roger prichard institute of parasitology mcgill university, montreal, canada
DESCRIPTION
2007 – 2009 publications Macrocyclic lactone resistance: 1. Understanding resistance mechanisms to different MLs 2. Progress with possible markers for ML resistances. Roger Prichard Institute of Parasitology McGill University, Montreal, Canada. Ligand-gated ion channels. - PowerPoint PPT PresentationTRANSCRIPT
2007 – 2009 publications
Macrocyclic lactone resistance:1. Understanding resistance mechanisms to
different MLs2. Progress with possible markers for ML
resistances
Roger PrichardInstitute of Parasitology
McGill University, Montreal, Canada
Ligand-gated ion channels
Fig. 4. Dose-response curves for L-glutamate at mutant H. contortusGluClα3B channels. , wild-type; , V235A; , ✖
E114G; , L256F. McCaverna et al. Mol. Pharmacol. 2009
H. contortus GluClα3B expressed in Xenopus oocytes
The effects of ivermectin resistance-associated mutations on the ability of glutamate to activate H. contortus GluCl3B channels (mean SEM)
Mutation EC50 for L-Glutamate Hill Number
Wild type 27.6 ±2.7 1.89 ± 0.35 E114G 31.5 ± 3.2 1.48 ± 0.31V235A 26.2 ± 2.5 1.97 ± 0.35L256F 92.2 ± 3.5*** 1.09 ± 0.16**T300S No channels
** p 0.01, *** p 0.001
Radioligand binding assays using[3H]ivermectin and membranepreparations from COS-7 cells transfected with wild-type and mutant H. contortus GluClα3B. The insets show the Scatchard plots for each mutant.McCaverna et al. Mol. Pharmacol. 2009
The effects of mutations in H. contortus GluClα3B on binding of 3[H]ivermectin to membrane preps from transfected COS-7 cells. Mean± SEM
Mutation Kd 3[H]Ivermectin Binding, nM
Wild type 0.35 ± 0.1E114G 0.39 ± 0.07V235A 0.32 ± 0.09L256F 2.26 ± 0.78***L256W 2.51 ± 0.7***L256Y 1.84 ± 0.49***L256V 0.79 ± 0.24*T300S 0.76 ± 0.25
* p< 0.05; *** p< 0.001
Annelies Van Zeveren in her Ph.D. studies looked for the L256F SNP, in GluClα3
homolog, in an ivermectin resistant strain of Ostertagia ostertagi that she and her
colleagues had experimentally selected, but did not find the L256F mutation.
L256F, in GluClα3, may be quite rare in different nematode isolates. However, if it does occur it does seem to produce an
‘IVM-resistance phenotype’
A dopamine-gated ion channel (HcGGR3*) from Haemonchus contortus is expressed in the
cervical papillae and is associated with macrocyclic lactone resistance
Rao VT, Siddiqui SZ, Prichard RK, Forrester SG.Mol Biochem Parasitol. 2009 Jul;166(1):54-61. Epub
