L'identification du rôle principal du gène pfcrt dans le mécanisme de chloroquino-résistance chez Plasmodium falciparum - Conférence de la 2e édition du Cours international « Atelier Paludisme » - FIDOCK David - Albert Einstein College of Medicine - USA - dfidock@aecom.yu.edu
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L'identification du rôle principal du gène pfcrt dans le mécanisme de chloroquino-résistance chez Plasmodium falciparum
1. Where are we in the fight against
Malaria?
• Drugs: Global spreading of resistance. Pharmaceutical
companies displaying renewed interest. Important
contribution by Gates, MMV, public/private partnerships.
• Vaccines: elusive (major research effort)
• Genetically engineered refractory mosquitoes:
interesting experimental approach, impractical?
• Insecticides: rapidly-growing resistance to DDT and
other insecticides
• Bednets: can reduce prevalence of malaria (how long?)
2. CQ: most widely used Chloroquine
antimalarial:
Safe, rapidly effective, Hemoglobin
affordable.
Digestive
vacuole
Acts in digestive Globin
digestion
vacuole.
Heme poly-
merization
Amino acids
Pigment
PARASITE
Red blood cell
3. Impact of chloroquine resistance
• Was first-line antimalarial, now fails frequently in
prophylaxis and treatment. Resistance associated with
increasing mortality in Africa.
• CQ no longer useful for presumptive diagnosis of
malaria.
• In partially immune individuals, symptoms may
resolve temporarily only to recur some days later; >
60% of patients may not return for treatment.
• Cost of drugs
$ 0.10 Chloroquine (CQ)
$ 0.13 Pyrimethamine / Sulfadoxine (PS)
$ 1.92 Mefloquine
$ 2 Artesunate (part of artemisinin combination therapy)
$40 Malarone (proguanil - atovaquone, cost to travelers)
5. Antimalarial Drug Policies in Africa
?
?
SP * = Declared “interim”
***
Chloroquine + SP *
*
Chloroquine ?
Chloroquine (with > 25% RII/RIII)
Source: Peter Bloland, CDC
6. Hypotheses on the CQR Mechanism:
Mostly based on observation that CQR parasites characterized
by reduced CQ accumulation and chemosensitization by
verapamil.
A. Due to drug efflux pump?? Similar to P-glycoprotein?
B. Due to reduced activity of CQ importer?
C. Due to pH gradient limiting influx of CQ?
D. Due to altered CQ metabolism or changes in heme receptor?
E. Due to reduction in heme receptor concentration or reduced
CQ access to heme?
7. pfmdr1: the first major candidate
CQR determinant
• Identified on basis of homology to mammalian multidrug
resistance (MDR) genes encoding P-glycoproteins, associated
with verapamil-reversible MDR. Parasite product (Pgh-1)
localized to digestive vacuole membrane.
• Point mutations in pfmdr1 associate with CQR in roughly half
of the published reports. Overexpression of Pgh-1 can lead to
increased susceptibility to CQ.
• Modification of pfmdr1 point mutations through allelic
exchange reduced the degree of CQR in a resistant line though
could not confer CQR to a sensitive line.
8. Mapping the CQR Determinant
in a P. falciparum Genetic Cross
CQ-sensitive CQ-resistant
clone clone
Mosquitoes
Chimpanzee
Clone Independent Progeny
and Determine Drug Responses
Map Genetic Locus
Identify Gene(s)
9. Location of pfcrt Linked to Chloroquine Resistance
crossover crossover
K
V V M M
C M C L KK K M NM M L M C LC L N
0 10 20 30 40 50 kb
hsp86 o1 o3 o2 cg8 cg4 cg3 pfcrt cg9 cg1 cg6 cg2 cg7
10. PfCRT Sequence and Polymorphic Positions
Polymorphic residues are indicated by their amino acid number.
Shaded regions delineate 10 predicted transmembrane segments.
Triangles indicate placement of introns in nucleotide sequence.
PfCRT predicted molecular mass is 49K.
11. Immuno-EM localizes PfCRT to digestive vacuole membrane
Hemozoin
Used affinity-purified rabbit IgG raised to PfCRT peptide
13. pfcrt Mutations Associated with CQR
PfCRT position & encoded amino acid
7
Parasite type & origin 72 74 75 97 220 271 326 356 371
6
Chloroquine sensitive
“wild type” C M N K H A Q N I R
106/1 (revertant?) C I E K H S E S I I
Chloroquine resistant
SE Asia & Africa E1a C I E T H S E S T I
SE Asia & Africa
C I E T H S E S I I
Papua New GuineaE1b
S M N T H S Q D L R
P1
South America W1a S M N T H S Q D L R
South America W1b C M N T H S Q D L R
South America W2 C M E T Q S Q N I T
Wellems & Plowe 2001: Fidock et al. 2000, Chen et al. 2001
14. To Test Role of pfcrt in Chloroquine Resistance
• Used 106/1 clone: CQ sensitive (IC50 of 8-15 ng/ml
versus 80-100 ng/ml for Dd2 and FCB).
