Mc4404.qxd

Conservation of a novel vacuolar transporter in Plasmodium
species and its central role in chloroquine resistance of
P. falciparum [AU:OK?]
Jane MR Carlton*, David A Fidock†, Abdoulaye Djimd釧¶,
Christopher V Plowe§ and Thomas E Wellems#
Chloroquine resistance in Plasmodium falciparum has recently 75–90 million cases of non-fatal malaria annually [2•], has been shown to result from mutations in the novel vacuolar recently become an area of increasing concern. transporter PfCRT. Field studies have demonstrated theimportance of these mutations in clinical resistance. Although Here, we review recent progress in deciphering CQ resis- a pfcrt homolog has been identified in Plasmodium vivax, there tance in malaria parasites. These developments include is no association between in vivo chloroquine resistance and the identification of mutations in a vacuolar transporter as codon mutations in the P. vivax gene. [AU:OK?] This is
the basis for CQ resistance in P. falciparum and the finding consistent with lines of evidence that suggest alternative of absolute selection of these mutations in clinical cases of mechanisms of chloroquine resistance among various malaria CQ treatment failure. These results are generating new hypotheses on the molecular mechanism of CQ resistance.
Investigations into CQ resistance in other malaria parasites Addresses
also provide evidence that mechanisms of resistance differ *National Center for Biotechnology Research, National Library of Medicine, National Institutes of Health, Building 45, 45 Center Drive,Bethesda, MD 20892-6510, USA Three distinct evolutionary clades of malaria
†Department of Microbiology and Immunology, Albert Einstein Collegeof Medicine of Yeshiva University, 1300 Morris Park Avenue, parasites
Malaria parasites are classified in the phylum ‡Malaria Research and Training Center, Faculty of Medicine, Apicomplexa, a large protist group consisting of almost Pharmacy and Dentistry, University of Mali, PO Box 1805, Bamako, Mali 5000 species. All apicomplexans are parasites and contain §Malaria Section, Center for Vaccine Development, University ofMaryland School of Medicine, 685 West Baltimore Street, HSF 480, an organellar structure, the apical complex, involved in host cell invasion. Within the phylum, the genus #Malaria Genetics Section, Laboratory of Parasitic Diseases, National Plasmodium includes ~200 known malaria species that par- Institute of Allergy and Infectious Diseases, National Institutes of asitize birds, reptiles, and mammals. The genus divides Health, Building 4, 4 Center Drive, MSC 0425, Bethesda, into three distinct and highly divergent evolutionary clades MD 20892-0425, USA; email: [email protected] [3,4]: the first includes P. falciparum and a closely related Current Opinion in Microbiology 2001, 4:XXX–XXX
parasite of apes, P. reichenowi; the second clade consists of P. vivax and monkey malaria species including P. knowlesi; 2001 Elsevier Science Ltd. All rights reserved.
and finally, the third clade includes rodent malaria speciessuch as P. berghei and P. chabaudi. [AU:OK?] Major differ- Abbreviations
CQ
ences in host specificity and disease manifestation occur P. falciparum chloroquine resistance transporter among species of these clades, as do wide variations ingenome composition and codon usage [5,6]. Because of the Introduction
difficulties of working with P. falciparum in the laboratory, [AUQ1: I have shortened the title slightly to make it more there has been support for the use of many of these relat- concise. I understand you want to emphasis the fact that ed species as models, for example, in studies of host cell the transporter plays a role in CQ resistance in P. falci- invasion [7], malaria vaccine development [8], and anti- parum and not P. vivax but I think this is covered in the malarial drug resistance (reviewed in [9•]).
abstract and it’s better THIS IS A PROOF The mechanism of chloroquine action
In human erythrocytes, P. falciparum supports its growth by
Malaria parasite resistance to the drug chloroquine (CQ) taking up host cell cytoplasm in an acidic digestive food poses a severe and increasing public health threat. This vacuole [10]. Toxic heme, in its hematin (µ-oxodimer) inexpensive and widely consumed drug has been the main [AU:OK?] form, is released in the vacuole by hemoglobin line of attack against the parasite, and its increasing failure digestion and crystallized into innocuous hemozoin, or accompanies a return of malaria-related morbidity and malaria pigment. CQ is proposed to interfere with this mortality levels not seen for decades [1•]. The problem is process by complexing with hematin [11,12], thereby cre- most acute in Plasmodium falciparum malaria, the species ating toxic complexes that cause parasite death. The actual responsible for the most severe form of the disease. The mechanism of toxicity [AUQ2: Is this toxicity of hematin or emergence of CQ-resistant P. vivax, a species that causes the CQ—hematin complex? Or both?] is still subject to Host–microbe interactions: parasites
debate, but hematin can increase membrane permeability [AU: New paragraph OK?] Of the 15 CQ-sensitive lines leading to cell lysis [13] and is known to inhibit parasite tested, [AU: What strains are being referred to here? Are enzymes [14]. Recent studies on the crystal structure of β- they from the original genetic cross (ref 25, 26) or are they hematin, a synthetic analog of malaria pigment, indicate clinical strains from around the world (ref 29)?] all but one that CQ is ‘chemiabsorbed’ onto hemozoin, capping crys- carried the pfcrt sequence of the CQ-sensitive HB3 parent.
tal growth that is required for hematin sequestration [15••].
