylcholine-gated Chloride
Channel Subunits in Schistosoma mansoni.
12:10-1:45
LUNCH
(Trottier Lobby)
Session IV: Immunity and Epidemiology
(Chair: Florence Dzierszinski)
(Trottier 0100)
2:00-2:25
Theresa W. Gyorkos, Serene A. Joseph, Mathieu Maheu-Giroux: Evidence
supporting deworming in children under 24 months of age.
2:25-2:50
Kasra Hassani, Elisabeth Antoniak, Marie-Josée Bellemare, Armando Jardim,
Martin Olivier: Temperature-induced protein secretion by Leishmania mexicana
modulates host macrophage signaling and functions.
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2:50-3:15
Felix Hugentobler, Karen K. Yam, Martin Olivier and Benoit Cousineau:
Development of a new generation of live vaccines against leishmaniasis using the
Gram-positive bacterium Lactococcus lactis.
3:15-3:30
COFFEE BREAK (Trottier Lobby)
3:30-3:55
Yovany Moreno, Joseph F. Nabhan, Jonathan Solomon, Charles D. Mackenzie
and Timothy G. Geary: Ivermectin disrupts the function of the excretory-secretory
apparatus in microfilariae of Brugia malayi.
3:55-4:20
Marie-Josée Bellemare, Marina Tiemi Shio and Martin Olivier: A New Vaccine
Adjuvant: Nanocrystal Hemozoin Embedded with TLR Ligands and Antigens.
4:20
CLOSING REMARKS AND PRIZES
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Posters
1. Marie-Christine Brotherton, Gina Racine, Aude L. Foucher, Jolyne Drummelsmith,
Barbara Papadopoulou, Marc Ouellette : Analysis of stage-specific expression of basic
proteins in Leishmania infantum.
2. Erin Dodd, D. Scott Bohle: Gallium Analogs of Hematin Anhydride: novel approaches to
exploring the structure and drug interactions of malaria pigment.
3. Colin Du, Nakita Haynes, Hamed Shateri Najafabadi, Reza Salavati, and Janet Yee:
Characterization of p105: A Putative Transcription Factor in Giardia lamblia
4. Carolina P.Gómez, Marina Shio, Martin Olivier, Albert Descoteaux : Role of the protein
SHP-1 during the process of phagocytosis in murine macrophages.
5. Stephanie Goyette, Momar Ndao, Brian J. Ward, and Florence S. Dzierszinski : Insights
into Toxoplasma gondii reactivation through biomarker studies.
6. Raya Mahbuba, Karen K. Yam, Felix Hugentobler, Benoit Cousineau: Generation and
evaluation of SMT-expressing lactococcal live vaccines against visceral leishmaniasis.
7. James McLean, Armando Jardim: Purification and characterization of the Leishmania
major glycosomal PTS2 receptor, Peroxin 7.
8. Nilmar S. Moretti; Luiz R. O. Tosi; Barbara Papadopoulou: Study the role of histone
deacetylation on the control of gene expression in the protozoan parasite Leishmania
9. Amber Olson, Kin Chan, Chi-Yip Ho, and Janet Yee: Gene expression profiling of
Giardia lamblia isolates.
10. Chase Reaume, Christine Ouellet, and Janet Yee: The application of counterflow
centrifugal elutriation and quantitative RT-PCR in the cell cycle analysis of Giardia
lamblia
11. Sarah J. Reiling, Petra Rohrbach: Analyzing transporters and ion channels of the
digestive vacuolar membrane of Plasmodium falciparum.
12. T. Scorza, D. Malu Tshikudi, O. Kevorkova, H. Romero, J. Lahssini, J. Sanchez
Dardon and R. Moreau: Changes in bone remodelling associated to Plasmodiuminduced and phenylhydrazine-induced haemolytic anaemia.
13. Hamed Shateri Najafabadi, Reza Salavati: A unified approach for identification of cooccurring features among all data types within biological networks
14. Dagobert Tazoo, David Scott Bohle and Ovidiu Oniga: Synthesis and antimalarial
activity of chalcones and thiophosphinic acids derivatives.
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Abstracts
Virginie Barrère, Roger Prichard, Luis Alvarez, Institute of Parasitology, McGill Univ.
Benzimidazole (BZ) resistance in sheep nematode Haemonchus contortus, Single Nucleotide Polymorphism
(SNP) associations on beta tubulin isotype 1 gene
Three mutations called Single Nucleotide Polymorphism (SNP) on beta tubulin isotype1 gene can be used as
markers to detect benzimidazole (BZ) resistance in Haemonchus contortus. The SNPs at codon 200 and 167 are
caused by a change from phenylalanine residue (TTC in BZ susceptible parasites) to a tyrosine residue (TAC in BZ
resistant parasites). The last mutation is localized at the residue 198; a glutamate residue (GAA for BZ susceptible
parasites) changed to alanine (GCA in BZ resistant parasites). Pyrosequencing is a technique which can be used to
detect the frequency of the BZ resistant and susceptible alleles in H. contortus. Infection of four groups of ten lambs
with 10 000 H. contortus larvae was performed and BZ treatments at recommended dose, three times and nine times
the recommended dose were applied on three different groups of animals. One group was kept untreated. Adult
parasites that survived the treatment were collected from the abomasum of lambs. DNA extraction was performed
on each worm. We amplified the beta tubulin isotype1 gene of every parasite and analyzed the frequency of the
three SNPs using pyrosequencing. The SNP198 did not show any variability among the whole population of
parasites tested. By comparing the frequency of resistant alleles in the different treatment populations, we observed
that, at the recommended dose, the parasites harboured mainly homozygous TTC at position 167 and homozygous
TAC at position 200. This association confers a high level of resistance to the anthelmintic; the few parasites that
survived nine times the recommended dose all harboured this association. No association between homozygous
TAC at both position 167 and 200 was detected. The different combinations that confer BZ resistance observed
among the three populations were analyzed and we conclude that only the 200TAC confers high level resistance.
Marie-Josée Bellemare, Marina Tiemi Shio and Martin Olivier, The Research Institute of the McGill University
Health Centre, Departments of Medicine, Microbiology and Immunology, McGill University, Montréal , QC.
A New Vaccine Adjuvant: Nanocrystal Hemozoin Embedded with TLR Ligands and Antigens
Hemozoin (HZ) is a heme-metabolic waste produced by Plasmodium spp during malarial infection. Several reports
have been showing the pro-inflammatory role of native and synthetically produced HZ. Recently, we, and others
demonstrated that HZ can activate the NLRP3-inflammasome resulting in the production of IL-1 by macrophages
similarly to the conventional aluminum hydroxide adjuvant. This similarity and the fact that we can synthesize
multicrystalline domain hemozoin prompt us the idea to intricate TLR ligands and antigens into the crystals and
evaluate their effect on antigen presentation and pro-inflammatory response. The protocol to generate the synthetic
HZ has been modified to include biomolecules such as LPS and CpG to target TLR receptors and ovalbumin (OVA)
as antigen. Numerous analytical techniques confirmed the presence of these biomolecules in the core of the crystals.
