Genetics and physiology of the tsetse fly bacterial endosymbiont Sodalis glossinidius - PROJECT SUMMARY Tsetse flies (Glossina spp.; Diptera: Glossinidae) are the vectors of Trypanosoma spp., parasitic protozoa and causative agents of human sleeping sickness and Animal African trypanosomiasis. Natural populations of tsetse flies can be infected with strains of the endosymbiotic bacterium Sodalis glossinidius (Sodalis). In tsetse flies, Sodalis undergoes both maternal and paternal transmission by invading tsetse cells and colonizing a wide range of tissues, including those inhabited by trypanosomes. Notably, Sodalis reaches high cell densities, replicating intra- and extracellularly in tsetse tissues, while neither triggering a tsetse immune response nor impairing the fitness of the fly. These observations indicate that Sodalis has the ability to invade, survive, replicate and egress from host cells. Whereas Sodalis invasion of insect cells depends on structural components of a protein secretion apparatus, to date, no secreted proteins have been identified. Moreover, the identity of genes and pathways required for intracellular survival, replication and host cell escape remains unknown. This poor understanding of Sodalis physiology has resulted from the lack of robust techniques to genetically modify this bacterium, hence hindering the development of Sodalis-based paratransgenesis of tsetse flies. We have recently developed two methods for the efficient delivery of exogenous DNA into Sodalis cells. Using one of these methods, we have now (1) implemented a powerful technique that combines CRISPR-Cas9 and bacteriophage recombineering to introduce precise modifications into the Sodalis genome, (2) expanded the Sodalis genetic code to enable incorporation of a noncanonical amino acid for protein tagging, and (3) introduced an efficient transposon mutagenesis procedure suitable for high-throughput insertion sequencing. Using these tools, this proposal seeks to establish foundational knowledge regarding molecular interactions between Sodalis and insect cells. Specifically, we seek to (1) define the repertoire of Sodalis proteins secreted inside insect cells, identifying which of these proteins promotes bacterium internalization, and (2) define the set of bacterial genes and pathways that are required for intracellular survival and replication, for escape from within insect cells, and for maintaining a persistent infection in the tsetse fly host.
