Genetic Diversity
Understanding the genetic diversity of falciparum malaria has important implications for the development and implementation of therapeutic interventions. It is this genetic variability that underlies the transmission success of the parasite and thwarts efforts to control disease. Although genetic variation in antigenic, drug resistance and pathogenesis determinants is known to be abundant, several studies have found a paradoxical lack of genetic diversity among silent (synonymous) sites in coding sequences (Rich et al) and among intron sequences (Volkman et al). Other groups are finding different estimates of genetic variation (Mu et al.), but to date we do not have a comprehensive survey of genetic variation across all 14 chromosomes or an assessment of genetic variation between coding and noncoding regions of the genome. In collaboration with Drs. Karen Day, Daniel L. Hartl, and Elizabeth Winzeler, our laboratory is taking a two-pronged approach. First, we are using high-density oligonucleotide arrays developed by Elizabeth Winzeler to identify single nucleotide polymorphisms (SNPs) across the genome. To support this effort, various regions of the genome are being assessed for genetic diversity in concert with the Day and Hartl groups. Second, we are targeting specific genes or gene families including the ATP Binding Cassette (ABC) genes, the AcylCoA Synthetases, and genes involved in the mutator phenotype (MutS) to test the hypothesis that genes under selective pressures may have a different level of polymorphisms than neutral genes in the genome. Currently we are engaged in the active investigation of genetic variation in both P. falciparum and P. vivax using a variety of approaches to further our understanding of the population structure of the parasite to optimize drug and vaccine design and implementation.
ABC Transporters
Author: Sarah K. Volkman
The genetic variability of potential targets of intervention strategies is an important determinant of its effectiveness. Understanding the level of variation at potentially important drug target sites is therefore important for designing effective drugs and vaccines. One prediction is that genes that are under selection may have an enrichment of SNPs relative to genes that are not under selection. To test this prediction, we have focused on several genes or gene families that are predicted to be under selective pressure in the parasite. One of these gene families is the ATP Binding Cassette or ABC gene family, which are known to be involved in the mediation of drug resistance in a variety of evolutionarily diverse organisms including P. falciparum. Initial analysis identifies over 15 members of this gene family in P. falciparum, some that contain single nucleotide polymorphisms (SNPs). Current efforts include use of this SNP analysis to determine if some of these P. falciparum ABC transporters are under selective pressures, which would argue for their biological importance in the parasite. As a consequence, these important gene products may provide new targets for the development of intervention strategies to help combat malaria.
AcylCoA Synthetases
Author: Lara Bethke
We began investigating acyl-CoA synthetase, an enzyme involved in fatty acid metabolism, because it had a high level of single-nucleotide polymorphisms in an oligonucleotide array analysis of the chromosome 2 genes of 5 Plasmodium falciparum isolates (Volkman et al., Science, 2002). We found 10 putative acyl-CoA synthetase paralogs, 8 of which are located in subtelomeric chromosomal regions, in the published P. falciparum genome sequence. One paralog, located on chromosome 12, was a hybrid form of the paralogs on chromosomes 13 and 14. We investigated the predicted chromosome 12 gene by PCR and Southern blot analysis, including direct sequence of the predicted crossover point, and confirmed the hybrid structure. This result implies that the gene on chromosome 12 was created by a recombination event that involved the subtelomeric regions of both chromosome 13 and 14. RT-PCR analysis demonstrates that all 10 of the paralogs are expressed in erythrocytic stages. Phylogenetic analysis suggests the subtelomeric paralogs are more closely related to each other than to the paralogs located at internal chromosomal sites. We analyzed 4 additional P. falciparum isolates by Southern blot using probes specific for the chromosome 13 and 14 genes, and found evidence for alternate banding patterns in 3 of the 4 isolates. These results are consistent with alternate recombination or deletion events that may be a consequence of the subtelomeric locations. The presence of a new acyl-CoA synthetase gene created by a double gene conversion event and the evidence for alternate recombination events in the subtelomeric regions further imply that mechanisms of recombination/gene conversion may play a major role in the generation of sequence diversity in Plasmodium falciparum.
ABC Transporters
Author: Sarah K. Volkman
The genetic variability of potential targets of intervention strategies is an important determinant of its effectiveness. Understanding the level of variation at potentially important drug target sites is therefore important for designing effective drugs and vaccines. One prediction is that genes that are under selection may have an enrichment of SNPs relative to genes that are not under selection. To test this prediction, we have focused on several genes or gene families that are predicted to be under selective pressure in the parasite. One of these gene families is the ATP Binding Cassette or ABC gene family, which are known to be involved in the mediation of drug resistance in a variety of evolutionarily diverse organisms including P. falciparum. Initial analysis identifies over 15 members of this gene family in P. falciparum, some that contain single nucleotide polymorphisms (SNPs). Current efforts include use of this SNP analysis to determine if some of these P. falciparum ABC transporters are under selective pressures, which would argue for their biological importance in the parasite. As a consequence, these important gene products may provide new targets for the development of intervention strategies to help combat malaria.
