Malaria parasite genome scan: insights into antimalarial resistance

Parasitol Res DOI 10.1007/s00436-010-1917-8

SHORT COMMUNICATION

Malaria parasite genome scan: insights into antimalarial resistance Bhavna Gupta & Gauri Awasthi & Aparup Das

Received: 14 April 2010 / Accepted: 7 May 2010 # Springer-Verlag 2010

Abstract Genome scan and genotype-phenotype association study offer excellent opportunities to unearth drug/ vaccine targets in human pathogens including malaria parasites. A recently conducted such study in worldwide isolates in the most devastating malaria parasite Plasmodium falciparum has reported important genomic information on genetic basis of antimalarial resistance. Several unknown genes were also found to be under strong influence of natural selection. The findings provide important insights into the malaria parasite genome evolution in general and to use this information to develop more focused malaria control strategies, in particular.

Evolutionary genomics: a new era in malaria research Recent rapid developments in whole genome sequencing technologies and parallel bioinformatic studies have revolutionized the field of evolutionary genomics. As a consequence, this subject now constitutes an important framework, not only to infer evolutionary history of functionally important genes but also to characterize hitherto unknown genes, possibly serving important functions in the genome. Moreover, availability of whole genome sequence information for several organisms of interest necessitates utilization of genome scan B. Gupta : G. Awasthi : A. Das (*) Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and Bioinformatics, National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi 110 077, India e-mail: [email protected]

approach and associated evolutionary analysis to identify important associations between phenotypes and genotypes in different animal taxa (Schrooten et al. 2004; Wang et al. 2006). This approach also helps in identifying genes targeted by Darwinian selection in the genome, opening the baselines to study unidentified genes that are serving important functions. For example, in human genome, more than 700 genetic variants that might be targets of recent natural selection during the past 10,000 years of human evolution could be detected by such an approach (Voight et al. 2006). However, in human pathogens, this type of study is very limited. Considering malaria as a highly fatal infectious disease in humans, and origin and rapid spread of drug-resistant malaria parasites is a major huddle to control malaria, application of genome scan followed by evolutionary analyses could help in retrieving new information on genetic mechanism of drug resistance and identification of new drug and vaccine targets (Fig. 1). Already available whole genome sequence information on the two most widely spread pathogens (Plasmodium falciparum and Plasmodium vivax; Carlton et al. 2008a; Gardner et al. 2002) and associated comparative genomic analyses (Carlton et al. 2008b; Das et al. 2009; Sharma et al. 2010) have divulged important information on genomic characteristics of these two species. Furthermore, genome-wide studies in P. falciparum to uncover genome diversity (Jeffares et al. 2006; Volkman et al. 2006), to identify vaccine targets (Mu et al. 2006), and to determine recombination hotspots and population structure (Mu et al. 2005) have already been conducted. The results have not only provided novel genomic information on malaria parasite genomics but opened up scopes for future research in two directions.

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Fig. 1 Schematic overviews of Plasmodium genome scan leading to drug and vaccine candidate discovery

Signatures of natural selection in P. falciparum genes A recent genome scan and associated evolutionary studies in the most devastating malaria parasite P. falciparum (Mu et al. 2010) has shown that how genome-wide identification of single nucleotide polymorphisms (SNPs) as genetic markers analyzed in different population genetic models could be of immense importance not only to obtain basic understanding on genome organization, but also to find ways for novel genetic information on important parasitic activities, e.g., antimalarial resistance. Mu and co-workers (Mu et al. 2010) have genotyped 3,354 SNPs spread across the genome of P. falciparum in 189 cultureadapted worldwide isolates and utilized these SNP markers to understand evolutionary dynamics of different genetic regions of this species. In order to correlate genetic diversity with geographic locations of different P. falciparum isolates, the authors used a Bayesian-based genetic partitioning statistic and found a perfect correlation between the distributions of genetic diversity with the geographical origin of P. falciparum isolates. However, there was an exception; the Cambodian isolates were falling to an entirely separate category. This feature was also supported by other statistical analyses. Additional investigations revealed that almost 75% of the SNPs in the Cambodian isolates were fixed which contribute to the formation of an entirely separate group. Furthermore, utilizing the SNP data of one SNP per 7 Kb of P. falciparum genome, the authors have detected low recombination events in the larger chromosome, corroborating similar findings in the yeast, Saccharomyces cerevisiae (Kaback et al. 1992; Kaback et al. 1989). Moreover, two cases of very high recombination events were detected in P. falciparum genome; one in the extreme end of the chromosome 1 (smallest chromosome in P. falciparum genome, Fig. 2) and the other in the

400-800 Kb 7th chromosomal region surrounding the Pfcrt (P. falciparum chloroquine-receptor transporter) gene (Fig. 2), known to be responsible for chloroquine (CQ) resistance. Interestingly, the central 100-Kb region (that included the Pfcrt gene) of this 400-800-Kb chromosomal fragment was found to have very low recombination activity. This is considered to be a very important finding, because the sole role of Pfcrt protein in fluxing out CQ from parasite cell is under cloud (Basco and Ringwald 1999; Durand et al. 1999; Su et al. 1997). Furthermore, the authors (Mu et al. 2010) have applied three different statistical tests to ascertain the extent of haplotype conservation in different genomic regions and found several genes bearing signature of natural selection. Among these, some important functional genes are; Pfcrt (CQ resistant), Pfama-1 (antigenic), ABC transporter (transporting iron into mitochondria), Pfsurfin (helps in binding and transportation of chemical compounds), and the putative drug and metabolite transporter (PF14_0260). However, only 11 genes located in different chromosomes (Fig. 2) have passed all the three statistical tests, thus to be considered under strong influence of Darwinian natural selection. Most of the genes identified are conserved in Plasmodium but have no known function, pending detail functional characterization. Thus, the genome scan approach could narrow down a handful of genes (of both known and unknown functions) and provide baseline for future research to understand P. falciparum genomics.