The one exception, 106/1, was found to encode all of the [AUQ3: Please clarify: according to this model, CQ doesn’t PfCRT mutations associated with CQ resistance except form a complex with hematin but with hemozoin, thereby the amino acid mutation at position 76, supporting a cen- preventing binding/crystallization of hematin to hemo- tral role for this residue in CQ resistance. Episomal transformation of 106/1 and two additional CQ-sensitivestrains with constructs expressing pfcrt from CQ-resistant The physiologic basis of chloroquine resistance
parasites resulted in transformed lines that grew at CQ A consistent characteristic of CQ-resistant P. falciparum concentrations tolerated only by naturally CQ-resistant parasites in vitro is their reduced accumulation of CQ in strains. Stepwise CQ pressure on the transformed 106/1 the digestive vacuole relative to accumulation of the drug parasites ultimately resulted in loss of the transfected in CQ-sensitive parasites [16–18]. Another characteristic of DNA and selection of a highly CQ-resistant line that had CQ-resistant parasites is their chemosensitization to CQ by undergone a single K76→I point mutation, providing addi- structurally diverse agents that include verapamil, a Ca2+ tional evidence for the central role of position 76 in CQ channel blocker [19]. [AUQ4: Does this mean that they became sensitive to CQ after exposure to verapamil?]Proposals to explain these features of resistant parasites The K76→T mutation has not been observed in the have included alterations in the intraerythrocytic parasite absence of mutations at other positions in PfCRT, that affect CQ uptake or efflux at the cytoplasmic mem- although the reverse situation has been documented (i.e.
brane, or change H+ or CQ concentration in the digestive mutations at other positions can occur without the pres- ence of K76→T, as in the 106/1 line). It is plausible thatmutations at other positions are required to maintain criti- Identification of the genetic determinant of
cal functional properties of the molecule in the presence of chloroquine resistance in P. falciparum
the K76→T change. The mutation A220→S may fulfill a To investigate the genetic basis of P. falciparum CQ resis- particular requirement in this regard, since this mutation tance, Wellems et al. [25] established a genetic cross has consistently been found to accompany K76→T in CQ- between a CQ-sensitive clone, HB3 from Honduras, and a resistant parasites from the different New World and Old CQ-resistant clone, Dd2 from Indochina. Linkage analysis World foci. The suggestion that K76→T cannot occur in of 16 independent progeny showed that the verapamil- the absence of other PfCRT point mutations may also reversible CQ-resistant phenotype segregated as a single explain the slow genesis of CQ resistance in the field as Mendelian trait that mapped to chromosome 7 [26].
well as the difficulties that have been experienced with Examination of further progeny localized this CQ resis- attempts to select CQ resistance in the laboratory. Indeed, tance determinant to a 36 kb segment on the chromosome the CQ-resistant line containing the K76→I point muta- [27]. A gene (cg2) initially identified as a probable CQ tion reported by Fidock et al. [29••] was obtained from the resistance candidate was ruled out by allelic-exchange CQ-sensitive 106/1 line that already contained six PfCRT mutations at other positions seen [AU:OK?] in SoutheastAsian and African parasites. Recently, Fidock et al. [29••] identified the pfcrt (P. falci-parum chloroquine resistance transporter) gene near cg2 in Characterization of the protein product of pfcrt
the 36 kb segment. In the CQ-resistant Dd2 parent, eight The protein product of pfcrt, PfCRT, belongs to a previ- point mutations (M74→I, N75→E, K76→T, A220→S, ously uncharacterized family of putative transporters, with Q271→E, N326→S, I356→T, and R371→I) were found in 10 transmembrane segments (Figure 1) but few other rec- ognizable features [31••]. Localization studies place it at pfcrt gene. Seven of these eight mutations were detected the membrane of the parasite’s digestive vacuole [29••].
