Oral Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

A genome-wide RNAi screen to identify host factors involved in Legionella pneumophila Dot/Icm effector translocation (#13)

Sze Ying Ong 1 , Ralf Schuelein 1 , Shivani Pasricha 1 , Kaylene Simpson 2 , Elizabeth Hartland 1
  1. Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  2. Peter MacCullum Institute, Victorian Functional Genomics Facility, Melbourne, VIC, Australia

The intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease, an acute form of pneumonia. After phagocytosis by lung macrophages, the bacteria replicate in a specialized vacuole termed the L. pneumophila containing vacuole (LCV) that resists fusion with late endocytic compartments and lysosomes and recruits membrane that resembles rough endoplasmic reticulum. The bacterial Dot/Icm Type IV secretion system (T4SS) is required for LCV biogenesis and is responsible for the translocation of ~300 bacterial effector proteins into host cells. Since the secretion of effectors by L. pneumophila does not occur in the absence of host cells, we hypothesized that Dot/Icm dependent translocation requires active participation by the host cell. The aim of this study was to identify host factors that contribute to Dot/Icm effector translocation and/or L. pneumophila entry into host cells, using a high-throughput genome-wide RNAi screen coupled with a FRET conversion fluorescence reporter assay for quantifying effector translocation. After extensive optimization, both the screen and the assay have been successfully established to provide maximum efficiency of siRNA-based knock-down of host factors and reliable, statistically consistent reporter assay output. The optimized parameters were then successfully transferred and adapted to robotics at the Victorian Centre for Functional Genomics (VCFG) genome screening facility. To date we have completed siRNA screening of the whole human genome and identified ~400 potential ‘hits’, that when silenced caused >30% reduction in Dot/Icm effector translocation. These 400 ‘hits’ are subjected to secondary screening for confirmation of reduced Dot/Icm effector translocation. At the conclusion of these validation studies, 3 ubiquitin-conjugating (E2s) and 2 ubiquitin ligases (E3s) are further identified to be of interest and are subjected to further characterization. We investigated their role in the function of the Dot/Icm system as well as their ability to support replication of L. pneumophila.