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

Abstract

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

Mitochondria and Wolbachia titers are positively correlated during maternal transmission

“…Mitochondria generate energy that is used by other organelles in the cell; we hypothesized that Wolbachia would rely on energy generated from mitochondria, and changes in the transport of ATP from mitochondrion to the cytoplasm would change Wolbachia titers, as suggested by White et al. (). Because we did not detect changes in Wolbachia titer in response to the likely reduction in ATP transport to the cytoplasm, Wolbachia may require little energy from the mitochondria or manipulates the host in some way to ensure its own persistence.…”

“…Both of the genes knocked down in mitochondria are associated with mitochondrial metabolism. Mitochondria generate energy that is used by other organelles in the cell; we hypothesized that Wolbachia would rely on energy generated from mitochondria, and changes in the transport of ATP from mitochondrion to the cytoplasm would change Wolbachia titers, as suggested by White et al (2017).…”

“…We used loci previously identified in a genomewide RNAi screen of Drosophila cell lines infected with Wolbachia (White et al., ). White et al.…”

“…Mitochondria may also benefit from these modifications. As mentioned previously, in a genomewide RNAi screen of Drosophila cell lines infected with Wolbachia, knockdown of several other genes involved in mitochondrial function also resulted in changes to Wolbachia titers (White et al, 2017). In White et al, the responsiveness of Wolbachia to changes in the gene expression of mitochondria could suggest interdependence, although White et al did not measure mitochondria response.…”

Wolbachia enhances insect‐specific flavivirus infection in Aedes aegypti mosquitoes

“…This may be particularly relevant for viruses that require lipids for attachment and entry into host cells and for replication (Lu, Cassese, & Kielian, 1999; Mackenzie, Khromykh, & Parton, 2007). Most recently, there is some evidence primarily from Drosophila that Wolbachia may be modifying host cellular structures or organelles rendering them less hospitable to viral replication (Rainey et al., 2016; White et al., 2017). A range of studies also point to a correlation between Wolbachia densities and the strength of blocking (Frentiu et al., 2010; Lu, Bian, Pan, & Xi, 2012; Osborne, Iturbe‐Ormaetxe, Brownlie, O'Neill, & Johnson, 2012), a trend that would be expected with any of the above explanations for blocking.…”