In Vivo vs. In Vitro
Authors: Johanna Daily, Ousmane Sarr, Dyann F. Wirth
The success of Plasmodium falciparum despite efforts aimed at reducing transmission through the development of novel drugs is due to its unique and complex biology. Its' complex life cycle and growth requirements have hindered the use of classic genetic approaches to study virulence and host pathogen interaction. Novel approaches using whole genomic exploration have been employed to investigate parasite biology. Recently, whole genomic approaches have characterized the changes in RNA and protein that correspond with the profound metabolic and structural changes that occur through its forty eight hour life cycle 1-4. Using these new approaches we set out to determine if we could define the expression profile of the parasite isolated directly from a human host. This analysis would provide more direct information about the biology of the parasite while it resides in this environment. In addition, expression differences between laboratory cultivated isolates and in vivo isolates may provide a subset of genes that are specifically required for parasite survival in the host environment and direct further analysis to genes critical in the host pathogen interaction.
The collaborative study of chloroquine resistance between Cheikh Anta Diop University and Harvard School of Public Health provided the infrastructure to evaluate parasite RNA harvested from mildly symptomatic patients with P. falciparum infection. A pilot study was conducted in 2002, with isolation of in vivo parasite RNA and characterization of transcript level of genes located on chromosome 2. The expression profile generated was compared to an expression profile generated from in vitro 3D7 ring stage parasites RNA. Preliminary analysis suggests this approach is valid and provides preliminary data for a larger study. This project is carried out through collaboration with Dr. Elizabeth Winzeler at Scripps Institute in La Jolla, California.
Related Publications
Ben Mamoun C, Gluzman IY, Hott C, MacMillan SK, Amarakone AS, Anderson DL, Carlton JM, Dame JB, Chakrabarti D, Martin RK, Brownstein BH, Goldberg DE (2001) Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis. Mol Microbiol.;39, 26-36.
Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ.(2002) A proteomic view of the Plasmodium falciparum life cycle. Nature. 3, 520-6.
Hayward RE, Derisi JL, Alfadhli S, Kaslow DC, Brown PO, Rathod PK.(2000) Shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria.
Mol Microbiol. 35,6-14.
Le Roch KG, Zhou Y, Batalov S, Winzeler EA.(2002) Monitoring the chromosome 2 intraerythrocytic transcriptome of Plasmodium falciparum using oligonucleotide arrays. Am J Trop Med Hyg. 67,33-43.
Pfmdr1/Pfcrt
Authors: Johanna Daily, Therese Dieng, Alissa Myrick, Ousmane Sarr, 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.
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.
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.
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.
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.