Antimalarial resistance and genomic association In order to correlate genome scan results with antimalarial resistance in P. falciparum, the authors first have deter-

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Fig. 2 Chromosomal location of 11 genes that are under natural selection as identified by three different statistical tests. a Schematic representation of all the 14 chromosomes of P. falciparum (scaled according to approximate sizes). The 11 genes have been positioned

on the respective chromosomes and represented by different colors. Position and size of the genes are in scale. b Enlarged view of the 7th chromosomal region that contains eight out of the total 11 genes targeted by natural selection (represented in different color boxes)

mined the IC50 values of seven antimalarials in 185 isolates out of the 189 used for genome scan. Uni-modal distributions in five antimalarials and bimodal distributions in two [CQ and sulphadoxine-pyrimethamine (SP)] indicate high level of antimalarial resistance developed for both CQ and SP in worldwide P. falciparum. Significantly, these two drugs are considered to be the first line antimalarials for malaria treatment (Mohapatra et al., 2005). Out of 143 total tested isolates from Thailand and Cambodia, only two and six isolates were found to be sensitive for CQ and SP, respectively (Mu et al. 2010). The lowest range of IC50 value was found for primaquine (PQ) followed by dihydroartemisinin (DHA) and amodiaquine (AMQ). Interestingly, all the isolates were found to be sensitive to PQ and DHA in in vitro assays. In general, Cambodian isolates were reported to be more resistant to all seven antimalarials. This is indicated by the fact that, in Cambodian isolates, statistically significant IC50 value of DHA was detected in comparison to other South East Asian and American isolates (Mu et al. 2010). This reflects that P. falciparum isolates from Cambodia have already started resisting the most reliable drug to treat malaria at present (Dondorp et al., 2009; Noedl et al. 2008). Moreover, high observed positive correlations between IC50 values of commonly used antimalarials mefloquine (MQ) and DHA, CQ and SP,

CQ and AMQ, CQ, and quinine suggested high incidences of combined antimalarial resistance which is really threatening for global malaria control program. Secondly, association studies between the drug response (IC50 values) and SNPs in different candidate genes have identified several associations including Pfcrt and Pfmdr-1 to CQ and quinine, respectively. Furthermore, a recent study indicated that in South America, amodiaquine and CQ, which were not in use for the last several years, have resulted in the complete fixation of a particular haplotype (SVMNT) of the Pfcrt gene (Sa et al. 2009). It is also reported that even in the absence of drug pressure, this particular haplotype provides equal fitness to the parasite (as was in presence of drug pressure) in comparison to another contemporary haplotype (CVIET) (Sa et al. 2009). Furthermore, in the absence of CQ, the CVIET haplotype-bearing P. falciparum are known to revert back to CQS type in the presence of CQ reversal verapamil, whereas the SVMNT bearing P. falciparum do not (Fidock et al. 2000). All these results corroborate earlier findings in such genotype-phenotype association in P. falciparum (Ferdig et al. 2004; Fidock et al. 2000) and complement with the finding on the role of natural selection in evolution of these two genes in several field-based molecular epidemiological studies (Wootton et al. 2002).

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How important are the results in the context of global malaria control? This malaria parasite genome scan with associated evolutionary genomics study in worldwide P. falciparum isolates have not only revealed several genomic and population genetic features of the most fatal human malaria pathogen around the world, but also presented interesting genomic features of drug resistance. Emergence and global spread of drug resistance in malaria is an important public health concern and evolutionary genomics has proved to be an important tool for understanding genetic basis of malaria drug resistance (Das and Dash 2007). Since genome scan is considered as an alternative and direct approach to identify genes targeted by natural selection (due to the adaptive advantage they provide to the organisms; Roberge et al. 2007), coupled with functional validation of the novel genes, could possibly provide new direction to malaria genomic research and help furthering our understanding in molecular epidemiology of P. falciparum malaria, which in turn contribute to device new genetic control measures. Such genome scan approaches and genotype-phenotype association studies should also be extended to understand highly genetically diverged antigenic gene families (e.g., var, stevor, rifin) in P. falciparum and to map population-specific alleles of these genes that could help in designing population specific vaccines—the ultimate hope to control malaria.

Conclusion and future prospects The most important findings out of the genome scan of worldwide P. falciparum isolates is the identification of as many as eight genes under strong Darwinian selection located in a tightly linked 100-Kb genetic region of chromosome 7 (Fig. 2). This 100-Kb region is important as very commonly studied Pfcrt gene (implicated in CQ resistance) is included in this region. For example, eight transporter genes are located in the 36-Kb genetic region surrounding the Pfcrt gene and one of these genes named cg2, placed downstream of the Pfcrt (Bajaj et al. 2008) has been shown to be phenotypically associated with CQ resistance (Su et al. 1997; Basco and Ringwald 1999; 2001; Durand et al. 1999). However, association of CQ resistance and cg2 genotype was not to an extent to completely justify exclusive role of cg2 gene in CQ resistance (Durand et al. 1999) Considering dubious role of Pfcrt and possible involvement of other nearby transporter genes, further evolutionary genetic work in this 100-Kb region could provide novel insights into genetic basis of P. falciparum drug resistance mechanism. This is not only important on an academic context, but also to effectively manage drug-resistant malaria.

Acknowledgments AD thanks the Indian Council of Medical Research (ICMR), New Delhi for intramural funding. BG and GA are Senior Research Fellow of ICMR. We thank all the members of the Evolutionary Genomics and Bioinformatics laboratory, National Institute of Malaria Research, New Delhi, and an anonymous reviewer for valuable suggestions.

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