MutS
Author: Lara 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.
Oligonucleotide Arrays
Author: Sarah K. Volkman
Oligonucleotide arrays have been shown to identify mutations in different organisms. To see if such an approach could be used to identify single nucleotide polymorphisms (SNPs) in P. falciparum, we worked in collaboration with Dr. Elizabeth Winzeler, who developed oligoarrays for falciparum malaria. As a pilot study, we used an array that contained oligonucleotides corresponding to the 210 genes across chromosome 2. From this pilot study we were able to identify SNPs within genes in a population of geographically diverse parasites. This analysis revealed that there was an enrichment of SNPs within genes predicted to be associated with the surface of the parasite environment and thus recognized by the host immune system. We also found a nonrandom distribution of SNPs along the chromosome, with an increase in mutations within genes localized to the subtelomeric regions of the chromosome. We are currently using this technology to address the distribution of SNPs among genes throughout the whole falciparum genome sequence.
Plasmodium vivax -Apical Membrane Antigen-1
Author: Anusha M. Gunasekera, Ph.D.
Parasite diversity has important implications for pathogenesis, epidemiology and control of malaria. We have carried out a pilot study in order to determine the population structure of the human malarial parasite P.vivax in Sri Lanka by examining the level of genetic diversity at the AMA-1 (Apical Membrane Antigen-1) locus among clinical isolates from this South Asian region.
Plasmodium vivax apical membrane antigen 1 (PvAMA-1) is an important malaria vaccine candidate. We have performed the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample. We found high allelic diversity within a Sri Lankan population of parasites and also evidence that this diversity is maintained by balancing selection at the PvAMA-1 locus, presumably to evade immune recognition by the host. We tested for selection in PvAMA-1 by traditional analyses, as well a novel approach for estimating population genetic parameters that is resilient to intra-locus recombination. In contrast to what has been observed at the AMA-1 locus of Plasmodium falciparum, the signature of diversifying selection is seen most strongly in Domain II of PvAMA-1, indicating that the different domains in each species may be subject to varying selective pressures and functional constraints.
We also find that recombination plays an important role in generating haplotype diversity, even in a region of low endemicity such as Sri Lanka. Our results may indicate that new haplotypes generated by recombination in areas of higher endemicity sweep into the Sri Lankan population, or that rare recombinant haplotypes generated within the Sri Lankan population are highly selectively advantageous. Alternatively, these observations may be related to the unique biological features of P.vivax infection, such as earlier gametogenesis and relapse, which increase the chance of mixed strain infections and sexual outcrossing, even in areas of low endemicity. Indeed approximately 13% of infections in the Sri Lanka population were multi-clonal in nature
Mapping of diversity and recombination hotspots onto a three-dimensional structural model of the protein indicates that one surface of the molecule may be particularly likely to bear epitopes for antibody recognition. Regions of this surface that show constrained variability may prove to be promising vaccine targets. Thus our findings may have important implications for immunological intervention strategies against malaria in endemic regions.
SNP Analysis
Authors: Sarah K. Volkman, Dyann F. Wirth
In P. falciparum, the issue of genetic diversity is controversial-genetic variation in proteins for antigenic determinants, drug resistance and pathogenesis is abundant, whereas DNA variation in silent (synonymous) sites in coding sequences appears absent. Nevertheless, sequence variation among small tandemly repeated sequences, called microsatellites (MS), within and among subpopulations is widespread. We began to address this apparent discrepancy by looking at the level of mutations within intron sequences from genes on chromosomes 2 and 3, the first two chromosomes to be fully sequenced and annotated (Volkman et al). From this analysis we found few single nucleotide polymorphisms (SNPs), but many MS polymorphisms. In fact, there was a significant enrichment in the number of SNPs within these MS regions, suggesting that the mechanisms that contribute to making MS sequences polymorphic also introduce SNP mutations. When we discounted SNPs within MS because these mutations were not being maintained in a neutral manner, we came up with estimates for the most recent common ancestor (MRCA) in the range of 6,300 to 23,000 years ago. This estimate suggests that sometime around the time when agriculture expanded, a single or few parasites spread throughout the human population and that the progeny of these ancestral parasites are found in humans today.
This estimate of the age of MRCA has important implications for the development of clinical interventions. Such an intervention (drug or vaccine) would target certain proteins in the parasite. If these proteins were variable, the parasite could easily outwit the effects of the drug or vaccine. This would require different strategies than if the protein targets did not change very much. Understanding the level of variation at potentially important target sites is therefore important for designing effective drugs and vaccines.
Currently our efforts have extended to different regions of the genome, both coding and noncoding on a variety of chromosomes. While our initial estimates, which focused on introns from mainly chromosome 2 and some from chromosome 3, showed little genetic variation, other studies (Mu et al) have arrived at different estimates for the age to MRCA. Work in progress includes the use oligonucleotide arrays or "chips" to assess the level of genetic diversity genome-wide, and surveys of different regions of the genome including 5' and 3' flanking regions, introns and exons for the levels and patterns of genetic diversity.