Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilises a type III secretion system (T3SS) to inject virulence effector proteins directly into intestinal cells. A number of T3SS effectors are known to subvert host cell signaling pathways. Among these effectors is NleB1 which inhibits death receptor (DR) signaling within host cells. DR signaling can result in inflammation or host cell death by apoptosis. Previously, we have shown that NleB1 is essential for the persistence of EPEC infection in the gut by binding and modifying the death domain (DD) of FADD, an essential signaling mediator of apoptotic signaling. This activity blocks the ability of host cells to undergo apoptosis via FAS or TNF signaling and essentially prolongs the life of the cell for the benefit of the bacteria. Protein prediction software revealed that NleB1 is highly similar to the GT-8 family of glycosyltransferases and contains a signature DxD catalytic motif which is essential for its function. We have shown that NleB1 functions as a novel enzyme which adds an N-acetyl glucosamine moiety to a conserved arginine residue within the DD of FADD rather than to serines, threonines and asparagines as other glycosyltransferases do. Here, we aim to investigate which region of NleB1 is important for binding to its substrate FADD by screening a library of transposon mutants of NleB1 for their ability to bind and N-GlcNAcylate FADD. Identification of potential substrate “docking” sites of NleB1 will contribute to a better understanding of the substrate specificity of this highly novel enzyme.