• 106/1 already has 6 of the mutations found in CQ
resistant parasites: hypothesized that only the
presence of the K76 residue prevented it from being
CQ resistant.
• Put pfcrt coding sequence from CQ resistant parent
Dd2 (containing the PFTCR T76 variant) under control of
P. falciparum regulatory elements -> electroporated
recombinant plasmid into 106/1 and selected on CQ.
15. Timetable with mutant pfcrt-transformed 106/1 line
Appeared Began Began Began Began
day 46 PT day 61 PT day 67 PT day 86 PT day 98 PT
CQ 18 CQ 36 CQ 60 CQ 75 CQ 90
Drug assay 56 Drug assay 61
day 73 PT day 108 PT
PT, post-transformation.
Note: CM drug-free line derived from CQ 18 line on day 60 PT.
18. Continuous Selection of Transformed106/1 Parasites
Produces Stable, Highly CQR Lines
• Continued CQ application (90 ng/ml) -> obtained CQR line.
• IC50, IC90 values consistently exceeded other CQR lines.
• PCR, Southern analyses: pNHSC plasmid not present.
Sequence of chromosomal
pfcrt gene: single point
mutation in highly CQR line
(34-1/E, “K76I”), precisely at
codon identified as critical by
linkage analysis. Encodes
novel 76I mutation.
19. Are pfcrt Point Mutations Responsible
for CQR Phenotype ?
• Pursued using allelic exchange strategy involving the
introduction of entire sets of pfcrt point mutations from
CQR parasites into sensitive parasite, to test for acqui-
sition of complete or partial CQR phenotype.
• Required two rounds of genetic modification (using
human dhfr and blasticidin S-deaminase markers) to
target desired region and introduce multiple alleles.
20. Transformation of C1GC03 with pfcrt Alleles
from CQR Strains of Distinct Geographic
Origins -> Clones Expressing Wild Type and
Mutant PfCRT Haplotypes.
PfCRT amino acids
Clones 72 74 75 76 97 220 271 326 356 371
GC03 C M N K H A Q N I R
C1GC03 C M N K H A Q N I R
C2GC03 C M N K H A Q N I R
C3Dd2 C I E T H S E S T I
C4Dd2 C I E T H S E S T I
Dd2 C I E T H S E S T I
C576I C I E I H S E S I I
106/76I C I E I H S E S I I
C67G8 S M N T H S Q D L R
7G8 S M N T H S Q D L R
21. CQS Parasites Expressing Mutant Old and New World pfcrt
Alleles Acquire Verapamil-Reversible CQR Phenotype
250 CQ IC50
Chloroquine
CQ+V P IC50
[CQ] (nM) 200
150
100
50
0
C3Dd2
C1GC03
C2GC03
C5K76I
C4Dd2
GC03
K76I
Dd2
7G8
C67G
8
Clones
Recombinant clones expressing mutant pfcrt show
reduced CQ accumulation (3H-CQ uptake assays)
22. Heme-Binding Antimalarial Structures
N(CH CH3)2
2 OH
OH N
NH N
H
OMe
o
Cl N N N
Chloroquine Quinine Quinidine
H
N CH3
H
H
NH OH CH3
OH O
O
O
CH2-N(CH CH3)2
2 H
H
O
Cl N N CF3 CH3
CF3 O
Amodiaquine Mefloquine Artemisinin
24. Evidence for stereospecificity of pfcrt-mediated CQR
200
180
AQ-13
AQ-26
160 CQ
AQ-33
140
AQ-40
120
100
80
60
40
20
0
C2GC03 C4Dd2 Dd2 C67G8 7G8
Clones
Diaminoalkane side chain analogs
N(CH CH3)2
2
NH - R - NEt2
Compound Side chain
AQ-13 (CH2)3
AQ-26 (CH2)4
Cl N
CQ CHMe(CH2)3
AQ-33 (CH2)6
AQ-40 (CH2)12
25. Spotlight on amino acid 76 as a candidate marker
of CQR in vitro and CQ treatment failure in vivo.
Amino acid 76 All CQR lines: Threonine (T) at 76
All CQS lines: Lysine (K) at 76
COOH
NH2
CQ treatment leads to parasite clearance in some
patients harboring the pfcrt T76 allele Other genes
required for resistance?? Result of antimalarial
immunity??
Is the K76T mutation critical for CQR?
26. Allelic-exchange strategy
Plasmid
Truncated pfcrt BSD
T76 Shortened UR
K76
Full-length pfcrt
Genome Endogenous UR DR
Endogenous UR Full-length pfcrt
BSD DR
Genome Truncated pfcrt Shortened UR
UR – upstream region; DR – downstream region; BSD – blasticidin-S-deaminase
27. Molecular characterization of recombinant clones
Southern blotting
Dd2
Dd276K
Dd276K
5.2
Dd2
kb
20.0
Dd276K
7.8 7.4 4.8 7.8
7.4
20.0
5.2
4.8
Wild-type
locus
PCR
Dd276K
Dd276K
Dd276K
Dd2
Dd2
Dd2
Truncated pfcrt Full-length pfcrt
BSD DR
Full-length UR Shortened UR
28. Allelic exchange strategy
• Replaced K for a T at PfCRT position 76 in Dd2.
