Reliance ofWolbachiaon High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen ofDrosophilaCells.
Wolbachiaare gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use aWolbachia-infectedDrosophilacell line and genome-wide RNA interference (RNAi) screening to identify host factors that influenceWolbachiatiter. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reducedWolbachiatiter and 41 that increasedWolbachiatiter. Host gene knockdowns that reducedWolbachiatiter 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 reducedWolbachiatiter. To test thein vivorelevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels ofWolbachiain theDrosophilaoocyte. The presence ofWolbachiain either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance ofWolbachiatiter relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated thatWolbachiais intimately associated with the host ER and dramatically alters the morphology of this organelle. GivenWolbachialack essential amino acid biosynthetic pathways, the reliance ofWolbachiaon high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioningWolbachiawith amino acids. In addition, the reliance ofWolbachiaon the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism forWolbachia'spotent ability to prevent RNA virus replication.
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064.
Pamela M
PM
White
Department of Biological Sciences, Florida International University, Miami, Florida.|Biomolecular Sciences Institute, Florida International University, Miami, Florida 33199.
Laura R
LR
Serbus
Institut Jacques Monod, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7592, University Paris Diderot, 75 205 Paris, France.
Alain
A
Debec
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064.
Adan
A
Codina
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064.
Walter
W
Bray
Institut Jacques Monod, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7592, University Paris Diderot, 75 205 Paris, France.
Antoine
A
Guichet
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064.
R Scott
RS
Lokey
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064 wtsulliv@ucsc.edu.
William
W
Sullivan
D000818:Animals; D002460:Cell Line; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation; D049750:Genome, Insect; D054884:Host-Pathogen Interactions / Q000235:genetics*; D050356:Lipid Metabolism; D008928:Mitochondria / Q000378:metabolism; D059748:Proteolysis; D034622:RNA Interference; D020577:Wolbachia / Q000472:pathogenicity*
D029721:Drosophila Proteins
Drosophila; RNAi; Wolbachia; oocyte; ubiquitin; virus