Frank H. Collins - US grants

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.

The broad aim of this Project Proposal is to develop the tools that can form the basis of a genetic control strategy for malaria in sub-Saharan African transmitted by the vector Anopheles gambiae. This TDRU Program Project involves 4 specific projects. The specific aims of Project 1 are the development of two different techniques for germ lines transformation of An. Gambiae, a transposon-based method and a site- specific recombinase method for introducing larger fragments of DNA into the genome. The aim of project 2 is to develop transgene constructs that can block transmission blocking antibody and another based on the small, antiparasitic peptides like cecropins and megainins. The aim of project 3 is to develop and test antiparasite transgene constructs based on An. Gambiae immune peptides that will be delivered to the hemocoel. The goal of Project 4 is to test the hypothesis that a meiotic drive system can be used as the necessary tool for driving a genetically engineered parasite refractory genotype (such as those produced in Projects 2 and 3) into the wild population.

The long term objective of this project is to develop two different methods for integrating new DNA into the genome of An. Gambiae. The more immediate of these objectives is to develop a germ line transformation method based on one or more Class II transposons,, transposons that move dia a DNA intermediate through a cut-and-paste mechanism. We will initially explore plasmid-to-plasmid transpositional activity of 4 such transposons that have already been used to transform non-drosophilid insects: Hermes, Minos, Mos1 mariner, and piggyBac. These transposons that show transpositional mobility in An. Gambiae embryos (this has already been documented for Hermes) will be examined in more detail in such assays in an effort to optimize plasmid- to-plasmid transposition by varying helper-to-donor plasmid ratios. At the same time, we will be conducting a gamma-ray mutagenesis screen of An. Gambiae in order to produce strains with a mutations in one or both of two genes that encode enzymes important for ommochrome eye pigment synthesis: tryptophan oxygenase (vermillion in D. melanogaster) and kynurenine hydroxylase (cinnabar in D. melanogaster). The resulting mutant strain(s) will then be the target of transformation experiments with constructs based on the most promising of the above Class II transposons. We will use wild type tryptophan oxygenase or kynurenine hydroxylase receptor constructs that already exit and have been validated in other Diptera (Ae. Aegypti and D. melanogaster). Finally, when a functional An. Gambiae transformation method based on a Class II transposons is available, we will undertake to develop a Cre/lox target site-specific recombination system for "docking" large fragments of DNA (in excess of 20 kb) in to the An. Gambiae genome. In this latter aim, we will be guided by the results of Drs. Lucy and Peter Cherbas at Indiana University who are currently developing such a method for D. melanogaster and Anthony A. James at UC Irvine who is doing similar work with Ae. Aegypti.

Malaria, tuberculosis, and AIDS are three major pathogen-specific causes of human mortality in the world. More than 90% of the world's malaria is in Africa, where the mosquito Anopheles gambiae is the primary vector. Human and malaria parasite genome projects are now underway and expected to be complete within the next year or two. This proposal seeks to initiate an A.gambiae genome project, with the expectation that the availability of the genomes of the parasite, human host, and the vector will lead to the development of novel malaria control strategies. The immediate aims of this proposal are the following: (1)to sequence 10,000 ESTs from normalized cDNA libraries made from each of five different mosquito tissues (salivary gland, midgut, fatbody, head, and immune- responsive cell lines), (2) to physically map to the mosquito's polytene chromosomes 2,000 genomic DNA Bacterial Artificial Chromosomes (BACs), (3) to sequence both ends of 25,000 genomic DNA BACs, and (4) to assess local scale synteny between the mosquito and Drosophila melanogaster by sequencing 3 megabases of sequence selected to be homologous with the well studied D. melanogaster Adh region. These 4 aims will provide sequences for direct gene discovery, for gene expression analysis (by microarray techniques), and for the determination of broad (polytene chromosome level) and local (megabase sequence level) synteny between D .melanogaster and A. gambiae. They will also facilitate a subsequent full genome sequencing project, either by a Sequence Tagged Connector strategy or by a shotgun strategy. The project is structured to involve the participation of both US and European institutions so as to make this an international effort. The long range is to encourage a broad range of funding agencies to join in supporting an effort to sequence the entire genome of this mosquito.

The objective of the Bioinformatics Resource Centers for Biodefense and Emerging/Re-emerging Infectious Diseases is to establish and maintain a web-based curated, stable, relational database to collect, populate, store, view, display, annotate, query and analyze genomic and related data and bibliographic information, providing a single robust point of access and user friendly interface for the scientific community. The contractor will develop comprehensive databases for multiple organisms considered agents of bioterrorism and/or causing emerging and re-emerging diseases.

The Bioinformatics Resource Centers for Infectious Diseases provide facilities, equipment, qualified personnel, and all necessary resources and services to collect, archive, update, integrate, and maintain a variety of research data from infectious diseases pathogens, and to provide for the query, analysis and display of such information through user friendly interfaces and computational analyses tools made freely available to the scientific community.

The Bioinformatics Resource Centers for Infectious Diseases provide facilities, equipment, qualified personnel, and all necessary resources and services to collect, archive, update, integrate, and maintain a variety of research data from infectious diseases pathogens, and to provide for the query, analysis and display of such information through user friendly interfaces and computational analyses tools made freely available to the scientific community.

The Bioinformatics Resource Centers for Infectious Diseases provide facilities, equipment, qualified personnel, and all necessary resources and services to collect, archive, update, integrate, and maintain a variety of research data from infectious diseases pathogens, and to provide for the query, analysis and display of such information through user friendly interfaces and computational analyses tools made freely available to the scientific community.

The Bioinformatics Resource Centers for Infectious Diseases provide facilities, equipment, qualified personnel, and all necessary resources and services to collect, archive, update, integrate, and maintain a variety of research data from infectious diseases pathogens, and to provide for the query, analysis and display of such information through user friendly interfaces and computational analyses tools made freely available to the scientific community.