Functional genomics in insect immunology: Identification and characterization of Rhodnius prolixus immune genes

Rhodnius prolixus is a bloodfeeding hemipteran insect and a vector of Trypanosoma cruzi, the etiologic agent of Chagas disease. This disease affects over 18 million people in Latin America, and there are no vaccines or efficacious drugs. Because of the unique lifecycle of T. cruzi within the vector, and its avoidance of the hemocoel where the primary immune response occur, we investigated the role of the innate immune response of R. prolixus to pathogens and parasites, including T. cruzi. Insect immunity is based on three principles: pathogen recognition, induction of appropriate regulatory pathways; and the production of molecules to eliminate the pathogens. In this dissertation, I use functional genomics (Suppressive Subtractive Hybridization) to identify and describe genes involved in the immune response of R. prolixus and the implications for T. cruzi. Initially we identified expressed sequence tags (ESTs) corresponding to genes that are differentially expressed in response to parasites and bacteria, and assigned putative gene functions based on sequence similarities. This thesis focuses on four genes of interest including a transcription factor (Dorsal), which may function in the regulation of expression of immune peptides, and three antimicrobial peptides (Defensin, Lysozyme and Prolixin) that serve directly to eliminate pathogens. Transcriptional factor binding motifs (NF-kappaB) present in the promoters of two of these genes (lysozyme and defensin) suggest they are transcriptionally regulated by Dorsal, whereas prolixin is not. We evaluated the temporal and spatial expression profiles of the antimicrobial peptide genes, using real time quantitative PCR, to establish molecular relationships between parasite and vector. Subsequently, we expressed dorsal and prolixin in bacteria and tested their functions. We concluded that invasion of the hemocoel of R. prolixus activates components of the immune system and the production of compounds lethal to T. cruzi, but the pathogen survives by living exclusively in the intestine avoiding vector responses. This study contributes to our overall knowledge of insect immunity, the arsenal of immune molecules available to different insects, and identifies novel and highly conserved immune molecules found in higher and lower insects.;Keywords. Rhodnius prolixus, functional genomics, innate immunity, Lysozyme, Defensin, Dorsal, NF-kappaB.