Furthermore, we observed that the crystals containing the biomolecules generated higher levels of IL-1 , NO and
TNF then pure crystals. In addition, to evaluate the adjuvant effect of the PAMP-coated or -intrinsic HZ, we use a
classical model of antigen presentation with OVA-loaded macrophages and OVA-specific T cells. We observed that
co-culture of T cells with macrophages loaded with intrinsic LPS/OVA-contained HZ showed a late and longer peak
of IL-2 production (306 pg/mL, at day 2 and the production was kept for 7 days) compare with LPS/OVA-coated
HZ (84 pg/mL, after 2 days). Therefore, these novel hemozoin microcrystals seem to present a very promising
avenue for a long lasting immune stimulation, at least in vitro. Research funded by a grant from CIHR.
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Berretta, F.1,3, Piccirillo C.A.2,3, and Stevenson M.M.1,3 1Department of Medicine, 2Department of Microbiology &
Immunology, and 3RI of the MUHC, McGill University, Montreal, QC, Canada
Decreased numbers of Foxp3+ regulatory T cells correlates with decreased IL-2 production and Th1
polarization of effector CD4+ T cells in P. chabaudi AS infected C57BL/6 mice.
CD4+Foxp3+ regulatory T cells (Tregs) play a pivotal role in immunity to infection including Plasmodium parasites,
the causative agent of malaria. We analyzed the role of Tregs in C57BL/6 (B6) mice infected with P. chabaudi AS.
B6 mice are resistant to this parasite and experience a peak parasitemia of ~35% parasitized RBC on days 8-10 post
infection (p.i.) and clear blood parasitemia about 3 wks p.i. The frequencies and numbers of Tregs in the spleen of
infected mice were observed to decrease significantly during early infection compared to naïve mice. FACS analysis
of CD4+Foxp3+Ki67+ cells revealed that the collapse of Tregs was partially due to reduced proliferation compared to
CD4+Foxp3- T cells. Since IL-2, produced mainly by activated effector CD4+ T cells, is necessary to promote the
expansion and survival of Tregs, we evaluated intracellular expression of IL-2 by CD4+Foxp3- T cells by flow
cytometry. An increase in IL-2 expressing CD4+Foxp3- T cells was observed during early infection prior to peak of
parasitemia. This early increase was followed by decreases in both the frequency and number of CD4 +Foxp3-IL-2+ T
cells which paralleled the decrease in Tregs and enhanced apoptosis of these cells. Moreover, decreased IL-2
expression by CD4+Foxp3- T cells correlated with increased expression by these cells of the Th1 cytokine IFN.
Altogether, our results suggest that the ability of resistant hosts such as B6 mice to survive P. chabaudi AS infection
is due to the tight balance between Tregs and effector T cells and that IL-2 might regulate this balance. Supported
by FQRNT (Quebec) and CIHR (MOP-81169).
Marie-Christine Brotherton, Gina Racine, Aude L. Foucher, Jolyne Drummelsmith, Barbara Papadopoulou, Marc
Ouellette, Centre de recherche en Infectiologie, CHUL, Université Laval.
Analysis of stage-specific expression of basic proteins in Leishmania infantum.
The protozoan parasite Leishmania has a dimorphic life cycle where it is found as an elongated flagellated
promastigote within the sandfly midgut, and as an intracellular non-motile round amastigote within mammalian host
macrophage. In silico studies of proteomes encoded by sequenced genomes revealed that intracellular organisms
encode for more basic proteomes than free-living ones. Indeed 58.5% of the L. infantum proteins are predicted to
harbor an isoelectric point (pI) greater than 7.0. We took advantage of the liquid–based Free-flow electrophoresis
(FFE) procedure to separate L. infantum basic proteins from the two life stages according to their pI. We compared
pooled FFE fractionated proteins resolved by 2D gels. We put pooled FFE fractions corresponding to pH 7.0 to 9.0
on pH 6.0 to 9.0 strips where we resolved up to 1823 protein spots and pooled FFE fractions corresponding to pH
over 9.0 on pH 6.0 to 11.0 strips where we resolved up to 646 protein spots. The comparative analysis revealed 33
unique or upregulated spots in promastigotes and 56 unique or upregulated spots in amastigotes. The identified
proteins were consistent with the main energy source for each life stage. Indeed some glycolytic enzymes were
found to be upregulated in promastigotes and some proteins implicated in neoglucogenesis and fatty acid βoxidation were upregulated in amastigotes. We also pinpointed six upregulated proteins in promastigotes that have
already been identified in T. brucei flagellum proteome. Moreover approximately 30% of the proteins identified in
this study were confirmed for the first time by mass spectrometry. Overall, the FFE fractionation method combined
with 2DE allowed us to separate basic proteins with high efficiency and represents a very useful strategy to discover
novel proteins implicated in Leishmania differentiation.
8
Normand Cyr, Armando Jardim, Institute of Parasitology, McGill University.
Biophysical characterization of the Leishmania donovani PEX14 and its role in the glycosomal translocation
machinery
Kinetoplastids, including the genus Leishmania, compartmentalize glycolysis and other vital metabolic pathways
inside an organelle called the glycosome, a microbody distantly related to the peroxisomes of higher eukaryotes.
Proteins, in order to be translocated across the impermeable glycosomal membrane, require a peroxisomal targeting
signal (PTS) recognized cytosolically by either peroxin 5 (PEX5) or peroxin 7 (PEX7). The cargo is then brought to
the surface of the glycosome where it binds to peroxin 14 (PEX14), a glycosomal membrane associated protein
directly implicated in the translocation mechanism. Previous studies have demonstrated that proper targeting of
proteins to the glycosome is essential for the viability of the parasite and therefore represents an interesting
therapeutic target. Quaternary structure analysis of the L. donovani PEX14 revealed that this protein forms a large
oligomeric complex. Domain mapping showed that elimination of a hydrophobic region and a coiled-coil motif
were necessary to disrupt oligomer formation. Furthermore, biophysical studies, including calorimetry, analytical
ultracentrifugation, circular dichroism and intrinsic fluorescence demonstrated that binding of LdPEX5 caused a
dramatic conformational change in the LdPEX14 complex, accompanied by a reorganization of a hydrophobic
segment in common to PEX14 proteins. Moreover, protein-membrane interaction studies using liposomes that
mimic the glycosomal membrane composition, showed that LdPEX14 was capable of binding to these lipid bilayers,
and to recruit the PTS1 protein-LdPEX5 complex to the liposomes. Further investigations indicated that deletion of
the hydrophobic segment implicated in the oligomerization of LdPEX14 abrogated the capability of LdPEX14 to
bind to liposomes. These studies will allow a more fundamental understanding of the glycosome biogenesis
machinery.
Erin Dodd, D. Scott Bohle, Department of Chemistry, McGill Univ.
Gallium Analogs of Hematin Anhydride: novel approaches to exploring the structure and drug interactions
of malaria pigment
The interaction between quinoline-type anti-malarial drugs and the drug target, a form of biomineralized heme
known as hemozoin, is very poorly understood at the molecular level. This interaction is notoriously difficult to
observe using iron heme due to insolubility and paramagnetism. In this work we explore the structural interactions
between anti-malarial drug and a model heme target outside the malaria organism. To this end, diamagnetic gallium
(III) protoporphyrin IX dimer has been developed as a model for the heme complex and tested against drug by NMR
and fluorescence methods. The data show dramatic structural rearrangements and allow determination of structural
interactions and binding affinity.
Colin Du1, Nakita Haynes2, Hamed Shateri Najafabadi3, Reza Salavati3, and Janet Yee1
1
Environmental and Life Sciences Graduate Program, 2Forensic Science Program, Trent University; 3Institute of
Parasitology, McGill University.
