This proposal focuses on synthesis of inducible antimicrobial functions by mosquito cells in culture. Preliminary data show that after treatment with heat-killed bacteria, Aedes albopictus and Aedes aegypti mosquito cells produce a secreted activity that resembles cecropins from other insects. The activity corresponds to a small, basic molecule that is heat stable, lacks methionine and cysteine, and lyses indicators E. coli after electrophoresis at low pH. Treatment of Aedes albopictus cells with heat-killed bacteria is also accompanied by increased synthesis of 4 larger proteins, ranging in size from 32 to 111 kDa, whose biological properties, other than coinducibility with the cecropin-like immunity activity, are unknown. The goal of this project is to characterize these proteins and their corresponding cDNAs. These efforts will advance our understanding of mosquito immunity functions, in anticipation of eventual manipulation of these functions to disrupt disease transmission by vector insects. Comparative studies with an Aedes albopictus and an Aedes aegypti cell line, each of which secretes cecropin-like activity, will be used to prioritize cloning efforts. We will focus on induced proteins l) common to both lines and/or 2) which interact to form multisubunit complexes. Cecropin activities will be standardized, and cecropin genes will be identified using an homologous nucleic acid probe, or, alternatively, by the classical derivation of oligonucleotide probes based on the amino acid sequence of the purified protein. Polyclonal antibodies to the larger proteins will be produced, and used to screen a cDNA expression library enriched for sequences corresponding to bacteria-induced transcripts. Antibody and cDNA probes resulting from cloning efforts will be used to identify tissues that synthesize the corresponding activities in the mosquito, using standard histochemical techniques. Finally, these probes will be used to examine the kinetics of synthesis of these proteins in cell culture. Evidence for coordinate expression will target candidates that may share common regulatory elements. This project will extend the genetic and biochemical advantages of mosquito cell culture to an important physiological system that interfaces with disease transmission cycles. Once the relevant genes are available, their manipulation in transfected cells will provide insights into gene function that cannot be obtained at the level of the organism, given the present status of transformation procedures for mosquitoes. We anticipate that what we can learn here with cultured cells will facilitate long-term efforts to control mosquito-borne disease through genetic manipulation of vectors.
