Reliance of <i>Wolbachia</i> on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of <i>Drosophila</i> Cells

Abstract

<i>Wolbachia</i> are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a <i>Wolbachia</i>-infected <i>Drosophila</i> cell line and genome-wide RNA interference (RNAi) screening to identify host factors that influence <i>Wolbachia</i> titer. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reduced <i>Wolbachia</i> titer and 41 that increased <i>Wolbachia</i> titer. Host gene knockdowns that reduced <i>Wolbachia</i> titer spanned a broad array of biological pathways including genes that influenced mitochondrial function and lipid metabolism. In addition, knockdown of seven genes in the host ubiquitin and proteolysis pathways significantly reduced <i>Wolbachia</i> titer. To test the <i>in vivo</i> relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of <i>Wolbachia</i> in the <i>Drosophila</i> oocyte. The presence of <i>Wolbachia</i> in either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance of <i>Wolbachia</i> titer relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated that <i>Wolbachia</i> is intimately associated with the host ER and dramatically alters the morphology of this organelle. Given <i>Wolbachia</i> lack essential amino acid biosynthetic pathways, the reliance of <i>Wolbachia</i> on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning <i>Wolbachia</i> with amino acids. In addition, the reliance of <i>Wolbachia</i> on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for <i>Wolbachia</i>'<i>s</i> potent ability to prevent RNA virus replication.