Genome-wide siRNA screening reveals XIAP as essential factor for NOD1-mediated innate immune signalling

Harald Bielig¹, Dr. Peter R. Braun², Isabella Gravenstein³, Maureen Menning⁴, Prof. Thomas F. Meyer⁵, Dr. Thomas A. Kufer⁶

1 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany

2 Max Planck Institute for Infection Biology, Berlin, Germany

3 Max Planck Institute for Infection Biology, Berlin, Germany

4 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany

5 Max Planck Institute for Infection Biology, Berlin, Germany

6 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany

Abstract

The innate immune system defines the first line of defence against invading microbes in mammals. Recognition of pathogens relies on detection of conserved non-self molecules (called pathogen-associated molecular patterns (PAMPs)), derived from the invaders, by pattern-recognition receptors (PRRs) on host cells. Important for bacterial recognition is the detection of bacterial peptidoglycan. The intracellular pattern recognition receptor NOD1 is the major PRR for detection of peptidoglycan derived from Gram-negative bacteria in humans. Activation of NOD1 refers pro-inflammatory responses that help to control infection. However, our knowledge on the underlying signal transduction processes is still fragmentary. To identify unknown components of the NOD1 signalling pathway, we performed a high throughput siRNA screen. A NF-kappaB-luciferase read-out to detect NOD1 activation triggered by the elicitor Tri-DAP in epithelial HEK293T cells combined with a counter-screen for TNF-mediated NF-kappaB activation was used to obtain NOD1 specific targets. Primary hits were validated independently in myeloid THP1 cells. Among most known components, we identified XIAP (X-linked inhibitor of apoptosis, also known as BIRC4) as being critical for NOD1-mediated NF-kappaB activation. XIAP was the highest ranking hit throughout our screening protocol, indicating a prominent role in NOD1-signalling. Knock-down of XIAP in several human cell lines strongly impaired Tri-DAP and Shigella flexneri induced NOD1-mediated NF-kappaB activation and Interleukin-8 secretion. XIAP is best studied as anti-apoptotic factor that acts by direct inhibition of effector caspases. Our findings propose a new essential function of XIAP in the innate immune response to bacterial pathogens. Ongoing studies, using S. flexneri as infection model, will further detail the role of XIAP in this process.This study is supported by the Deutsche Forschungsgemeinschaft, grant SFB670-NG01.

DOI^®: 10.3288/contoo.paper.1652