Wednesday, April 2, 2025
4/2/2025
Defining the molecular interface of Wolbachia Ankyrin-host interaction - PROJECT SUMMARY/ABSTRACT Wolbachia pipientis is an obligate intracellular alpha-proteobacterium that infects 40-60% of insect species on the planet. Wolbachia infection inhibits RNA virus replication in insects, a phenomenon known as pathogen blocking. Therefore, Wolbachia infected mosquitos are being released in many parts of the world to control the spread of human diseases. Importantly, although the mechanism behind Wolbachia’s virus inhibition is not known, Wolbachia must manipulate host biology to infect new hosts, persist, and be transmitted to the next generation. Our long-term goals are to identify the molecular toolkit used by Wolbachia to establish infection and the host targets of these tools; to define the co-evolution of that molecular interface. To that end, we focus on the ankyrin repeat containing proteins found in Wolbachia proteomes. Our hypothesis is that via these effectors, the host cell is modified, allowing Wolbachia to invade and persist. Our preliminary data identified multiple ankyrin repeat containing proteins in Wolbachia (WARPs) and established their phenotypes in Drosophila melanogaster. Wolbachia strain wMel encodes 25 such proteins and we see striking phenotypes upon overexpression in the fruit fly. The phenotypes are dependent on the ankyrin repeat domain, establishing its importance in binding host targets. These results led to our central hypothesis that Wolbachia uses WARPs to modify the host cell environment and that these ankyrin repeat proteins have evolved across the clade, as Wolbachia has spread to new host backgrounds. Towards this hypothesis, we have begun several large-scale screens to identify host targets for the WARPs and suppressors of toxicity for these secreted effectors. We have already identified some direct interactions between WARPs and Drosophila proteins and look to further define the molecular interface between symbiont and host using evolution-guided chimeras and molecular biological assays such as bacterial-2-hybrid and reciprocal co-IPs. Guided by strong preliminary data, we propose to pursue three Specific Aims to identify and characterize WARP-target interaction, and their co-evolution, using the power of the Drosophila system. We will (1) determine WARP targets (2) identify the relevance of WARPs and their targets to Wolbachia and host biology, and (3) define the co-evolution of the host-symbiont molecular interface. Studies of Wolbachia - host interactions are still in their infancy despite the recognized contributions of endosymbiotic associations to insect reproduction and evolution, and the ability to alter vector competence. These proposed studies will significantly advance our understanding of how Wolbachia manipulates host biology to establish infection, a necessary prerequisite to its use in vector control.
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