Characterization of p105: A Putative Transcription Factor in Giardia lamblia
Giardia lamblia is a unicellular eukaryotic parasite of both evolutionary and medical importance. This pathogen
causes giardiasis in humans, a disease characterized by severe diarrhea and abdominal cramping. A 15 bp conserved
DNA sequence called him for the histone motif, is found to be unique to the promoters of Giardia core histone genes
and is essential for their maximal expression. We speculate that this motif may be involved in the coordinated
expression of Giardia core histone genes during the cell cycle by acting as the binding site for a transcriptional
regulatory complex. Previous work in our laboratory identified several potential him-binding proteins from Giardia
nuclear extracts including p105, a putative 105 kDa transcription factor. INTERLOG analysis of the Giardia
genome database yielded a protein-protein interaction network that predicts a direct interaction of p105 with a Nterminal acetyltransferase (NAT) and an indirect interaction with G2/Mitotic-specific cyclin B.
The aim of this research is to identify and characterize the DNA-binding activity of p105 in gel-shift assays, and the
interaction of p105 to NAT and cyclin B in co-immunoprecipitation assays. Preliminary results from this research
will be presented. This work is supported by a NSERC Discovery Grant.
9
Daniel Feingold, Joseph A. Dent and Laura Nilson Dept. of Biology, McGill University
Three novel Cys loop ligand-gated ion channels in Drosophila melanogaster: Potential pesticide targets?
Cys-loop ligand gated ion channels are pentameric neurotransmitter receptors that are ubiquitous in both vertebrate
and invertebrate nervous systems. Their great diversity as well as their central role in mediating rapid synaptic
transmission has made these channels attractive molecular targets for a number of pesticides. Despite the widespread
use of such pesticides, issues regarding resistance and off target toxicity continue to pose problems in regions that
rely on pesticides for crop protection and disease prevention. We are characterizing three novel Cys- loop LGIC
subunits - CG7589 and CG6927 and CG11340 – in Drosophila melanogaster in order to determine their validity as
putative pesticide targets. These genes are of particular interest because they exhibit little homology among other
invertebrate or vertebrate species (Dent 2006). We generated putative knockouts of all three genes. Viability assays
conducted on CG7589 and CG11340 mutants suggest that deletions in these genes are semi lethal. These results
suggest that antagonists of the channels formed by these subunits might make potent insecticides. In situ
hybridization experiments reveal that CG7589, CG6927 and CG11340 have unconventional expression patterns in
that they do not appear to be expressed in the nervous system. CG7589 and CG11340 are expressed in the gut and
Malpighian tubules while CG6927 appears to be expressed in the salivary glands and tracheal tissue. Consistent with
this expression data, the channels formed by these putative subunits fail to respond to a wide array of
neurotransmitters that typically elicit responses in Cys-loop LGICs.
Based on the semi-lethal phenotype of CG7589 and CG11340, as well as the expression of all three putative channel
subunits in important secretory tissues, CG7589, CG6927 and CG11340 are predicted to represent promising new
pesticide targets.
Carolina P.Gómez1, Marina Shio2, Martin Olivier2, Albert Descoteaux1, 1Institut National de la Recherche
Scientifique- Institut Armand Frappier, 2McGill University.
Role of the protein SHP-1 during the process of phagocytosis in murine macrophages.
The process of phagocytosis and phagosomal maturation involves the recruitment of several proteins that participate
in the formation of phagosome, and in the acidification and/or fusion with other vesicules in order to either recycle
cellular compounds or degrade cellular debris and parasites. Among these proteins, phosphatases are known to be
involved in such processes (p. ex: SHIP-1), and in some cases, are used by the intracellular parasites to delay the
maturation process to their advantage (p.e: M. tuberculosis). However, the role of the protein SHP-1 in such process
has yet to be determined. Using murine macrophage cell lines defficient for SHP-1 (motheaten [Me SHP-1-/-]), and
their wild type littermate we studied the kinetics of the recruitment of the Lysosomal-associated membrane protein 1
(LAMP1), a known marker for phagosomal maturation. Using confocal microscopy and Western Blot we noticed a
delayed recruitment in SHP-1-/- cells. Also, we noticed that the active form of Cathepsin D (Cath D), which is also
related to the maturation process in phagosomes, is presented earlier in the phagosome isolation extracts from SHP1+/+ cells, and this is not due to an absence of the precursor in the cells. Furthermore, there seems to be a higher
recruitment of the precursor in the SHP-1-/- cells.
10
Stephanie Goyette1,2, Momar Ndao2, Brian J. Ward2, and Florence S. Dzierszinski1 , 1Institute of Parasitology,
McGill University; 2MUHC, McGill University.
Insights into Toxoplasma gondii reactivation through biomarker studies.
T. gondii is a ubiquitous parasite and a global zoonotic pathogen. Upon infection, lytic forms called tachyzoites
multiply and spread throughout the host to reach target organs, where they differentiate into dormant encysted
bradyzoites. Bradyzoites sustain the chronic phase of infection, are drug-resistant, and persist for the lifetime of the
host. Although T. gondii infection typically causes mild disease in healthy adults, toxoplasmosis can be a
debilitating and/or life-threatening disease in the congenitally-infected fetus or immunocompromised host. Much of
the morbidity and mortality associated with this organism occurs as a result of reactivation, but the mechanisms that
lead to cyst rupture and recrudescent infection are poorly understood. In order to investigate the mechanisms that
trigger tissue cyst reactivation, we have established a murine model of reactivation, in which chronically infected
mice are depleted of interferon-gamma until they show signs of disease. To isolate parasite and/or host factors that
arise in the early and late stages of reactivation and may ultimately influence the outcome of infection, proteomic
profile analysis of sera and cerebrospinal fluid from experimentally reactivated mice are obtained by SELDI Tof
MS. In order to determine whether parasite-derived biomarkers can actually be detected using this method, we have
engineered transgenic T. gondii lines that secrete the model antigen ovalbumin (OVA) in the tachyzoite stage only,
or in the bradyzoite stage only, or constitutively in both stages.
SG is a recipient of a CIHR doctoral scholarship. This work is partly supported by a CRC award to FD, and a CIHR
award to MN and BW.
Theresa W. Gyorkos, Serene A. Joseph, Mathieu Maheu-Giroux, Division of Clinical Epidemiology, Research
Institute of the McGill University Health Centre and the Department of Epidemiology, Biostatistics and
Occupational Health, McGill University.
EVIDENCE SUPPORTING DEWORMING IN CHILDREN UNDER 24 MONTHS OF AGE
Background: Large-scale deworming programs targeting pre-school children in endemic areas are recommended by
WHO, PAHO and other agencies. In South America, of 31 endemic countries, only 5 countries reported deworming
of preschool children in 2008, with only 2 having coverage rates over 70%. Empirical evidence supporting
deworming in pre-school children is limited. Objective: To determine the prevalence and intensity of soiltransmitted helminth (STH) infection in children under 24 months of age. Methods: A baseline nutrition and
parasitological survey was conducted in the Peruvian Amazon between July 2007 and February 2008. The study
population included all children between the ages of 7-9 months and between 12-14 months living in Belen, a
community of extreme poverty. The Kato-Katz method was used to obtain epg counts. Ethics approvals were
obtained from both Canada and Peru. Results: A total of 164 7-9 month-olds and 185 12-14 month-olds were
recruited. Parasite infections were first detected among 8-month-olds (Ascaris 4.4%). At 9 months, all three STH
infections were present: Ascaris 2.0%; Trichuris 4.0% and hookworm 4.0%. By 14 months of age, prevalences had
increased to 14.8%, 30.0% and 1.9%, respectively. The highest Ascaris and Trichuris intensities were recorded in
the 12-14 month-old group: 42,840 epg and 2,640 epg, respectively. Conclusion: STH infection is present in very
young pre-school children, and intensities can be in the moderate category. This empirical evidence supports
deworming programs targeted to pre-school children in highly endemic areas. Because of the predominance of
Ascaris and Trichuris infections in this age group, mebendazole, rather than albendazole, may be the deworming
drug of choice.
