Drug Resistance

Drug resistance is a major obstacle to the prevention and treatment of malaria infections worldwide. Our laboratory studies specific loci that may modulate drug resistance or response, and uses genomic tools to identify new information about how the parasite responds to drug pressure and develops drug resistance. Specifically we study the relationship between mutations in candidate drug resistance genes (pfcrt and pfmdr1) and clinical drug resistance in Senegal. Our studies suggest that there are additional genes required for a drug resistant phenotype, and identification of novel markers of drug resistance or response is underway. Other possible mechanisms of drug resistance are being investigated; including the role of putative ABC transporters in Plasmodium falciparum and characterization of mismatch repair pathways in Plasmodium species. In support of our efforts to understand how genetic mutations in candidate drug resistance genes contribute to drug resistance, we study a homologue of pfmdr1 in Leishmania species called lemdr1. We have demonstrated that LeMDR1 is localized intracellularly and that mutations in this gene alter drug resistance. To understand the effects of chloroquine pressure on gene expression in Plasmodium falciparum, we use genomic methods including Serial Analysis of Gene Expression (SAGE) and high-density oligonucleotide array approaches to identify genetic loci that have differential expression under drug pressure. Through these efforts our goal is to better understand how drug resistance occurs and to develop new approaches to combat this global problem.

Gene Expression in Response to Chloroquine

Author: Anusha Munasinghe

By far the most significant factor contributing to the resurgence of malaria is the development of resistance by these forms to cheap and formerly efficacious drugs, such as the quinoline compound chloroquine.  Transcript profiling following drug exposure can be useful for uncovering new leads for drug development, dissecting mechanisms of drug action and resistance, as well as identifying processes linked to the toxic consequences of drug, such as cell death. To this end, chloroquine-induced alterations in transcript levels were preliminarily investigated by SAGE. 

Chloroquine accumulates in the acidic food vacuole of asexual blood stages, where digestion of host hemoglobin generates toxic free heme (FP).   The antimalarial is thought to function primarily by binding FP and preventing its sequestration.  FP-CQ complexes, in turn, may permeabilize membranes; interfere with free radical detoxification; and block protein synthesis, to name a few.

We observe the differential regulation of over one hundred unique genes in the drug-sensitive 3D7 strain.  The SAGE results were validated by Northern analysis and semi-quantitative RT-PCR in a number of cases. A few differentially regulated genes detected in the present study, such as those involved in protein synthesis, oxidative stress responses, and signal transduction, fell into categories previously shown to be directly targeted by chloroquine.  Specifically, PfMDR1, which encodes the P-glycoprotein homologue and modulates resistance in conjunction with other loci was upregulated.  Previous studies have all assayed PfMDR1 expression in resistance strains.  This is the first demonstration of its induction in a chloroquine sensitive strain following drug exposure.

An unanticipated response to drug in the current study was a significant decrease in transcripts involved in mitochondrial metabolism. Chloroquine is not known to target the mitochondria directly; hence the observed dampening of gene expression from this category may reflect early responses to chloroquine's effects on cell viability.  Other unexpected categories of differentially regulated loci revealed by SAGE include those encoding cytoskeletal elements and cell surface antigens.  Intriguingly a large number of unknown ORFs were also modulated by chloroquine treatment.  Such responses may represent largely unexplored consequences of chloroquine treatment in the parasite and as such, provides us with first hints at new avenues that could be exploited therapeutically.

Further investigations into chloroquine-induced alterations in gene expression, following varying exposures and doses of drug, will help delineate early responders, likely to be involved in direct action, from delayed responders, expected to mediate toxic consequences of drug.  In fact we are currently utilizing Affymetrix malaria chips to study 3D7 responses to varying doses of chloroquine, after both a 30 min and 6 hour exposure.

In summary, the ability to assay transcript levels in parallel provides a unique view of gene expression in Plasmodium, affording novel insights into the parasite transcriptome as a whole, and its modulation in response to external stress.  This study together with previously published expression analyses in Plasmodium will set the stage for large-scale cross sectional analyses of the malarial genome.