2009 Mar 4.
Electrophysiology of HcGGR3 in Xenopus laevis oocytes
Response to dopamine
Response to other amines
HcGGR3 forms a homomeric channel and is primarily gated by dopamineRao et al. Mol. Biochem. Parasitol. 2009
Expression of HcGGR3 in lab macrocyclic lactone (ML)-selected strains
of Haemonchus contortus (♀)
qPCR: Standard curve relative quantification methodFold change as compared to expression in PF23 strain
(normalized with 18srRNA)
anova: P<0.0001
HcGGR3 is down regulated in lab strains of ML- selected worms
Level of expression in PF23 strain
-34
-29
-24
-19
-14
-9
-4
1
IVF23 MOF23F
old
ch
an
ge
as
co
mp
are
d
to th
e P
F2
3 s
tra
in
Macrocyclic lactone selected lab strains
PF23: ML sensitive strainIVF23 & MOF23: Lab selected strains
Rao et al. Mol. Biochem. Parasitol. 2009
0
20
40
60
80
100
120
PF23 IVF23 MOF23
Allel
e freq
uenc
y (%)
Strains of H. Contortus
Genotyping of Hcggr3 for the region encoding 3’ UTR(single ♂s)
N = 30
Selection of HcGGR3 allele in Macrocyclic lactone resistance
GGTGGGTAAACGATAGATCAACTATATCGCACATAAAAAATACAATGCAAACACATATTTTGTAAACGTTAATTCCACCGTAGCCAATTAACCGTATAAATATGCAATTCAACCTAATTGAGTTGCATTATCACATTCGTTGATTTCTCTAAATGTAGAGAGTTGAGGAG
PF23: ML sensitive strainIVF23 & MOF23: Lab selected strains
Heterozygous: T/CHomozygous: T/T
Rao et al. Mol. Biochem. Parasitol. 2009
Pharyngeal pump rates of wild-type C. elegans after 2.5 h exposure to 2-fold serial dilutions of IVM and MOX (mean ± SD). A circle ( ) represents pumping rate in
non-treated worms, a triangle ( ) indicates pumping rate in IVM exposed worms and a square ( ) indicates pumping rate in MOX exposed worms.
Ardelli et al. 2009 Vet Parasitol
Motility phenotype of wild-type C. elegans after exposure to 2.5 nM of drug (mean ± SD). A circle ( ) represents velocity in non-treated worms, a triangle ( ) indicates velocity in IVM exposed worms and a square ( )
indicates velocity in MOX exposed wormsArdelli et al. 2009 Vet Parasitol
Transcriptional profiles of GluCl genes in wild-type C. elegans after exposure to 2.5 nM of IVM or MOX for 2h (mean ± SD). The black bars represent gene transcription after exposure to IVM and the grey bars
represent gene transcription after exposure to MOX. The y-axis represents the fold change in transcription relative to non-treated controls and listed
along the x-axis is the C. elegans gene. Changes that are statistically different between the drug treatments (p < 0.05) following IVM or MOX
exposure are indicated with .Ardelli et al. 2009 Vet Parasitol
ABC transporters
Effect of the ivermectin (IVE) and selamectin (SEL)] on mitoxantrone (MXR) accumulation mediated by human ABCG2 and murine Abcg2. Transduced MDCKII cells were preincubated with or without Ko143 (1 µM) or the other tested compounds (50 µM). Mean MXR fluorescence is shown in terms of relative arbitrary units. The bars indicate the means ± S.D. ** p< 0.01, comparing the difference between human ABCG2- and murine Abcg2- transduced MDCKII cells. §§, p< 0.01, comparing the difference between IVE and SEL in murine Abcg2-transduced cells. §§§, p< 0.001, comparing the difference between IVE and SEL in human ABCG2-transduced cells.
Merino et al. Drug Met. Disp. 2009
Natural Allelic Variants of Bovine ATP-Binding Cassette Transporter ABCG2: Increased Activity of the Ser581 Variant and Development of Tools for the Discovery of New ABCG2 Inhibitors
Selamectin was a significantly more potent inhibitor of the Tyr581 variant compared with the wild-type Ser581
Half-transporters
Cross-resistance to anthelmintics. C. elegans L1s in M9 buffer were incubated in serial dilutions of moxidectin (MOX), levamisole (LEV), albendazole (ALB) or pyrantel (PYR), and their fold-resistance determined in IVR6 (solid) and IVR10 (hatched) relative to Bristol N2 strain (1; dotted line). James & Davey, Int. J. Parasitol. 2008
IVM selection on C. elegans
ABC transporter gene expression in ivermectin-resistant Caenorhabditis elegans. pgp-1, pgp-2, mrp-1, mrp-2, mrp-5 and mrp-6 were amplified from cDNA using gene-specific primers in IVR6 (solid) and IVR10 (hatched) strains cultured with ivermectin. James & Davey, Int. J. Parasitol. 2008.