11
Kasra Hassani1, Elisabeth Antoniak1, Marie-Josée Bellemare1, Armando Jardim2, Martin Olivier1, 1Department
Microbiology and Immunology, McGill University, 2Institute of Parasitology, McGill University
Temperature-induced protein secretion by Leishmania mexicana modulates host macrophage signaling and
functions.
Leishmania are protozoan parasites and causative agents of leishmaniasis disease. During infection, Leishmania
parasites can deactivate the host macrophage and propagate within its phagolysosome. When entering the
mammalian host through sandfly bite, Leishmania parasites encounter an elevation in temperature (from ambient
temperature to 37°C). We have observed that this temperature shift (TS) results in a dramatic increase in protein
secretion for Leishmania mexicana in as short as 4 hours. We have identified 126 proteins that are released by L.
mexicana parasite (exo-proteome). Since, most of the identified proteins lacked a signal peptide, we hypothesized
that un-conventional secretion pathways are involved in protein release. To evaluate this theory, we first performed
scanning electron microscopy and observed a rapid increase in budding of exo-vesicles from parasite surface. Using
an un-conventional protein secretion inhibitor, glibenclamide, we were able to partially inhibit protein secretion by
L. mexicana and also observed the absence of budding exo-vesicles from the surface of the parasite. Furthermore,
since we had identified potential and known virulence factors of Leishmania, we were interested to look at their
effect on macrophage signaling. Interestingly, we observed that similar to Leishmania parasites, the exo-proteome of
L. mexicana upon TS is also able to modulate the protein tyrosine phosphatases (PTPs) of the macrophage and to
inhibit LPS-induced nitric oxide (NO) production. Therefore, we can conclude that upon entry to the mammalian
host, L. mexicana secretes proteins that are able to modulate and partially paralyze macrophage signaling and
functions, thus facilitating establishment of infection by this parasite.
Felix Hugentobler, Karen K. Yam, Martin Olivier and Benoit Cousineau, Department of Microbiology &
Immunology, McGill University, Montreal, QC
Development of a new generation of live vaccines against leishmaniasis using the Gram-positive bacterium
Lactococcus lactis
Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the
dairy industry. L. lactis was given Generally Recognized As Safe (GRAS) status by the American Food and Drug
Administration. Leishmania is a human parasite affecting over 12 million individuals worldwide, however no
vaccine is currently available. Protective immunity in mice against various forms of leishmaniasis depends on the
activation of T-helper (Th) type 1 cells induced by the essential cytokine interleukin 12 (IL-12). To generate a
Leishmania live vaccine, we engineered L. lactis strains expressing one of two known protective Leishmania
antigens, LACK or A2, alone or in combination with mouse IL-12. We found that mice, subcutaneously immunized
with live L. lactis expressing LACK anchored to the cell wall in combination with L. lactis secreting mouse IL-12
displayed antigen specific humoral immune responses. Serum antibody titers indicated more prominent Th1 immune
responses and the animals were protected against Leishmania major infection. Similarly, high antibody titers and
protection were obtained against Leishmania donovani challenge following mouse immunization with L. lactis
expressing A2 along with L. lactis secreting mouse IL-12.
12
Fikregabrail A. Kassa1, 3, Marina Tiemi Shio1, 3, Marie-Josée Bellemare1, 3, Momar Ndao2,3 and Martin Olivier1, 3
1
Department of Microbiology and Immunology; 2National Reference Centre for Parasitology; 3The Research
Institute of the McGill University Health Center, McGill University, Montreal.
Identification of inflammation-related biomarkers from human serum adhering to the malaria pigment
hemozoin
Malaria is one of the most prevalent infectious diseases worldwide with more than 250 million cases and one million
deaths each year. One of the well characterized malarial antigens is hemozoin (HZ), which is a dark-brown crystal
formed by the parasite and released into the host during the burst of infected red blood cells. Our laboratory has
demonstrated that HZ has a stimulatory effect on the host immune system such as its ability to induce the proinflammatory cytokine IL-1β in a NLRP3-dependent manner and to be responsible for some of the malaria related
clinical symptoms such as fever. However, the host serum proteins interacting with malarial HZ as well as how this
interaction modifies its recognition by phagocytes remained elusive. Using proteomic (LC-MS/MS) and
immunochemical approaches, we compared the serum protein profiles of 12 malaria patients and six healthy
individuals. Particularly, we utilized the malarial HZ itself to capture HZ-binding serum proteins in vitro, enabling
us to identify several proteins such as apolipoprotein E (ApoE), serum amyloid A (SAA), gelsolin, complement
factor H and fibrinogen. Of particular interest is LPS binding protein (LBP), which is reported herein for the first
time in the context of malaria. LBP is usually produced during innate inflammatory response to gram-negative
bacterial infections. The exact role of LBP in the acute phase response to malaria in general and HZ in particular
remains to be investigated. The identification of these inflammation-related biomarkers in malaria paves the way to
utilize them collectively as diagnostic and therapeutic targets. Research funded by an operating grant from CIHR to
Dr. Martin Olivier.
Pranav Kumar, Éric Madore, Frédéric Raymond, Michel J. Tremblay, Centre de Recherche en Infectiologie, Centre
Hospitalier de l'Université Laval, Québec, Canada
Gene expression modulation in nelfinavir–resistant Leishmania donovani amastigotes is associated with gene
amplification, gene deletion, formation of supernumerary chromosomes and chromosome loss.
Visceral leishmaniasis has now emerged as an important opportunistic disease in patients infected with human
immunodeficiency virus type-1 (HIV-1). Recently, we have found that the HIV-1 protease inhibitor nelfinavir
induces programmed cell death in Leishmania amastigotes by a caspase-independent mechanism. In order to get
more insight into the mechanism of drug action on the parasite, we have developed in vitro nelfinavir resistant
amastigotes by direct drug pressure in culture. In the present study, we performed RNA expression profiling
analyses of closely related Leishmania species. Leishmania infantum was used as a screening tool to compare
nelfinavir-resistant and -sensitive Leishmania donovani in order to identify candidate genes involved in the
molecular mechanisms of nelfinavir resistance. Several genes were found to be differentially expressed between the
two strains. We also carried out comparative gene hybridization (CGH) analyses of nelfinavir-resistant and sensitive Leishmania donovani using whole-genome 70-mer oligonucleotide microarrays. RNA expression profiles
and the CGH study of nelfinavir resistant vs sensitive Leishmania donovani amastigotes suggested several
mechanisms by which parasites modulate their gene expression. These include gene deletion, formation of
extrachromosomal circular amplicons and chromosome aneuploidy. In the case of gene deletions or formation of
extrachromosomal circular amplicons, gene rearrangement occurred by homologous recombination between
conserved repeated sequences. Interestingly, gene expression modulation of the entire chromosome occurred in the
nelfinavir-resistant mutant. CGH and RNA expression analyses revealed that this was either due to the presence of
supernumerary chromosomes, or to the loss of one chromosome. This study provides further insights into the
mechanisms of drug resistance in Leishmania amastigotes. This work is supported by a Strategic new initiative team
grant to M. J. Tremblay and Marc Ouellette from the FQRNT Centre for Host-Parasite Interactions.