• Replaced K for a T at PfCRT position 76 in 7G8 and also substituted
other residues proximal to this mutation in transmembrane domain I.
• Introduced Dd2 mutations in transmembrane domain I proximal to
position 76 into the 7G8 functional pfcrt allele.
pfcrt haplotype of recombinant and wild-type clones
PfCRT amino acid polymorphisms
Line Rec. Parent Construct 72 74 75 76 97 220 271 326 356 371
Dd2 (CQR) C I E T H S S S T I
Dd2_T76K Yes Dd2 T76K C I E K H S S S T I
Dd2_control Yes Dd2 Control C I E T H S S S T I
7G8 (CQR) S M N T H S Q D L R
7G8_T76K Yes 7G8 T76K C I E K H S Q D L R
7G8_control Yes GC03 Control S M N T H S Q D L R
7G8_Dd2_TMI Yes 7G8 Dd2_T76 C I E T H S Q D L R
GC03 (CQS) C M N K H A Q N I R
3D7 (CQS) C M N K H A Q N I R
Rec., Recombinant.
All recombinant lines were cloned and their phenotypes established
29. Drug Susceptibility Profile: Dd2 Background
* p< 0.05, ** p< 0.01, *** p< 0.001
Chloroquine Mono-desethyl chloroquine
Clones Clones
Removal of K76T mutation ablates all CQR and leads
to
total loss of verapamil reversibility.
30. Drug Susceptibility Profile: Dd2 Background
Quinine Artemisinin
Quinidine Mefloquine
Amodiaquine • Tendency towards reduced
susceptibility to quinine,
quinidine and mefloquine,
statistically non-significant.
• Some reduction in cross-
resistance to amodiaquine.
31. Drug Susceptibility Profile : 7G8 Background
* p< 0.05, ** p< 0.01
Chloroquine Monodesethyl chloroquine
Clones Clones
Removal of K76T mutation also negates CQR and
verapamil reversibility on 7G8 (S. American) background.
Modest reduction in susceptibility to quinine and
amodiaquine.
32. Summary of K76T study
• Previous clinical studies have implicated PfCRT K76T as
CQR marker.
• Certain pfcrt mutations postulated to affect degree of
VP reversibility.
• In this study, we used allelic exchange to prove that
K76T mutation is necessary for CQR mechanism.
• Loss of this mutation ablates resistance (to CQ and
side-chain analogs), and negates VP reversibility.
• Mutations in TM domain I of PfCRT appear to
determine degree of reversibility.
• Data may suggest physical interaction of mutant PfCRT with
CQ and VP.
33. OVERALL SUMMARY
• Genetic cross, field isolates implicate pfcrt as a key
determinant of CQR. Clinical studies: pfcrt mutations
associated with increased risk of CQ treatment failure.
• Allelic exchange studies demonstrate that pfcrt mutations
confer verapamil-reversible CQR!
• CQR likely arose in multiple endemic areas via mutations in
pfcrt. Degree of CQR probably influenced by changes in
additional genes including pfmdr1.
• PfCRT mutations affect susceptibility to multiple heme-
binding antimalarials. Drug transport? Indirect pH effect?
34. Clinical data support central role for pfcrt in
CQR
• Trial in Mali: gave CQ to 400 patients with uncomplicated
falciparum malaria: CQ treatment failure recorded in 60.
• Every case of CQ treatment failure found to harbor the
PfCRT K76T mutation exclusively, compared to background
prevalence of 40%.
• Lesser selection also observed for pfmdr1 mutations (50%
background, 86% in CQ treatment failures).
• Some patients carried PfCRT K76T marker and were
cured, indicating either lack of a second genetic determinant
or the involvement of other factors (immunity, concomitant
infection?).
35. Evidence for age- and immunity-dependent clearance
of P. falciparum infections (Mali, Djimde et al.)
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
<1 1 2 3 4 5 6 7 8 9 10 11 12 13+
Age in years
36. Evidence for chloroquine-selected sweep of mutant
pfcrt alleles throughout Asia and Africa
a, b, Allelic diversity for CQS (red) and CQR (black) isolates from Africa (a) and Asia (b). Peaks
Represent regions with reduced diversity. c, d, allelic diversity ratio comparing CQR and CQS
isolates from Africa (c) and Asia (d) respectively. A highly significant peak was identified for pfcrt
(chromosome 7), demonstrating the power of this approach for detecting drug-resistance genes in
malaria parasites. ADR < 3, not statistically significant. From Wootton et al. Nature (2002) 18: 320.
37. Acknowledgments
Albert Einstein College of Medicine
Amar bir Singh Sidhu, Dominik Verdier-Pinard,
Rebecca Muhle, Viswanathan Lakshmanan,
Pedro Moura, Stephanie Valderramos
Myles Akabas
Malaria Genetics Section, LPD, NIAID, NIH
Thomas Wellems
Roland Cooper, Michael Ferdig, Xin-zhuan Su
Georgetown University
Paul Roepe