Trypanosome cAMP signaling mediates parasite-vector interaction - PROJECT SUMMARY/ABSTRACT African trypanosomes (Trypanosoma brucei) and related trypanosomatid parasites are responsible for vector-borne diseases that cause great human suffering and economic burden in endemic countries. T. brucei is transmitted between humans and other mammalian hosts by the tsetse fly, which is not merely a vessel for moving parasites between hosts, but an integral part of the parasite's developmental life cycle necessary for sustained transmission. In the absence of a vaccine to prevent infection in the mammalian host, targeting parasite development within the insect vector is considered an option for reducing disease transmission, though little is known of parasite interactions in the vector necessary for transmission. To survive, develop, and be transmitted, T. brucei must sense and respond to changing environmental signals as it moves through tissues within the insect vector. Little is known about parasite signaling pathways and vector-derived factors that control parasite migration - this is a critical knowledge gap and potential target of new transmission-blocking agents. Trypanosome cAMP signaling, originally shown to be critical for parasite chemotaxis in vitro, has recently been connected to progression of parasites through the tsetse, in particular, migration from the midgut (MG) to the proventriculus (PV). Initiation of cAMP signaling is controlled by an expanded protein family of adenylate cyclases (ACs) that differ in their extracellular putative ligand-binding domains and exhibit tissue-specific expression profiles during parasite migration through the tsetse. Trypanosome cAMP signaling is therefore an attractive target for transmission-blocking agents, however, very little is known about regulation of AC activity and downstream targets of cAMP signaling. This proposal brings together a multidisciplinary team of investigators with collective expertise in trypanosome biology, cAMP signaling, transcriptomics, and genetic manipulation of parasites, as well as tsetse biology, fly infections, and interactions between parasite, vector, and microbiome. Our specific aims are to (1) identify endogenous, tsetse fly-derived modulators of T. brucei chemotaxis; (2) define parasite cAMP effector genes responsible for parasite migration from the tsetse midgut to proventriculus; and (3) define parasite receptors that perceive chemotactic signals in the fly. We will leverage our established chemotaxis assay to test tsetse-derived factors for impacts on parasite chemotaxis (Aim 1). To define genes required for MG ➔ PV migration in the tsetse, we will employ a MG ➔ PV defective trypanosome mutant to identify MG-induced, cAMP-dependent transcriptome changes associated with movement out of the MG (Aim 2). Finally, we will implement systems for genetic manipulation of fly-transmissible T. brucei to allow functional assessment of trypanosome receptors and additional cAMP signaling genes during tsetse infection (Aim 3). Completing these aims will increase understanding of parasite-vector interactions, provide new avenues for targeting the pathogen within the arthropod vector, and identify promising targets to consider for transmission-blocking vaccines or small molecule inhibition.