l'identification du rôle principal du gène pfcrt dans le mécanisme de...
DESCRIPTION
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 - [email protected]TRANSCRIPT
Where are we in the fight against Where are we in the fight against Malaria?Malaria?
• Drugs: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?)
CQ: most widely usedantimalarial:Safe, rapidly effective,affordable.
Acts in digestivevacuole.
Amino acids
Hemoglobin
Pigment
Globindigestion
Heme poly-merization
Digestive vacuole
Red blood cell
PARASITE
Chloroquine
Impact of Impact of chloroquinechloroquine resistanceresistance• 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)
Chloroquine Resistance Arose IndependentlyIn the Old and New World
Antimalarial Drug Policies in Africa
Chloroquine
SP
Chloroquine + SP
Chloroquine (with > 25% RII/RIII)
?
?
?
* = Declared “interim”
** *
**
Source: Peter Bloland, CDC
Hypotheses on the CQR Mechanism:
Mostly based on observation that CQR parasites characterized Mostly based on observation that CQR parasites characterized by reduced CQ accumulation and by reduced CQ accumulation and chemosensitizationchemosensitization by by verapamilverapamil..
A.A. Due to drug efflux pump?? Similar to PDue to drug efflux pump?? Similar to P--glycoprotein?glycoprotein?B. B. Due to reduced activity of CQ importer?Due to reduced activity of CQ importer?C. C. Due to pH gradient limiting influx of CQ?Due to pH gradient limiting influx of CQ?D. D. Due to altered CQ metabolism or changes in Due to altered CQ metabolism or changes in hemeheme receptor?receptor?E. E. Due to reduction in Due to reduction in hemeheme receptor concentration or reducedreceptor concentration or reduced
CQ access to CQ access to hemeheme? ?
pfmdr1: the first major candidate CQR determinant
• Identified on basis of homology to mammalian multidrugresistance (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.
Mapping the CQR Determinantin a P. falciparum Genetic Cross
CQ-sensitive clone
CQ-resistant clone
Mosquitoes
Chimpanzee
Clone Independent Progenyand Determine Drug Responses
Map Genetic Locus
Identify Gene(s)
0 2 00 501 0 40 3 kb
M
hsp86 o1 o2 cg4 cg3 cg6 cg2
K MV
L NKCC MV
MKM L CK
M CM
L L N
cg7
crossovercrossover
cg9o3 cg8 cg1pfcrt
Location of pfcrt Linked to Chloroquine Resistance
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.
Immuno-EM localizes PfCRT to digestive vacuole membrane
Used affinity-purified rabbit IgG raised to PfCRT peptide
Hemozoin
PfCRT
NH2
COOH
K76
N75M74
C72
H97
A220N326
Q271 R371
I356
TINQSQTEMCSouth America W2RLDQSHTNMCSouth America W1bRLDQSHTNMSSouth America W1aRLDQSHTNMSPapua New Guinea
P1
IISESHTEICSE Asia & Africa E1b
ITSESHTEICSE Asia & Africa E1aChloroquine resistant
IISESHKEIC106/1 (revertant?)RINQAHKNMC“wild type”
Chloroquine sensitive371356326271220977
6757472Parasite type & originPfCRT position & encoded amino acid
Wellems & Plowe 2001: Fidock et al. 2000, Chen et al. 2001
pfcrt Mutations Associated with CQR
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 -> electroporatedrecombinant plasmid into 106/1 and selected on CQ.
Timetable with mutant pfcrt-transformed 106/1 line
CQ 18
Appeared day 46 PT
PT, post-transformation. Note: CM drug-free line derived from CQ 18 line on day 60 PT.