Lemdr1

Author: Matthew A. Dodge

The approach our research has taken is to examine the problem of drug resistance in the protozoan parasite Leishmania as a model for Plasmodium drug resistance from independent but complimentary perspectives.  A cell biological, biophysical, and genetic, approach was used; each aimed at elucidating the problem of chemotherapeutic failure.

First the cellular localization of LeMDR1, an ABC-transporter involved in the transport of a broad spectrum of compounds including important chemotherapeutics, was examined.  Novel localization of this transporter to a new organelle in L. enriettii, the MVT, using microscopy of GFP and HA tagged versions of the protein explains an initially contradictory drug resistance phenotype.  This intracellular localization could have important ramifications for duel drug therapy regimes.

Second, indirect evidence of LeMDR1 transport activity is provided by FACS and microscopy using several known MDR substrates, but in order to verify that LeMDR1 transports vinblastine we have begun a collaboration with the Woods Hole Molecular Biology Institute.  We are going to directly measure LeMDR1 transport ability by either expressing the protein in Xenopous oocytes or by reconstituting the purified protein in liposomes.  Both measurements will be accomplished with new highly sensitive amperometric detection using vinblastine specific probes that may be used in the future to elucidate resistance caused by other MDR transporters.

Finally, we are examining how drug resistance may develop in these parasites.  We have identified a homologue of MutS in L. major, the gene responsible for the classic "mutator" phenotype, using a combination of bioinformatics and confirmed by RT-PCR.  Because attempts to reduce MutS expression using anti-sense RNA were unsuccessful, we are currently making a MutS gene disruption in Leishmania so that we may assay the phenotype.  It is possible that, like in many other organisms, dysfunction in MutS could be allowing parasites to achieve drug resistance at a more rapid rate than normally achievable.  In fact, evidence from this lab suggests that a single-point mutation in the LeMDR1 gene can drastically alter the drug resistance phenotype of Leishmania.

MutS

Author: Laura Bethke

The DNA mismatch repair pathway has been shown to be involved in point mutation-mediated drug resistance in many organisms, including yeast and bacteria.  Our laboratory has previously demonstrated the presence of the post-replicative mismatch repair pathway in the human malaria parasite, P. falciparum, and in the rodent malaria species, P. berghei.  We have shown that genes with significant sequence homology to MutS and MutL of S. cerevisiae are present in the P. falciparum genome, and that these genes are expressed in a cell cycle-dependent manner, suggesting that they are functional.  Analysis of PfMutS and PfMutS2, two P. falciparum MutS homologues, by expression in bacterial cells indirectly indicates a role for the PfMutS2 gene in mismatch repair activity.  Current research involves an investigation of the role of the MutS homologues in drug resistance of malaria parasites by development of MutS null mutants in the rodent malaria parasite P. berghei.  Future work may involve further investigation of the DNA mismatch repair pathway in P. falciparum, in order to better understand the role of this pathway in parasite adaptation and survival.

Pfmdr1/Pfcrt

Authors: Johanna Daily, Therese Dieng, Alissa Myrick, Ousmane Sarr, Susan Thomas, Dyann F. Wirth

Chloroquine resistance in Plasmodium falciparum is established in many parts of Africa and is becoming more prevalent in areas of West Africa such as Senegal.  Resistance to chloroquine was first reported in Dakar in 1988 and more recent treatment failure rates of 13% have been reported in Senegal.¹  This site is ideal to study the correlation of genetic polymorphisms and chloroquine resistance secondary to the prevalence of single clone infections typical of hypoendemic transmission and in addition, chloroquine remains the first line therapy. 

There has been recent progress in the identification of genetic markers of chloroquine resistance with the discovery of mutations in the P. falciparum pfcrt gene.²   Genetic mutations inpfcrt are strongly associated with both in vitro and in vivo drug resistance.  Resistance defined as in vivo may be confounded by factors such as host immunity.   In contrast, in vitro methods can provide a direct measurement of parasite survival in response to drug treatment.  Thus, resistance of different parasite strains can also be defined by the 50% inhibitory concentration or IC₅₀.  As measured by this in vitro standard, the mutation in pfcrt K76T is still highly correlated with resistance.  Specifically, the presence of the K76T mutation is found in the overwhelming majority of isolates that display in vitro resistance in studies reported from PNG, Thailand, Indonesia, Cameroon and Senegal ^3-5. 

