Poster Presentation Lorne Infection and Immunity 2013

Mapping of the substrate binding domains of the type III effector NleD (#113)

Kristina Creuzburg 1 , Sabrina Mühlen 1 , Elizabeth L Hartland 1
  1. University of Melbourne, Parkville, VIC, Australia

Enteropathogenic Escherichia coli (EPEC) is an extracellular human pathogen, which employs a type III secretion system to deliver effector proteins into eukaryotic cells. There, the translocated effector proteins subvert host cell signaling pathways to facilitate the survival of the pathogen. Several effector proteins have recently been shown to inhibit the innate immune response of the host by targeting different components of the inflammatory signaling pathways. The type III effector protein NleD is a zinc metalloprotease that specifically cleaves the stress-activated protein kinases (SAPKs) JNK and p38. This cleavage results in blocking the activity of the transcription factor AP-1.

The aim of this study was to map the protein domains of NleD, which confer substrate binding. For this purpose, a random transposon-based mutagenesis approach was used. An in-frame five amino acid insertion library was generated and the resulting mutants were screened for their ability to cleave JNK and p38 when transfected into mammalian cells. Our results suggest that the substrate binding domain in NleD is located within the C-terminal region of the protein close to the catalytic site.