CQ 36
Began day 61 PT
CQ 60
Began day 67 PT
CQ 75
Began day 86 PT
CQ 90
Began day 98 PT
Drug assay 56 day 73 PT
Drug assay 61 day 108 PT
0102030405060708090
100
0 8 16 24 30 36 48 60 72 84 96[chloroquine] (ng/ml)
CQ 18/VPCQ 36/VPCQ 60/VPCM/VP106/1/VP
0102030405060708090
100
0 8 16 24 30 36 48 60 72 84 96[chloroquine] (ng/ml)
CQ 18CQ 36CQ 60CM106/1
Drug Assay 56. CQ ± VP response of pNHSCtransformed 106/1 lines
without verapamil with verapamil
Assay 61. CQ ± VP response of CQ pressured transformants
with verapamilwithout verapamil
0102030405060708090
100
0 9 20 46 103 231
[chloroquine] (ng/ml)
CQ 60/VPCQ 75/VPCQ 90/VPCQ 120/VPCM/VP106/1/VPFCB/VPDd2/VP
0102030405060708090
100
0 9 20 46 103 231
[chloroquine] (ng/ml)
CQ 60CQ 75CQ 90CQ 120CM106/1FCBDd2
Continuous Selection of Transformed106/1 ParasitesProduces 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.
• 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.
Are pfcrt Point Mutations Responsible for CQR Phenotype ?
Transformation of C1GC03 with pfcrt Alleles from CQR Strains of Distinct Geographic
Origins -> Clones Expressing Wild Type and Mutant PfCRT Haplotypes.
Clones 72 74 75 76 97 220 271 326 356 371GC03 C M N K H A Q N I RC1GC03 C M N K H A Q N I RC2GC03 C M N K H A Q N I RC3Dd2 C I E T H S E S T IC4Dd2 C I E T H S E S T IDd2 C I E T H S E S T IC576I C I E I H S E S I I106/76I C I E I H S E S I IC67G8 S M N T H S Q D L R7G8 S M N T H S Q D L R
PfCRT amino acids
0
50
100
150
200
250 CQ IC50CQ+VP IC50
GC
03
C1G
C03
C2G
C03
C3D
d2
C4D
d2
Dd2
C5K
76I
K76
I
7G8
C67
G8
[CQ
] (nM
)
Clones
Chloroquine
Recombinant clones expressing mutant pfcrt show reduced CQ accumulation (3H-CQ uptake assays)
CQS Parasites Expressing Mutant Old and New World pfcrtAlleles Acquire Verapamil-Reversible CQR Phenotype
N
N
OHH
OMe
Quinine
N
N
o
OH
Quinidine
N
CF3
CF3
N
H
OH
H
Mefloquine
N
NHN(CH2CH3)2
Cl
Chloroquine
O
CH3
CH3
O
O
OOCH3
H
H
H
Artemisinin
N
NH OH
CH2-N(CH2CH3)2
Cl
Amodiaquine
Heme-Binding Antimalarial Structures
0
20
40
60
80
100 MFQIC50MFQ+VPIC50
* ** ** *** * **
****
* ***
** * ****
*
0
10
20
30
40
50 ADQIC50ADQ+VPIC50
*
0
10
20
30
40
50
60 ARTIC50ART+VPIC50
* ***
*****
** *** *
Mutant pfcrt SequencesIncrease ParasiteSusceptibility to Quinine,Mefloquine and ArtemisininAnd do not DramaticallyDiminish AmodiaquineEfficacy.