However, it has been also noted that some parasites that were cleared after administration of chloroquine also had the K76T mutation.  This observation has been reported in several studies, including in the original report in which 41% of all isolates contained the K76T mutation, though only 14% of these isolates exhibited in vivo resistance to chloroquine ¹.   A study done in Cameroon using in vitro methods for testing chloroquine susceptibility reported nine isolates with the K76T mutation that had chloroquine IC₅₀s in the sensitive range³.  Both the in vivo and in vitro data suggest that pfcrt K76T mutation is necessary for chloroquine resistance, but a subset of isolates from various field studies do not manifest this correlation which may suggest that for these isolates, mutations in addition to the pfcrt K76T mutation are needed to confer chloroquine resistance. 

Analysis of these chloroquine resistance mutations was studied in Pikine Dakar, the location of a collaborative field study between Cheikh Anta Diop University and Harvard School of Public Health.  This study, funded through Fogharty allows for training of Senegalese scientists, technology transfer and scientific exchange with HSPH malaria experts to establish an independent malaria research unit in Dakar.  The pfcrt T76 allele was found in >90% of in vitro resistant isolates, while isolates with wild type K76 allele were almost completely chloroquine sensitive.  However a number of isolates harboring the T76 allele were also in vitro chloroquine sensitive and therefore the K76T polymorphism did not significantly correlate with resistance in this study (p=0.18 Mann-Whitney U test)⁶.  

Pfcrt has additional polymorphisms at codons A220S, Q271E, N326S and R371I associated with in vivo chloroquine resistance.  A second study set out to determine whether pfcrt has acquired sequential mutations leading to greater in vitro chloroquine resistance.   The presence or absence of polymorphisms at codons K76T, A220S, Q271E, N326S and R371I were found to be almost completely linked.  Therefore, no correlation of chloroquine IC₅₀ and these additional alleles could be established⁷. 

The field site had expanded its study population size and incorporated an in vivo outcome arm to the chloroquine resistance study in 2001 and 2002. The correlation of polymorphisms in pfmdr1 and other genes and chloroquine resistance, particularly high level resistance will be evaluated.  Long term goals include identifying alternative mutations that correlate with chloroquine resistance and exploring the genotypic resistance to second line agents such as sulfa.

Related publications:

Basco, L.K. & Ringwald, P.  (2001).  Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon.  Journal. of Infectious Disease 183, 28-31.Gaye, O., Soumare, M., Sambou, B., Faye, O., Dieng, Y., Diouf, M., Bah, I.B., Dieng, T., N'dir, O. & Diallo, S.  (1999).  Heterogeneity of chloroquine resistant malaria in Senegal.  Bulletin de la Societe de Pathologie Exotique 92, 149-52.

Chen, N., Russell, B., Staley, J., Kotecka, B., Nasveld, P. & Cheng, Q.  (2001).  Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum.  Journal of Infectious Disease 183, 1543-5.

Daily J , Roberts, C,. Thomas, S., O. Ndir, O.,  Dieng T., Mboup S Wirth, D. (2003)  Prevalence of Plasmodium falciparum pfcrt Polymorphisms and in vitro Chloroquine Sensitivity in Senegal.  Parasitology in press.

Djimde, A., Doumbo, O.K., Cortese, J.F., Kayentao, K., Doumbo, S., Diourte, Y., Dicko, A., Su, X.Z., Nomura, T., Fidock, D.A., Wellems, T.E., Plowe, C.V. & Coulibaly, D.  (2001a).  A molecular marker for chloroquine-resistant falciparum malaria.  New England Journal of Medicine 344, 257-63.

Maguire, J., Susanti, A.I., Krisin, Sismadi, P., Fryauff, D.J. & Baird, J.K.  (2001).  The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia.  Annals of Tropical Medicine and Parasitology 95, 559-72.

Thomas, S. Ndir ,O., Dieng, T., Mboup, S., Wypij, D., Maguire, J. & Wirth, D.  (2002).  In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal.  American Journal of Tropical. Medicine and Hygiene 66, 474-80.