Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2017

Using and abusing the host: genome-wide siRNA screen reveals host factors important for effector translocation in Coxiella burnetii (#151)

Patrice Newton 1 , Nicole Lau 2 , Shawna CO Reed 3 , Piyush B Madhamshettiwar 4 , Kaylene J Simpson 4 , Craig R Roy 3 , Hayley J Newton 2
  1. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  2. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  3. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America
  4. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

Infection with the unique intracellular pathogen Coxiella burnetii via the inhalation of contaminated aerosols results in the zoonotic disease Q fever. This illness presents with a broad range of clinical manifestations that extend from acute asymptomatic flu-like disease to chronic endocarditis. The main reservoir for human infection are ruminants such as dairy cows, goats and sheep.

 

Upon internalisation within alveolar macrophages, C. burnetii resides within an acidic lysosome-derived organelle, termed the Coxiella-containing vacuole (CCV). The bacteria depend on a functional Type IVB Dot/Icm Secretion System (T4SS) and the subsequent delivery of effector proteins (approximately 150) into the cytoplasm to successfully establish this replicative niche. Since this secretion system is only active once the bacteria reach an acidic lysosomal compartment, Coxiella also relies on passively trafficking through the host canonical endocytic pathway. Indeed, silencing of either Rab5A or Rab7A, proteins that control membrane fusion with early and late endosomes, respectively, results in a significant decrease in translocation of effector proteins.

 

In this study, we utilized a high-throughput siRNA screen combined with a FRET‑based translocation assay to investigate the role of host factors for translocation of effectors by C. burnetii. Using pooled siRNA against 18120 individual protein-coding genes the effect on translocation of MceA (a known effector of C. burnetii) into HeLa 229 cells was measured using CCF2-AM. 400 potential targets were chosen for a secondary validation screen in which de-convoluted siRNA were used to confirm a translocation defect for approximately 60% of the selected targets. A small subset of potential hits was further characterised for their role during Coxiella infection.

 

Collectively, our results elucidate the extensive number of host proteins that are important for efficient translocation of bacterial proteins, many of which play a role in the endocytic pathway as well as lysosome biogenesis and function. Additionally, these results can begin to reveal insights into a possible mechanism for T4SS activation.