Mosquito-born pathogens continue to have a major impact on the health of human populations throughout the world, and malaria and dengue fever are considered two of our most important reemerging diseases. All pathogens transmitted by mosquitos are ingested with the blood meal and consequently they must survive within the mig-gut environment and be able to penetrate the peritrophic matrix and mid-gut epithelium to reach their site of development. Despite the importance of the midgut as a determinant for vector competence, little specific information is available regarding the genetic regulation of critical physiological processes that occur within the midgut following blood feeding. One of these processes is the formation of the peritrophic matrix (PM) that surrounds the blood bolus nd physically separates it from the midgut epithelium. We have cloned a full- length cDNA for Aedes aegypti glutamine synthetase (GS) and determined that it is mid-gut specific and induced by blood feeding. Our hypothesis is that GS is critical for chitin synthesis in PM formation y providing the glutamine necessary for the glutamine necessary for the glutamine: fructose-6-phosphate aminotransferase (GFAT) catalyzed production of glucosamine-6-phosphate. Enzyme assay analyses also indicate that GFAT and chitin synthase (CS) play regulatory and critical roles in this biochemical process. Because very little is known concerning the biosynthetic pathway involved in PM chitin formation, or on the genetic regulation of this pathway, we propose herein biochemical and molecular studies of GS, GFAT, and CS that should provide a cleared understanding of this process. Specifically, we will (a) obtain DNA clones for GS, GFAT, and CS, and nucleotide and putative translation production sequences will be compared with those from other organisms, (2) use cDNA clones of these three enzymes to assess timing and location of transcriptional activity, using northern analysis and in situ hybridization, and to obtain recombinant proteins for antibody production and subsequent immunolocalization and enzyme inhibition studies, and (3) map and sequence the genomic structures for GS, GFAT and CS in order to identify and characterize those mechanisms regulating gene expression. We will use a number of techniques including extensive studies of promoter sequences, transgene mosquitos, electrophoresis mobility shift assay, DNase I footprinting, and physiological studies to evaluate potential promoter and enhancer sequences. None of these genes has been isolated from mosquitos and we anticipate satisfying our specific aims will provide us with a much clearer understanding of chitin synthesis associated with PM formation. A more complete understanding of biochemical events operating in the mid-gut following blood feeding also as the potential of providing new approaches for disrupting pathogen transmission. Likewise, the goal of using transformation strategies to engineer pathogen-resistant mosquitos will require the availability and thorough understanding of promoters that are induced at the right time and at the right place.