13
Louis-Philippe Leroux, Manami Nishi, and Florence Dzierszinski. Institute of Parasitology, McGill University:
Subversion of the MHC-II pathway by Toxoplasma gondii: surprising discoordinate expression of MHC-II and
the Invariant chain (Ii).
Like many intracellular pathogens, the protozoan parasite Toxoplasma gondii interferes with MHC-II antigen
presentation to dampen the CD4 T cell response. We have shown that T. gondii uses redundant mechanisms to this
end. For example, although IFNg-induced MHC-II transcription is downregulated and surface MHC-II is low in
infected cells, significant intracellular levels of MHC-II in the form of SDS-resistant dimers are detected, suggesting
post-translational regulatory mechanisms. Although expression of MHC-II genes (MHC-II, invariant chain (Ii) and
H2-DM) is typically coordinated, Ii was surprisingly induced in T. gondii-infected cells, even in the absence of
IFNg. Ii chaperones MHC-II in the endocytic pathway and protects the MHC-II groove from unspecific peptide
loading before it is processed by endosomal proteases. Ii accumulated in intracellular compartments (mainly ER but
also endosomes), but not at the cell surface, from 20hrs post-infection until host cell lysis. Ii did not accumulate in
Leishmania or Salmonella-infected cells. Ii upregulation occurred in infected macrophages and dendritic cells from
the spleen, mesenteric lymph nodes, and peritoneal exudate of infected mice, validating this phenotype in vivo. Ii
processing analysis in FACS-sorted infected cells compared to non-infected cells revealed the accumulation of both
p31 and p41 Ii isoforms as well as the p10 cleavage product. Preliminary analysis of the endosomal proteases
involved in the processing of p10 into CLIP indicated that expression of cathepsin (Cat) L, but not CatS and CatF,
was affected in infected cells. Together, this phenotype represents a novel mechanism of host cell subversion by T.
gondii. LPL is a recipient of the Lynden Laird Lyster Memorial Fellowship. This work is supported by March of
Dimes and CRC awards to FD.
Kevin MacDonald and Paula Ribeiro, Institute of Parasitology, McGill Univ.
Silencing Acetylcholine-gated Chloride Channel Subunits in Schistosoma mansoni
Drugs targeting worm motility have been particularly effective against helminth parasites, as evidenced by current
treatment for schistosomiasis, Praziquantel. An analysis of the recently sequenced genome of S. mansoni has
revealed that the two major neuroreceptor classes controlling muscle movement, G-protein-coupled receptors
(GPCRs) and ligand-gated ion channels (LGICs), are present. Moreover, it appears that several of these receptors
may be responsive to acetylcholine (ACh), which has been demonstrated to act as an inhibitory neurotransmitter in
nematodes. Here, we present a bioinformatics analysis of the putative ACh receptors found within the genome of S.
mansoni. A total of 2 GPCRs and 9 LGIC subunits were found. One of the GPCRs was truncated, missing the fourth
and fifth transmembrane domains, while the other appears to be full length. A majority of the putative ACh-gated
ion channel subunits display an EA substitution at the boundary of the M2 region, indicating that they are anionselective. 5 of the anion-selective subunit sequences were used to generate siRNA and to transfect newly
transformed S. mansoni schistosomulae. Schistosomulae were then screened for behavioral phenotypes after 8 days,
then treated with 10 mM ACh and screened again on day 9. All screening was done using Simple PCI motion
tracking software to quantify worm motility. Silencing of each subunit produced a highly significant (P<0.01)
increase in contraction rate and body length in 8 day-old schistosomulae when compared with the negative control.
After treatment with ACh, two of the silenced subunits continued to show a highly significant increase in
contraction rate and three of the subunits produced a highly significant change in body length/width ratio. These
preliminary findings suggest that ACh may act through LGICs as an inhibitory neurotransmitter affecting worm
motility.
14
Raya Mahbuba, Karen K. Yam, Felix Hugentobler, Benoit Cousineau, Department of Microbiology and
Immunology, McGill University.
Generation and evaluation of SMT-expressing lactococcal live vaccines against visceral leishmaniasis.
Visceral leishmaniasis (VL) is caused by species of the Leishmania donovani complex. There are 500,000 new cases
of VL every year and more than 50,000 deaths resulting from the parasite infection. The disease affects internal
organs such as the liver, spleen and bone marrow and eventually causes death if left untreated. However, current
treatments are expensive, often deleterious to the patient and may also be ineffective especially due to drug-resistant
parasites. As yet, no vaccines against VL have been licensed for human use. We have engineered a strain of the
Gram-positive, non-colonizing, non-pathogenic bacterium, Lactococcus lactis, to heterologously express the
protective Leishmania antigen sterol 24-c-methyltransferase (SMT) in the cytoplasm. This strain of L. lactis was
assessed for its protective capability as a live vaccine against Leishmania donovani in female BALB/c mice. Mice
that were subcutaneously immunized with live SMT-expressing L. lactis demonstrated antigen-specific antibody
responses. These mice also demonstrated lower parasite load in the liver and significantly lower hepatomegaly
following L. donovani infection challenge. Overall, we demonstrate that L. lactis is a suitable vehicle for the
delivery of vaccine antigens and the generation of protective immune responses.
Laura-Isobel McCall, Greg Matlashewski, Dept. of Microbiology & Immunology, McGill Univ.
Involvement of the Leishmania donovani virulence factor A2 in the parasite stress response
Although Leishmania major and Leishmania donovani are closely related, they lead to very different disease
manifestations: L. major causes cutaneous leishmaniasis and L. donovani visceral disease. The mechanisms leading
to this difference are not yet understood, but the L. donovani protein A2 has been implicated. A2 is expressed
mainly in the intracellular amastigote form of L. donovani, whereas it is a non-expressed pseudogene in L. major.
Introducing A2 into L. major increased L. major survival in the visceral organs and A2 has been shown to play a key
role in L. donovani survival in the mammalian host. However, the function of A2 remains unknown. Here, we
provide evidence that A2 is a stress response protein. A2 was induced at neutral pH by several stress signals,
including 37°C or 40°C heat-shock, ethanol and misfolded protein stress induced by a proline analog. L. donovani
and L. major ectopically expressing A2 also survived 40°C heat-shock significantly better than control-transfected
or wild-type L. major. A2 was not secreted by L. donovani and was localized to the endoplasmic reticulum (ER)
both during macrophage infection and in heat-shocked promastigotes. Finally, A2 interacted with the abundant ER
chaperone BiP but not with other abundant proteins such as the cytoplasmic HSP83 or with other ER-localized
proteins, including the classically-secreted gp63 or secreted acid phosphatase SAcP. These results suggest that A2
may play a role in the parasite stress response and help L. donovani survive the higher temperatures associated with
the visceral organs and with the fever observed in visceral leishmaniasis. Overall, these observations provide
valuable insight into the function of A2 and into the L. major-L. donovani dichotomy.
This work was supported by the CIHR and FRSQ.
James McLean, Armando Jardim, Institute of Parasitology, McGill Univ.