‘Natural’ IVM resistance in C. elegans results in overexpression of a number of ABC transporter genes
(Pgps & mrps)
The expression of gcs-1 and gstp-1 (B) was examined in IVR6 (solid) and IVR10 (hatched) strains cultured on ivermectin. Bars show means ± SD of the fold change in gene expression relative to the Bristol N2 strain from 2 independent experiments. * indicates P≤0.05; ** indicates P≤0.01. James & Davey, Int. J. Parasitol. 2008.
c-glutamylcysteinesynthetase (gcs-1), the rate-limiting enzyme for glutathione synthesis was overexpressed in IVR10,
while a glutathione transferase π (gstp-1) was overexpressed in IVR6
Allelic variation in an Onchocerca volvulus ABC transporter was reduced in IVM-treated human populations in Ghana in 1999
Ardelli & Prichard, Trans R Soc Trop Med Hyg. 2007, 101:1223
05
101520253035404550
AA CC A
A
AA CG A
A
AA GG T
T
AG CC A
A
AG CG A
A
AG CG A
T
AG CG T
T
AG GG A
A
AG GG A
T
AG GG T
T
GG CG T
T
GG GG A
T
GG GG T
T
Frequency
P=0.0077
P=0.048 P=0.048
P=0.029
P=0.054
BEFORE IVM
AFTER IVM 13x
PLP (half-transporter) Genotype before IVM and after 13X IVM: Onchocerca
volvulus
Fisher’s exact test Pre IVM= 66Post IVM=35
Female worms
Allelic selection after 13 three-monthly doses of IVM in female wormsLoss of polymorphism. Bourguinat et al. 2008 Mol Biochem Parasitol
Position 1 =AA/AG/GG Position 2 =CC/CG/GGPosition 4 =AA/AT/TT
Mrp expression in C. elegans following exposure to 2.5 nM IVM, compared with wild-type worms not exposed to drug
Mrp expression in C. elegans following expsure to 2.5 nM MOX, compared with wild-type worms not exposed to drug
B Ardelli & Prichard J. Helminthol. in press
Analysis of H. contortus showed that expression of both
thioredoxin 12-kDa (HcTrx1) and the 16-kDa (HcTrx3)
genes were increased in an IVM-resistant strain relative to
a sensitive strain.
Sotirchos, Hudson , Ellis & Davey. Free Radic Biol
Med. 2008
Thioredoxins and IVM resistance
β-tubulin and ML resistance
H. contortus: Comparison of the frequency of 200TTC-phenylalanine, 200TTC/TAC-phenylalanine/tyrosine and 200TAC-tyrosine in unselected
(PF), IVM selected (IVF) and MOX selected (MOF) strains
Tyr at amino acid 167 or 200: PF = 11.1%; IVF = 48.1%; MOF = 51.9%
Mottier & Prichard 2008 Pharmacogenetics & Genomics
β-tubulin
BEFORE TXAFTER TX
Onchocerca volvulus: β- tubulin Female worms after 13 x IVM
N before=183N after 4 doses=59N after 13 doses=39
IVM selection caused a significant change in β-tubulin genotype
Bourguinat et al. 2007 PLoS NTD
Before and after 13 three-monthly doses
0
20
40
60
80
aa ab bb
Genotype
Fre
qu
en
cy
Fisher’s exact test
P=1e-6
P=1e-8
TAC/TTC in H. contortus β-tubulin in Swedish flocks which had been under BZ or ML treatment
“An allele frequency of ≥65% was detected in one of the two flocks in 13 (29%) of the 45 farms examined. On many farms (24, 25, 33, 36, 37, 39, 42, 43 and 44) the allele frequency was similar in both the BZ and ML treated flocks”
Höglund et al. Vet Parasitol, 2009
Conclusions• ML resistances appear to be multigenic
• Phenotypic effects of IVM & MOX markedly different
• Differences in genes implicated in IVM & MOX resistances
• Possibly GluCls involved in ML resistances, but data not conclusive
• ABC transporters induced & overexpressed in ML resistances, but not same between IVM & MOX
• MLs select on β-tubulin
• Thioredoxins may also be overexpressed in ML resistances