A role for the VPS retromer inBrucellaintracellular replication revealed by genome-wide siRNA screening

Abstract

26Brucella, the causing agent of brucellosis, is a major zoonotic pathogen with worldwide 27 distribution. Brucella resides and replicates inside infected host cells in membrane-bound 28 compartments called BCVs (Brucella-containing vacuoles). Following uptake, Brucella 29 resides in eBCVs (endosomal BCVs) that gradually mature from early to late endosomal 30 features. Through a poorly understood process that is key to the intracellular lifestyle of 31 Brucella, the eBCV escapes fusion with lysosomes by transitioning to the rBCV (replicative 32 BCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the 33 notion that this complex intracellular lifestyle must depend on a multitude of host factors, a 34 holistic view on which of these components control Brucella cell entry, trafficking and 35 replication is still missing. Here we used a systematic cell-based siRNA knockdown screen in 36HeLa cells infected with Brucella abortus and identified 425 components of the human 37 infectome for Brucella infection. These include multiple components of pathways involved in 38 central processes such as cell cycle, actin cytoskeleton dynamics or vesicular trafficking. Using 39 assays for pathogen entry, knockdown complementation and co-localization at single-cell 40 resolution, we identified the requirement of the VPS retromer for Brucella to escape the 41 lysosomal degradative pathway and to establish its intracellular replicative niche. We thus 42 validated a component of the VPS retromer as novel host factor critical for Brucella 43 intracellular trafficking. Further, our genome-wide data shed light on the interplay between 44 central host processes and the biogenesis of the Brucella replicative niche. 45 46 3 Importance 47With >300,000 new cases of human brucellosis annually, Brucella is regarded as one of the 48 most important zoonotic bacterial pathogen worldwide. The causing agent of brucellosis 49 resides inside host cells within vacuoles termed Brucella containing vacuoles (BCVs). 50Although few host components required to escape the degradative lysosomal pathway and to 51 establish the ER-derived replicative BCV (rBCV) have already been identified, the global 52 understanding of this highly coordinated process is still partial and many factors remain 53 unknown. To gain a deeper insight into these fundamental questions we performed a genome-54 wide RNA interference (RNAi) screen aiming at discovering novel host factors involved in the 55Brucella intracellular cycle. We identified 425 host proteins that contribute to Brucella cellular 56 entry, intracellular trafficking, and replication. Together, this study sheds light on previously 57 unknown host pathways required for the Brucella infection cycle and highlights the VPS 58 retromer components as critical factors for the establishment of the Brucella intracellular 59 replicative niche. 60 61 62Cellular invasion is a common strategy shared by many bacterial pathogens of human and 63 animals in order to escape host defenses and to establ...