Purification and characterization of the Leishmania major glycosomal PTS2 receptor, Peroxin 7
Leishmania are Trypanosomatid parasites that infect roughly 12 million people in tropical countries. This neglected
tropical disease causes debilitating and often fatal consequences in the absence of chemotherapeutic intervention.
Consequently, there is a need to identify new drug targets to combat the increasing incidence of resistance to current
treatments. An attractive drug target in these parasites is the glycosome, a unique microbody organelle that
compartmentalizes several essential enzymatic pathways. Peroxin 7 (PEX7) is a protein directly involved in the
import of this enzymatic machinery across the impermeable glycosome membrane. This receptor is known to be
necessary for parasite viability. PEX7 is predicted to have a hydrophobic outer surface that has made production of
the recombinant protein in E. coli challenging. To investigate the biophysical role of PEX7 in the trafficking and
import of protein into the glycosome, we have developed a strategy to express and purify recombinant Leishmania
PEX7 in the absence of detergent. Biochemical studies have confirmed that the recombinant PEX7 is functionally
active and, like the native protein binds PEX5, PEX14, and the PTS2 ligand.
15
Yovany Morenoa, Joseph F. Nabhana, Jonathan Solomona, Charles D. Mackenzieb and Timothy G. Gearya, aInstitute
of Parasitology, McGill Univ. bDepartment of Pathology, Michigan State Univ.
Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi.
In vivo, ivermectin (IVM) treatment of filarial infections is characterized by a rapid drop in the levels of circulating
microfilariae (Mf) followed by the long term suppression of their production. Nevertheless, the direct effect of this
drug on the Mf of most filarial nematodes is still debatable as there is no clear in vitro evidence of its
microfilaricidal action. IVM acts upon binding to nematode Glutamate-gated Chloride Channels (GluCl), resulting
in disruption of the neurotransmission processes that are regulated by the activity of these channels. To identify the
physiological effects of IVM on Mf, we cloned and localized two AVR-14 subunits from Brugia malayi, which
constitute their only putative IVM-sensitive GluCl subunits. Bma-AVR-14 subunits co-localized with a muscle
structure surrounding the Mf- Excretory-Secretory (ES) vesicle. It suggests that under the control of GluCl, protein
release in the ES apparatus is driven by the contraction of this vesicle. Consistently, in vitro IVM treatment led to a
decrease in total protein released from Mf. Protein release decreased in 0.1 µM IVM up to 58, 68 and 42 % the
amount released by the control at 24, 48 and 72 h, respectively. To understand how IVM can affect secretion of
proteins released by the parasite, we identified 3 different localization patterns among a group of 5 known Mf- ES
products; suggesting that either the parasite surface or the ES apparatus are probable anatomical pathways for
physiological protein release. Nevertheless, the presence of muscle association with the ES-vesicle and the low
permeability characteristic of the Mf sheath points to the ES-apparatus as the main source of Mf protein delivery to
the mammalian host. Mf treatment with IVM targets the ES apparatus, which constitutes the main source of parasite
protein release to the mammalian host. This leads to the inference that rapid Mf clearance related to IVM treatment
in vivo reduces the secretion of immunomodulatory parasite proteins and thus induces a parasiticidal effect of the
host immune system. This work was supported by a grant of NSERC and CRC.
Nilmar S. Moretti2; Luiz R. O. Tosi1; Barbara Papadopoulou2
1- Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos – Faculdade de Medicina de Ribeirão
Preto – Universidade de São Paulo
2- Centre de Recherche en Infectiologie – Université Laval - Canadá
Study the role of histone deacetylation on the control of gene expression in the protozoan parasite Leishmania
An important aspect of the organization and expression of eukaryotic genomes is the demarcation of
transcriptionally active and inert regions. This is mainly achieved by controlling chromatin accessibility for the
transcription machinery. Posttranslational histone modifications play a central role in chromatin remodeling and
consequently in transcriptional regulation. In the protozoan parasite Leishmania, control of gene expression occurs
mainly at the post-transcriptional level. To better understand the possible epigenetics mechanisms in this parasite,
we generated an overexpressor of the Saccharomyces cerevisae Sir2 homologous protein. Sir2 belongs to the NAD +dependent histone deacetylase (HDAC) family and its implication in histone modification has been widely
documented and Sir2 also has roles in gene silencing, DNA Repair, cell cycle progression and ageing. Microarray’s
experiments performed comparing the wild type and the Sir2 overexpressor cell lines, indicate several changes in
mRNA expression, including genes involved in transcription regulation, translational control, chromatin
modifications and processing of small non-coding RNAs. Our preliminary data in Leishmania and published data in
the closely related parasite Trypanosoma brucei suggest the existence of epigenetic control of gene expression that
could be operating at the level of transcription. A new set of experiments is planed to verify the position of downregulated genes in the Sir2 overexpressor are part of chromosomal regions with condense chromatin where histones
are acetylated and whether on the other hand up-regulated genes are the result of an increase transcript stability due
to changes in the expression of some RNA-binding proteins, which are under the control of Sir2.
16
Amber Olson1, Kin Chan2, Chi-Yip Ho2, and Janet Yee1. 1Biochemistry Program, Trent University; 2Microarray
Laboratory, Samuel Lunenfeld Research Institute, University of Toronto.
Gene expression profiling of Giardia lamblia isolates.
In Canada, the majority of the waterborne disease outbreaks linked to protozoa is caused by the enteric parasite
Giardia lamblia. Isolates of Giardia can be classified into one of seven major genetic assemblages (A – G). Giardia
in assemblage A or B are capable of infecting humans, wildlife, companion animals, and livestock — thus, have a
potential for zoonosis. However, there is no information available on the dynamics of gene expression among
Giardia isolates.
In this study, microarray analyses were performed on three Giardia isolates representing two assemblages. The WB
isolate originated from a human patient and belongs in assemblage A. The GS isolate also originated from a human
patient but belongs in assemblage B. The third isolate originated from a beaver and belongs in assemblage A.
Fluorescent-labeled cDNA were generated from laboratory cultures of the three Giardia isolates and used in
hybridization experiments with microarray slides containing oligonucleotides corresponding to 9115 genes and
ORFs within the Giardia genome. More than 1000 genes with statistically different expression among the three
isolates were determined by Significant Analysis of Microarray (SAM). The expression of several genes identified
in the microarray analyses are currently being validated by qPCR analyses. These results and their significance will
be discussed.
This work is supported by a NSERC Discovery Grant (JY) and a Graduate Scholarship from the Walkerton Clean
Water Centre (AO). The microarray slides are provided by PFGRC.
Patocka, Nicholas, Ribeiro, Paula, Institute of Parasitology, McGill Univ.
