A role for VPS35 in Brucella intracellular replication revealed by genome-wide siRNA screening

Brucella, the causing agent of brucellosis, is a major zoonotic pathogen with worldwide distribution. Brucella resides and replicates inside infected host cells in membrane-bound compartments called BCVs (Brucella-containing vacuoles). Following uptake, Brucella resides in eBCVs (endosomal BCVs) that gradually mature from early to late endosomal features. Through a poorly understood process that is key to the intracellular lifestyle of Brucella, the eBCV escapes fusion with lysosomes by transitioning to the rBCV (replicative BCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the notion that this complex intracellular lifestyle must depend on a multitude of host factors, a holistic view on which of these components control Brucella cell entry, trafficking and replication is still missing. Here we used a systematic cell-based siRNA knockdown screen in HeLa cells infected with Brucella abortus and identified 426 components of the human infectome for Brucella infection. These include multiple components of pathways involved in central processes such as cell cycle, actin cytoskeleton dynamics or vesicular trafficking. Using assays for pathogen entry, knockdown complementation and co-localization at single-cell resolution, we identified the requirement of VPS35 for Brucella to escape the lysosomal degradative pathway and to establish its intracellular replicative niche. We thus validated a component of the retromer as novel host factor critical for Brucella intracellular trafficking. Further, our genome-wide data shed light on the interplay between central host processes and the biogenesis of the Brucella replicative niche.