in 15 CQ-resistant parasite strains collected from diverse Moreover, PfCRT mutations are associated with a decrease regions of Asia and Africa (the remaining mutation (acidification) in the pH of the digestive vacuole of CQ- I356→T was detected in some strains). CQ-resistant resistant parasites by some 0.3–0.5 units compared with strains from South America were found to harbor distinct the pH of the digestive vacuole of CQ-sensitive parasites sets of PfCRT mutations but shared the K76→T and [29••]. This result might appear paradoxical given that vac- A220→S mutations in common with the Asian and African uolar acidification predicts increased CQ accumulation in strains. These findings suggested that PfCRT mutations the digestive vacuole on the basis of Henderson- arose separately in association with CQ resistance in South Hasselbach equilibrium [18,32], whereas CQ-resistant America and Asia/Africa, a result consistent with the inde- parasites are known to exhibit reduced CQ accumulation.
pendent genesis of CQ resistance in these regions [30]. CQ accumulation in the digestive vacuole, however, is dri- A novel vacuolar transporter in Plasmodium species Carlton et al.
The schematic structure of the proteinproduct of the pfcrt gene, PfCRT, showing theten predicted transmembrane domains. Thepositions of the mutations identified from theanalysis of over forty geographically diverse isolates are indicated by filled circles.
[AUQ17: In the text it says there are eight
point mutations but there are ten shown
here. Please clarify.] The K (lysine) to T
(threonine) change at position 76 (indicated
by the arrow) is critical to CQ resistance in
P. falciparum.
ven to a large extent by binding of CQ to hematin [AUQ5: mutation in vivo by CQ treatment. [AUQ10: I don’t under- See AUQ3] [17,22], and recent data have shown a steep stand why this demonstrates a selection for the K76→T pH-dependence in the conversion of soluble hematin- mutation by CQ treatment. 41% of the infections had the receptor [AUQ6: What is this receptor?] to hemozoin K76→T mutation (i.e. CQ resistant) yet only 14% of the [12,23••]. These results have suggested a model whereby infections were not successfully treated with CQ. Is the alterations in PfCRT could cause increased acidification of take home message the fact that 100% of these failed treat- the digestive vacuole, resulting in reduced levels of acces- ments had the mutation?] The presence of K76→T at the sible hematin with a consequent reduction in time of treatment was strongly associated with subsequent CQ—hematin complexes [AUQ7: See AUQ3] and toxicity. failure of CQ treatment [37••]. Moreover, the ability ofindividuals [AU:OK?] to clear infections carrying the The above theory is, however, difficult to reconcile with K76→T mutation in this highly endemic area was strongly the reported effectiveness of CQ analogs with substituted associated with increasing age. These data suggest that or shortened side chains against CQ-resistant parasites immunity against P. falciparum acquired with age con- [33–36]. Such findings support a second theory: that tributed to successful treatment outcomes of some PfCRT mutations alter CQ flux across the digestive vac- individuals harboring parasites with the K76→T mutation uole membrane. The predicted structure of PfCRT places amino acid substitutions K76→T and K76→I within atransmembrane region that may be involved in transport of [AUQ11: New paragraph OK?] Although it is possible that diprotic [AUQ8: What does “diprotic” mean?] CQ or parasite genetic factors other than pfcrt may modulate another charged substance. Both of these changes involve in vitro or in vivo levels of CQ resistance and that host fac- loss of a positive charge at position 76 in the molecule.
tors other than acquired immunity may affect the clearanceof CQ-resistant parasites, such factors have yet to be clear- PfCRT mutations and their association with
ly demonstrated and understood in the context of failure of chloroquine treatment
treatment failures. The identification of PfCRT K76→T mutation as a key molecular marker of CQ resistance offers evidence that mutations in PfCRT were critical for CQ new opportunities for diagnosis and public health surveil- resistance in vivo [37••]. In this trial, CQ treatment lance of P. falciparum infections.
responses were followed in 469 cases of uncomplicated fal-ciparum malaria. CQ failed to treat 14% of these cases.
Effects of pfmdr1 and other secondary genes
[AU:OK?] In every case of treatment failure, the K76→T on chloroquine resistance levels
mutation, in concert with other PfCRT mutations, was Although the association of pfcrt alleles with CQ resistance exclusively present in the post-treatment infection. This in vitro and in vivo is evident, the roles of other genes, such compared with a baseline prevalence of 41% of infections as the multidrug resistance gene pfmdr1 [38,39], are less carrying the K76→T mutation in a random sample of 116 clear. Impetus for the isolation of pfmdr1 came from the patients, [AUQ9: Were these patients from the CQ effica- finding that verapamil, which inhibits P-glycoprotein cy trail?] demonstrating absolute selection for this mediated multidrug resistance in mammalian tumor cells, Host–microbe interactions: parasites
Evidence for another chloroquine resistance
mechanism in P. vivax
Since its introduction, CQ has been the drug of choice for
eliminating not only P. falciparum blood-stage parasites but also infections caused by the three other human parasites P. ovale, P. malariae and P. vivax. To date, no reports of CQ- resistant P. ovale and P. malariae have been confirmed [46].
