Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Hochstrasser, Mark

Abstract

Wolbachia is a vertically transmitted intracellular bacterium that infects a large number of arthropods and filarial nematodes. Many strains of Wolbachia manipulate host reproduction through cytoplasmic incompatibility (CI). In its simplest form, CI is a phenomenon where male host infected with Wolbachia cannot produce viable offspring with uninfected females; this apparent sterility is restored when the male host mates with Wolbachia-infected females—a phenomenon called rescue. Since Wolbachia is transmitted through the female germline, CI helps Wolbachia propagate in the host population. These properties of CI have been utilized in Wolbachia-based methods to control mosquito-borne diseases such as Dengue and Zika and these methods have proven to be effective in field trials.In recent years, pairs of Wolbachia proteins expressed by two syntenic genes have been discovered as CI factors (Cifs) that is responsible for CI induction and rescue. They are able to recapitulate the CI-like defect and rescue phenomena when expressed in transgenic Drosophila, mosquito and yeast S. Cerevisiae. Different functional models have been proposed to explain how CI factors induce and rescue CI. However, the detailed molecular mechanism remains elusive. This thesis describes projects aimed at elucidating the mechanism of Wolbachia-induced CI. In the first two projects, we aim to identify the enzymatic target of CidB—a CI factor possessing deubiquitylase (DUB) activity. DUB is an enzyme that specifically cleaves ubiquitin, an important post-translational modifier protein, from its substrates. We first developed and characterized an unbiased tool based on OtUBD to purify ubiquitylated proteins from biological samples. Using OtUBD pulldowns and proteomics, we were able to screen for candidate DUB substrates of CidB in yeast. OtUBD also proves to be a versatile, efficient, and economical new tool for general ubiquitin-related research with specific advantages over other methods, such as in detecting monoubiquitylation or ubiquitin linkages to noncanonical sites. In another project, we investigated the role of the interactions between the cognate Cif pair—CinA and CinB—in CI rescue. Through collaboration, we solved the crystal structure of the CinA-CinB complex. With structurally guided mutagenesis, we demonstrated that the binding between CinA-CinB is crucial for rescue in yeast and transgenic Drosophila systems. These experiments provide evidences to differentiate between different CI models. In summary, the studies described here offer valuable insights towards understanding of the mechanism of CI as well as a novel method to study ubiquitin-related topics in general.

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