Mechanism of a Male Killing Toxin in a Drosophila endosymbiont - PROJECT SUMMARY/ABSTRACT The co-evolution of bacterial protein toxins and their targets has led to the discovery of many novel protein modifications and the development of applications in molecular biology. Arthropods, the most diverse phylum on Earth, are hosts to many bacterial symbionts that secrete diverse toxins of unknown function during infection. The maternally transmitted, intracellular parasite Spiroplasma poulsonii encodes one such toxin (Spiroplasma androcidin or Spaid) that causes male killing in its natural host Drosophila melanogaster. This proposed project aims to understand how the bacterial toxin Spaid recognizes the host cellular environment to specifically kill males. To that end, we use the Spiroplasma-Drosophila model system and a combination of genetics and molecular biology to identify specific host proteins targeted by Spaid and how the toxin binds these proteins. Previous work has suggested that Spiroplasma, via Spaid, detects the presence of the male specific lethal complex in flies. This complex is responsible for dosage compensation in male flies, which lack a second copy of the X. It is formed during early development in males and is part of the Drosophila sex determination pathway. Given this result, we hypothesized that natural resistance to Spaid would exist in Drosophila populations, that the target of Spaid is part of the dosage compensation complex (dcc), and that Spaid directly binds to dcc components, altering their activity. Our strong preliminary data suggest that we are correct, as we find natural variants of Drosophila melanogaster (within the DGRP), which produce males in the presence of Spaid and/or Spiroplasma. We also find that tissue specific knockdown of dcc components partially suppresses Spaid toxicity in male tissue. Towards this hypothesis, and guided by strong preliminary data, we propose to pursue two Specific Aims to identify host proteins that interact directly with the Spaid toxin and characterize how variation within host proteins and Spaid affect male killing at the molecular and cellular level. We will (1) Identify Spaid targets using a yeast 2-hybrid screen and co-immunoprecipitation, (2) identify suppressors of Spaid toxicity using natural variation in Drosophila and a genetic screen. Results from these experiments will illustrate how a bacterial toxin manipulates the host cellular environment.