CQ-resistant P. vivax, however, was first reported from Papua New Guinea in 1989 [47] and since then has been an increasing problem in other countries.
To investigate whether similar mechanisms of CQ resis- Depiction of the factors that contribute to the failure of chloroquinetreatment (clinical resistance) in uncomplicated P. falciparum malaria.
tance exist in P. falciparum and P. vivax, pfcrt homologs Mutations in pfcrt confer the CQ resistance (CQR) phenotype to were identified in P. vivax, as well as in other Plasmodium P. falciparum malaria parasites. In the presence of these mutations, species, and assessed for possible relationship with CQ immune status is a critical factor in therapeutic outcome.
resistance. Results from this study showed that pfcrt hashighly conserved homologs in all of the Plasmodium clades also chemosensitized CQ-resistant P. falciparum strains [31••]. Homologs of pfcrt from P. vivax, P. knowlesi and [19]. The pfmdr1gene encodes an ATP-dependent trans- P. berghei were sequenced, revealing the gene family to be membrane protein, Pgh-1, that has also been localized to highly conserved in composition and structure across all the parasite’s digestive vacuole [40]. Evidence from differ- three lineages. Regions of the orthologous P. vivax gene, ent studies has sometimes shown associations between CQ pvcg10, were sequenced from 20 geographically distinct resistance and pfmdr1 copy number [38] or mutations [41], laboratory lines and field isolates of P. vivax. No association most notably at position 86 in the protein where mutation between codon mutations in pvcg10 and in vivo CQ of an asparagine residue to tyrosine has frequently been response could be demonstrated, indicating that the mole- documented (N86→Y; ‘K1 allele’); however, many excep- cular events underlying CQ resistance in P. vivax differ tions to these associations have been established both from from those in P. falciparum [31••].
a genetic cross [25] and from field surveys (reviewed in[42•]). In this light, it is useful to consider laboratory models ofmalaria and ask what information they may provide of rel- Concomitant with mutant pfcrt selection in clinical cases of evance to the mechanisms of CQ resistance in human malaria, Djimdé et al. [37••] found an increase of the Pgh-1 malaria species. Although little can be said with regard to N86→Y mutation from a baseline prevalence of 50% to a P. vivax at this point, available data suggest that mecha- prevalence of 86% in cases of CQ treatment failure. A total nisms of CQ resistance in the rodent malaria parasites, of 30% of infections from the treatment failure group car- P. chabaudi and P. berghei [AU:OK?], have notable differ- ried the wild-type Pgh-1 N86 (16% as mixed parasite ences from the mechanism in P. falciparum. CQ-resistant populations with the N86 and Y86 codons). Furthermore, lines of P. chabaudi have been selected with relative ease in the presence of parasites with the mutant N86→Y in addi- the laboratory [48], in contrast to the difficulties in obtain- tion to the PfCRT K76→T mutation did not increase the ing CQ-resistant P. falciparum lines [49]. Quantitative trait relative risk of treatment failure when compared with mapping of progeny from crosses between CQ-resistant infections carrying only the PfCRT K76→T mutation and CQ-sensitive P. chabaudi clones produced evidence for before treatment. Prediction of CQ susceptibility in clini- a combined role of several genes on different chromo- cal cases of malaria was therefore not possible through somes in conferring CQ resistance [50], unlike the major monitoring of pfmdr1 genetic alterations. genetic locus identified in P. falciparum [26,27]. An unsta-ble form of CQ resistance in P. berghei has been associated [AUQ12: New paragraph OK?] Interestingly, recent allelic- with reduced malaria pigment formation [51], whereas showed that, although pfmdr1 mutations there are no obvious differences in the quantity of hemo- could not confer resistance to CQ-sensitive parasites, zoin in CQ-resistant and CQ-sensitive P. falciparum [52].
removal of three pfmdr1 mutations S1034→C, N1042→D,and D1246→Y from a CQ-resistant parasite modified the The fact that mechanisms of CQ resistance among differ- in vitro measures of resistance [43••]. Mutations in pfmdr1, ent Plasmodium species can vary has several implications.
and in other as yet undefined modulator genes, may thus Clearly, results from one species and studies that utilize represent secondary adaptations that enhance parasite fit- laboratory models of malaria should be extrapolated with ness in the presence of pfcrt mutations. Such adaptations care. In particular, similarity between Plasmodium species would be analogous to the compensatory alterations pro- in terms of conserved molecular mechanisms of drug duced in response to acquisition of central resistance response and resistance may depend on the class of anti- determinants shown in other microbial systems [44,45•].
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