A yeast-based approach identifies Legionella pneumophila effectors that target membrane trafficking pathways

The remarkable ability of Legionella pneumophila to invade and replicate inside mammalian and protozoan cells is a cornerstone of the pathogenesis of the severe pneumonia known as Legionnaires' Disease. Legionella modulates numerous host trafficking processes to its own benefit and survival. These processes include uptake, phagosome maturation, recruitment of endoplasmic reticulum and egress. An intact Type IVB Secretion System (TFBSS) known as Icm/Dot is absolutely required for the intracellular survival of the bacteria and formation of a replicative vacuole. The Icm/Dot transporter translocates effector proteins into the host cell milieu and it is these effectors that are predicted to target host pathways and collectively contribute to intracellular fitness. Although numerous effectors have been identified, their role in the intracellular lifecycle is unclear. We developed a novel approach using the model organism Saccharomyces cerevisiae to detect effector genes. We hypothesized Icm/Dot effectors that target host trafficking pathways in host cells could also inhibit traffic in yeast. By screening a random Legionella genomic library for genes capable of causing vacuolar protein sorting (vps) defects in yeast, we identified 3 novel genes, vipA, vipD and vipF (V&barbelow;ps I&barbelow;nhibitor P&barbelow;rotein). These genes encode proteins that inhibit Vps pathways in yeast by different mechanisms. The Vip proteins are translocated into host cells via the Icm/Dot TFBSS during Legionella infection. VipA is predicted to contain a coiled-coil domain, VipD is predicted to contain a patatin/phospholipase domain and VipF is predicted to contain an acetyltransferase domain. All of them are dispensable for intracellular growth, a feature common to all Icm/Dot effectors. Co-precipitation studies with VipA identified beta-actin as a direct binding partner. Linker mutagenesis of vipA identified a 30 amino acid region important for causing a Vps- phenotype in yeast. Significantly less actin co-precipitated with a VipA linker mutant protein compared with wild-type VipA. Thus, the ability of VipA to inhibit traffic and bind actin are linked. The involvement of VipA in targeting the cytoskeleton is an example of how Legionella modulates host processes in an Icm/Dot dependent manner. The use of the yeast Vps system has proven itself as a reliable methodology to identify and characterize virulence factors that target host trafficking pathways during microbial pathogenesis.