50
100
150
200
250 CQIC50CQ+VPIC50
*
*
*
*
*
*
**
* **
*
**
*
* **
*
* * *
**
****
Chloroquine
0
C67
G8
GC
03C
1GC
03
C2G
C03
C3D
d2
C4D
d2
Dd2
C5K
76I
K76
I
Clones
7G8 0
50
100
150
200
250
300
350 QNIC50QN+VPIC50
*
**
*
*
*
**
** ****
****
C67
G8
GC
03C
1GC
03
C2G
C03
C3D
d2
C4D
d2
Dd2
C5K
76I
K76
I
Clones
7G8
Quinine
C67
G8
GC
03
C1G
C03
C2G
C03
C3D
d2
C4D
d2
Dd2
C5K
76I
K76
I
Clones
7G8
Amodiaquine
GC
03C
1GC
03
C2G
C03
C3D
d2
C4D
d2
Dd2
K76
I
Clones
7G8
C67
G8
C5K
76I
Mefloquine
GC
03C
1GC
03
C2G
C03
C3D
d2
C4D
d2
Dd2
K76
I
Clones
7G8
C67
G8
C5K
76I
Artemisinin
N
NHN(CH2CH3)2
Cl
- R - NEt2Diaminoalkane side chain analogs Compound Side chainAQ-13 (CH2)3AQ-26 (CH2)4CQ CHMe(CH2)3AQ-33 (CH2)6AQ-40 (CH2)12
0
20
40
60
80
100
120
140
160
180
200
C2GC03 C4Dd2 Dd2 C67G8 7G8
Clones
AQ-13 AQ-26 CQ AQ-33AQ-40
Evidence for stereospecificity of pfcrt-mediated CQR
Amino acid 76
Spotlight on amino acid 76 as a candidate marker of CQR in vitro and CQ treatment failure in vivo.
COOH
NH2
All CQR lines: Threonine (T) at 76All CQS lines: Lysine (K) at 76
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?Is the K76T mutation critical for CQR?
Allelic-exchange strategy
Shortened URTruncated pfcrt
Plasmid
Genome
BSD
Endogenous UR
DR
Full-length pfcrtEndogenous UR
Full-length pfcrt
BSD
DR
Truncated pfcrtGenome
T76K76
Shortened UR
UR – upstream region; DR – downstream region; BSD – blasticidin-S-deaminase
Molecular characterization of recombinant clones
PCR
DRFull-length pfcrt
Full-length UR Shortened URBSD
Truncated pfcrt
Dd2
76K
5.2
7.87.4
4.8
20.0
Dd2
kbDd2
76K
Dd2
Dd2
76K
Dd2
Dd2
76K
Wild-type locus
Dd2
Dd2
76K
Southern blotting
Dd276K
20.07.8 7.4 4.8
Dd2
5.2
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 IDd2_T76K Yes Dd2 T76K C I E K H S S S T IDd2_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 R7G8_T76K Yes 7G8 T76K C I E K H S Q D L R7G8_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.
PfCRT amino acid polymorphisms
pfcrt haplotype of recombinant and wild-type clones
• 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.
All recombinant lines were cloned and their phenotypes established
Allelic exchange strategy
Drug Susceptibility Profile: Dd2 Background
Chloroquine Mono-desethyl chloroquine
Clones Clones
Removal of K76T mutation ablates all CQR and leads to
total loss of verapamil reversibility.
* p< 0.05, ** p< 0.01, *** p< 0.001
QuinineQuinidine
ArtemisininMefloquine
Amodiaquine
Drug Susceptibility Profile: Dd2 Background
• Tendency towards reduced susceptibility to quinine, quinidine and mefloquine, statistically non-significant.
• Some reduction in cross-resistance to amodiaquine.
Drug Susceptibility Profile : 7G8 Background
Chloroquine Monodesethyl chloroquine* p< 0.05, ** p< 0.01
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.
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.
OVERALL SUMMARYOVERALL SUMMARY
• Genetic cross, field isolates implicate pfcrt as a keydeterminant 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?
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?).
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<1 1 2 3 4 5 6 7 8 9 10 11 12 13+
Age in years
Evidence for age- and immunity-dependent clearance of P. falciparum infections (Mali, Djimde et al.)
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). PeaksRepresent 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 inmalaria parasites. ADR < 3, not statistically significant. From Wootton et al. Nature (2002) 18: 320.
AcknowledgmentsAcknowledgments
Malaria Genetics Section, LPD, NIAID, NIHThomas Wellems
Roland Cooper, Michael Ferdig, Xin-zhuan Su
Georgetown UniversityPaul Roepe
Albert Einstein College of MedicineAmar bir Singh Sidhu, Dominik Verdier-Pinard,
Rebecca Muhle, Viswanathan Lakshmanan, Pedro Moura, Stephanie Valderramos
Myles Akabas