Download powerpoint - McGill University

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
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
Wild type
E114G
V235A
L256F
T300S
EC50 for L-Glutamate
27.6 ±2.7
31.5 ± 3.2
26.2 ± 2.5
92.2 ± 3.5***
No channels
** p 0.01, *** p 0.001
Fig. 4. Dose-response curves for L-glutamate at
mutant H. contortus
GluClα3B channels. , wild-type; ✖, V235A; ,
E114G; , L256F.
McCaverna et al. Mol. Pharmacol. 2009
Hill Number
1.89 ± 0.35
1.48 ± 0.31
1.97 ± 0.35
1.09 ± 0.16**
Radioligand binding assays using
[3H]ivermectin and membrane
preparations 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
E114G
V235A
L256F
L256W
L256Y
L256V
T300S
0.35 ± 0.1
0.39 ± 0.07
0.32 ± 0.09
2.26 ± 0.78***
2.51 ± 0.7***
1.84 ± 0.49***
0.79 ± 0.24*
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 dopamine
Rao et al. Mol. Biochem. Parasitol. 2009
Expression of HcGGR3 in lab macrocyclic lactone (ML)-selected strains
of Haemonchus contortus (♀)
qPCR: Standard curve relative quantification method
Fold change as compared to expression in PF23 strain
(normalized with 18srRNA)
Fold change as compared
to the PF23 strain
1
IVF23
MOF23
-4
Level of
expression in
PF23 strain
-9
-14
-19
-24
-29
-34
Macrocyclic lactone selected lab strains
PF23: ML sensitive strain
IVF23 & MOF23: Lab selected strains
anova: P<0.0001
HcGGR3 is down regulated in lab strains of ML- selected worms
Rao et al. Mol. Biochem. Parasitol. 2009
Genotyping of Hcggr3 for the region encoding 3’ UTR
(single ♂s)
GGTGGGTAAACGATAGATCAACTATATCGCACATAAAAAATACAATGCAAACACATATTTTGTAAA
CGTTAATTCCACCGTAGCCAATTAACCGTATAAATATGCAATTCAACCTAATTGAGTTGCATTATCA
CATTCGTTGATTTCTCTAAATGTAGAGAGTTGAGGAG
Homozygous: T/T
Heterozygous: T/C
Allele frequency (%)
120
100
Rao et al. Mol.
Biochem.
Parasitol. 2009
80
60
40
20
0
PF23
IVF23
MOF23
Strains of H. Contortus
PF23: ML sensitive strain
IVF23 & MOF23: Lab selected strains
N = 30
Selection of HcGGR3 allele in Macrocyclic lactone resistance
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 worms
Ardelli 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
Half-transporters
Merino et al. Drug Met. Disp. 2009
Natural Allelic Variants of Bovine ATPBinding 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
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.
IVM selection
on C. elegans
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
‘Natural’ IVM
resistance in C.
elegans results in
overexpression of
a number of ABC
transporter genes
(Pgps
& mrps)
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.
c-glutamylcysteine
synthetase (gcs-1), the
rate-limiting enzyme for
glutathione synthesis was
overexpressed in IVR10,
while a glutathione
transferase π (gstp-1)
was overexpressed in IVR6
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.
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
Frequency
PLP (half-transporter) Genotype before IVM
and after 13X IVM: Onchocerca volvulus
50
45
40
35
30
25
20
15
10
5
0
P=0.054
Female worms
P=0.029
P=0.0077
P=0.048
P=0.048
Position 1 =AA/AG/GG
Position 2 =CC/CG/GG
Position 4 =AA/AT/TT
Fisher’s exact test
BEFORE IVM
AFTER IVM 13x
Pre IVM= 66
Post IVM=35
Allelic selection after 13 three-monthly doses of IVM in female worms
Loss of polymorphism. Bourguinat et al. 2008 Mol Biochem Parasitol
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
Thioredoxins and IVM resistance
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
β-tubulin and ML resistance
β-tubulin
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
Onchocerca volvulus: β- tubulin Female
worms after 13 x IVM
Before and after 13 three-monthly doses
BEFORE TX
AFTER TX
P=1e-8
Fisher’s exact test
80
P=1e-6
N before=183
N after 4 doses=59
N after 13 doses=39
Frequency
60
40
20
0
aa
ab
bb
Genotype
IVM selection caused a significant change in β-tubulin genotype
Bourguinat et al. 2007 PLoS NTD
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