Malaria, which causes between 2 and 3 million deaths each year, is the second most important pathogen-specific cause of mortality in the world today after tuberculosis. Most malaria deaths occur in infants or young children in African, where the mosquito Anopheles gambiae is the major vector. Because of the widespread emergence of drug-resistant strains of malaria parasites and insecticide resistant strains of the vector, new strategies for malaria control are urgently needed. This has led to a renewed interest in the study of the mosquito-parasite interaction as a possible source of knowledge that could contribute to new forms of control. Within the framework of this long term objective, I have selected a strain of A. gambiae that is able to encapsulate and kill most species of malaria parasites to which this mosquito is normally susceptible. Preliminary microsatellite mapping of susceptibility to the monkey parasite Plasmodium cynomolgi B has linked one major and two minor genes to this phenotype. The broad objective of this project are to clone the genes responsible for this encapsulation phenotype and to determine the manner in which those gene products confer refractoriness to malaria parasites. The work will focus initially on the major encapsulation gene. To accomplish this objective, I will address 4 specific aims: (1) the genes that enable the refractory A. gambiae strain to encapsulate and kill the parasite P. cynomolgi B will be genetically mapped using microsatellite markers to within regions of genomic DNA that are no more than a few hundred kilobases of DNA or loss (my target is less than 300 kb); (2) a set of overlapping contiguous clones (contigs) which span the genetic interval defining the major gene will be identified; (3) the genes within these intervals will be identified by a combination of genomic sequencing and by screening cDNA libraries produced from mRNA isolated from O. cynomolgi B-infected, adult female refractory and susceptible strain mosquitos at the time when encapsulation occurs; (4) expression patterns (level of mRNA and size of the products) and sequences of the genes in these intervals will be determined by qualitative RT-PCR analysis of mRNA isolated from P.cynomolgi B-infected refractory and susceptible strain mosquitoes and comparison of sequences the cDNAs of the two strains. If a technique for germ line transformation of A. gambiae becomes available within the time frame of this project, this technique will be used to confirm the function of candidate genes.