Exploring Serotonin Signaling in Schistosoma mansoni: Transporter and Receptor
Serotonin (5:hydroxytryptamine: 5HT) is an important modulator of neuromuscular function and metabolism in
flatworms, including the bloodfluke Schistosoma mansoni. Exogenous application of 5HT to intact schistosomes
causes contraction of the body wall musculature and a robust increase in motor activity. It is unknown at present
whether the effect of exogenous serotonin is mediated by activation of surface (tegumental) receptors leading to
downstream signaling via the worm’s sensory nervous system, or if 5HT is transported by a surface carrier to act on
internal receptors. Previous work showed the presence of a 5HT - specific transporter (SERT) in S. mansoni. The
parasite SERT was shown to mediate the uptake of exogenous 5-HT in live parasites (Patocka and Ribeiro, 2007),
suggesting it may be located on the surface. In addition to this transporter, we have recently identified two 5HT-like
receptor sequences in schistosomes. One of these receptors was cloned and shown to respond to 5HT through the
activation of cAMP when expressed in mammalian cells. This response was specific to serotonin with minimal
response to any of the other biogenic amines. Immunolocalization of the transporter showed it to be in both
neuronal and tegumental structures. Similar experiments showed the receptor to be localized subtegumentally in
both female and male adult worms. In order to test the importance of these proteins in vivo, parasites were treated
with siRNA or specific drugs to block activity and then analyzed for changes in behaviour. Treatment with siRNA
against the SERT as well as treatment with known SERT antagonists produced a increase in motility, consistent with
an elevation in extracellular serotonin. Long-term treatment with anti-SERT drugs produced decreases in viability,
suggesting that SmSERT is essential for survival.
17
Chase Reaume1, Christine Ouellet2, and Janet Yee1,2. 1Environmental and Life Sciences Graduate Program,
2
Biochemistry Program, Trent University
The application of counterflow centrifugal elutriation and quantitative RT-PCR in the cell cycle analysis of
Giardia lamblia
Giardia lamblia is a protist found in freshwater lakes and streams worldwide. Ingestion of this parasite via
contaminated water or fecal-oral contact can lead to the onset of giardiasis, or “Beaver Fever”, a disease
characterized by acute and severe diarrhea. The Giardia cell cycle is of research interest due to its influence on the
pathogenesis and transmission of giardiasis. In order to study the changes in gene expression that are associated
with each stage of the cell cycle, synchronized cultures of Giardia are required. However, previous methods that
used drugs, such as aphidicolin, to obtain synchronization of Giardia cultures also damaged the DNA of the cells
and affected their metabolic functions. We have recently developed the technique of counterflow centrifugal
elutriation to obtain fractions of Giardia cultures enriched in cells from every stage of the cell cycle. This
presentation will provide an overview of the counterflow centrifugal elutriation procedure and our results from
quantitative RT-PCR analysis of the cell cycle fractions obtained from this procedure.
This work is supported by a NSERC Discovery Grant and funding from Trent University.
Sarah J. Reiling, Petra Rohrbach, Institute of Parasitology, McGill University.
Analyzing transporters and ion channels of the digestive vacuolar membrane of Plasmodium falciparum.
Malaria is one of the most studied parasitic diseases and yet the parasite is always a step ahead. Widespread
treatment with antimalarial drugs has lead to a dangerously emerging number of resistant parasites. The molecular
mechanisms of solute transport by PfMDR1, a main digestive vacuolar membrane transporter involved in drug
resistance, are investigated in this research project. A novel live cell surrogate assay, based on fluorescent substrate,
was developed to determine the PfMDR1 transport activity of Plasmodium falciparum strains with different pfmdr1
polymorphisms that expressed differing drug-responsiveness. The flourochrome Fluo-4 acetomethylester (AM) was
found to be a substrate of PfMDR1 and accumulated in the parasite’s digestive vacuole, suggesting that PfMDR1
pumps Fluo-4 into this compartment and has a unidirectional transport route for that substrate. The enrichment of
Fluo-4 in the digestive vacuole was prevented by P-gp inhibitors, which provided further evidence for the hypothesis
that PfMDR1 transports solutes into this parasitic compartment. In addition, the P. falciparum chloroquine-sensitive
HB3 strain showed a broader dispersion of Fluo-4 compared to the chloroquine-resistant strain Dd2, which implies a
correlation between PfMDR1 polymorphism, drug-responsiveness and Fluo-4 distribution. A novel technique that
utilizes fluorescent channel-blockers will provide us with further information on ion channels and transporters in the
digestive vacuolar membrane that have not been fully characterized to date. Fluorescence recovery after
photobleaching will be used for analysis of intracellular molecular dynamics. This knowledge is crucial to
understand how new drugs need to be designed to maximize their efficiency against this parasitic disease.
Ruiz-Lancheros Elizabeth, Geary Timothy, Institute of Parasitology, McGill University.
Novel approach for receptor deorphanization in the model nematode, Caenorhabditis elegans
With the increase of anthelmintic resistance in parasitic nematodes, rational discovery of new drugs with new
mechanisms of action is urgently needed. The nematode neuropeptidergic system is an excellent source of targets,
since it is exclusive of invertebrates and plays critical roles in worm biology. Unfortunately, few components
(receptors or signalling pathways) of this system are known. We designed a novel strategy to match neuropeptides
(FMRFamide-related peptides; FLPs) with their cognate receptors in C. elegans bioassays. By testing peptides in
dissected wild-type (WT) worms, we identified characteristic phenotypes produced by 7 different FLPs. We then
screened receptor using FLPs as baits and reverse genetics to interrogate the association of a FLP-phenotype with a
specific receptor. We screened 28 candidate G protein-coupled receptors (GPCRs) selected by phylogenetic studies
for which individual knockout strains are available. Each FLP was matched with one or two GPCRs, based on the
specific loss of the FLP-phenotype in knockout mutant strains. The ligand–receptor associations were confirmed by
expressing some of the identified GPCRs in modified strains of Saccharomyces cerevisiae for receptor activation
bioassays. Data from homologous and heterologous systems suggest that these FLPs selectively activate specific
GPCRs and thus control locomotion. The identification of FLP-GPCRs for future studies allows further
characterization of neuropeptide receptor function and the investigation of their roles in nematode viability,
behaviour and maintenance of parasites in their hosts. Activation of essential FLP-GPCRs is likely to be a useful
anthelmintic mechanism of action. This work was supported by NSERC, Canada Research Chairs and a Tomlinson
Fellowship from McGill.
18
T. Scorza, D. Malu Tshikudi, O. Kevorkova, H. Romero, J. Lahssini, J. Sanchez Dardon and R. Moreau.,
Department of Biological Sciences, UQAM, Montreal, Canada.
Changes in bone remodelling associated to Plasmodium-induced and phenylhydrazine-induced haemolytic
anaemia.
Haemolytic anaemia (HA) is a disorder resulting from enhanced destruction of red blood cells (RBC). Patients with
chronic HA develop osteoporosis and have high index values of oxidative stress and low antioxidant defences. Since
the plasmatic concentrations of free heme, a potent oxidant, are elevated during haemolytic disorders, we
hypothesize that the alterations in bone metabolism during HA, resulting in bone loss, are concurrent in part to
heme-sustained redox-dependent modifications in bone remodelling. Our preliminary data with the Plasmodium
chabaudi adami DK malaria model in BALB/c mice, characterized by acute HA and inflammation, and with
phenylhydrazine (PHZ)-treated mice, which suffer from short-term acute HA of non-inflammatory origin, reveal
important drops in bone resorption and bone formation markers in the sera. Thus, levels of osteocalcin, tartrateresistant acid phosphatase 5b and procollagen type I N-terminal propeptide are significantly lower, and reactive
oxygen species are significantly elevated in bone marrow cell suspensions from Plasmodium-infected and PHZtreated mice. In addition, PHZ-treated mice, which developed more severe HA, have lower bone mass density.
Importantly, moderate bleeding leads to anaemia but fails in modifying bone remodelling markers in sera,
suggesting that haemolysis is required for the inhibition.
This work has been partially funded by the New Initiatives Program (Centre Host-Parasite Interactions,
FQRNT).
Hamed Shateri Najafabadi, Reza Salavati, Institute of Parasitology, McGill University
A unified approach for identification of co-occurring features among all data types within biological networks
Biological networks encompass a wealth of information about functional elements that shape the behaviors of genes
and proteins and, in a broader sense, cells and organisms. However, no generalized method exists to extract this
information efficiently. Here, we present a unified approach that can virtually scan all data types for extraction of
co-occurring features in biological networks based on their mutual information. We show that this approach has an
exceptionally wide applicability in solving different network-related biological questions, from de novo discovery of
protein motifs that mediate protein-protein interactions to identification of diseases that result from abnormalities in
the same protein complex. We have applied this method to different coexpression and physical interaction networks
in human and yeast, and have validated the found co-occurring features, including regulatory elements and
functional protein motifs, using available databases. We also discuss denoising and prediction of networks based on
co-occurring features, and their application in identification of functionally/physically interacting proteins in
parasites. Our approach establishes a universal framework for network-based analysis of functional elements, genes,
proteins, and their corresponding phenotypes.
Strasser, Rona, Pilar, A.V., and Jardim, A. Institute of Parasitology, McGill.
Leishmania donovani receptor-cargo protein complexes form in the cytosol and dock at glycosomal protein
Peroxin14, modulating its conformation.
The glycosomes of Leishmania donovani are unique microbody organelles that compartmentalize a variety of
metabolic pathways essential for parasite survival. Trafficking and import of newly synthesized proteins to the
glycosome is dependent on the receptor proteins peroxin 5 (LdPEX5) and peroxin 7 (LdPEX7), and the docking
protein peroxin 14 (LdPEX14), a peripheral membrane protein anchored to the surface of the glycosomal membrane.
To understand the molecular event required for the sorting and trafficking of nascent polypeptides to the glycosome
we employed a variety of techniques to examine the structure of native LdPEX5 and LdPEX7. Biochemical
analysis revealed that in the cytosol of L. donovani, LdPEX5 and LdPEX7 form heteromeric complexes that are
loaded with PTS1 and PTS2 cargo proteins. These structurally diverse arrays of heteromeric complexes are
responsible for trafficking of proteins into the glycosome. At the glycosome surface these receptor-cargo complexes
dock to LdPEX14, which triggers conformational changes that facilitate the import of proteins across the membrane
of the organelle.
19
Dagobert Tazoo1, David Scott Bohle1and Ovidiu Oniga2, 1Department of Chemistry, McGILL University, Montreal
(QC), Canada, 2Department of pharmaceutical Chemistry, University of Medicine and Pharmacy of Cluj-napoca,
Cluj-napoca, Romania
Synthesis and antimalarial activity of chalcones and thiophosphinic acids derivatives.
The synthesis of novel chalcones derivatives, novel thiophosphinic acids and antimalarial activity against cultured
Plasmodium falciparum and hemoglobin hydrolysis are described. Chalcones were prepared via Claisen–Schmidt
condensation from available substituted chromones with thiazoles (Scheme 1), while thiophosphinic acids were
obtained by the reaction of the corresponding phosphinic acid with the Lawesson’s reagent (Scheme 2).
Antiplasmodial activity of the chalcones showed that they blocked P. falciparum development at low micromolar
concentrations and these compounds delayed the progression of malaria but did not eradicate infections. The
thiophosphinic acids are very potent inhibitors of the aminopeptidases against M1AAP and M17LAP. We developed
new inhibitors related to hPheP[CH2]Phe which have both improved cellular uptake by the parasite and stronger
binding to the aminopeptidases after their uptake.
Tshikudi M. Diane, Désautels François, Satoskar Abhay and Scorza Tatiana, Université du Québec à Montréal
MIF: a down-regulator of early T cell-dependent IFN responses in Plasmodium chabaudi adami (DK)
infected mice.
Macrophage Migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine readily secreted in malaria and
contributing to anaemia. MIF is involved in various pathologies as sepsis, asthma, rheumatoid arthritis, and has been
shown to increase the expression of Toll-like receptor (TLR) 4 in various cell types. In addition, activated T helper
(Th) 2 cells secrete MIF and neutralization of MIF suppresses antibody production and enhances cytotoxic T
lymphocytes responses and IFN secretion in vivo. As neutralization of MIF during P. c. adami (DK) infection in
BALB/c mice significantly reduced peak parasitemia, we hypothesized that MIF modulated the early events leading
to T cell activation through a TLR-dependent mechanism. Herein we show that MIF deficient (KO) mice develop
lower peak and cumulative parasitemia and have a delay in the resolution of infection, which we speculate relies on
modifications of the helper (Th)-1 and 2 profiles of responses. Interestingly, splenic T cells purified from MIF KO
mice at day 4 post-infection secrete more IFN and have higher expression of the early activation marker CD69 than
wild type cells. However, no effects were measured in TLR2 or TLR4 expression in these cells. Following semi
quantitative PCR and Western Blot analysis to assess the gene expression and protein levels of T-Bet and GATA 3,
our data revealed a higher constitutive expression of T-Bet in naive MIF/KO T cells but this difference was lost
following infection or in vitro stimulation. As purified T cells from MIF KO naive mice secrete more IFN  and
significantly less IL-4 than wild type T cells when stimulated with anti-CD3/anti-CD28 monoclonal antibodies, we
hypothesize that in absence of MIF T cells may preferentially differentiate into Th-1 cells. Taken together, these
results suggest that besides its pro-inflammatory properties, MIF down-regulates early activation of IFN-secreting
T cells in malaria by a mechanism that may involve control of T-bet expression.
Claudia M. Wever, Patrick Janukavicius, Claire Bottomley, Danielle Farrington, Igor Putrenko and Joseph A. Dent,
Department of Biology, McGill University.
Validating Acetylcholine-Gated Chloride Channels as Novel Nematocide Targets.
Nematode parasites are the cause of many severe diseases, for example river-blindness and heartworm in humans
and animals respectively. Although there are successful anti-parasitic drugs, resistance appears to be reducing their
effectiveness (Osei-Atweneboana et al., 2007). We have identified a novel class of ligand gated ion channels, the
acetylcholine-gated chloride channels (ACCs), in Caenorhabditis elegans (C. elegans). This family of channels is
specific to nematodes (Dent, 2006) and is not a target of previously known nematocides. The class of ACCs
comprises 8 subunit genes; a beneficial characteristic since a gene family that represents multiple targets of a single
drug may slow the onset of resistance. To test the validity of these ACC subunits as targets for nematocides, we
determined the expression pattern of the 8 ACC subunit genes using promoter::GFP fusion constructs and have been
characterizing the electrophysiological properties of ACC channels formed by the different subunits. Most of the
subunits are expressed in a non-overlapping subset of approximately 20 neurons in C. elegans. We have also shown
that both ACC-3 and K10D6.1 subunits form functional homomeric channels that respond to acetylcholine. Due to
the expression of ACCs in a significant fraction of the nervous system, a drug that targets these channels by overactivating them promises to have highly deleterious effects on nematode physiology. We have begun testing the
effects of chronically inhibiting ACC-1 expressing tissues, and have preliminary data suggesting that this is lethal to
the worm. Because of this, and the fact that the ACCs are nematode-specific, we conclude that ACCs merit further
investigation as anti-parasitic drug targets and we hypothesize that a drug that targets these channels would be a safe
and effective nematocide. This work is supported by NSERC and Chemtura Co.
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