Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

A unique glycosyltransferase effector from enteropathogenic Escherichia coli that targets innate immune signalling proteins (#65)

Cristina Giogha 1 2 , Nichollas E Scott 3 , Tania Wong Fok Lung 4 , Georgina L Pollock 1 , Marina Harper 5 , Ethan D Goddard-Borger 6 , Jaclyn S Pearson 1 2 , Elizabeth L Hartland 1 2
  1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  2. Department of Molecular and Translational Science, Monash University, Clayton, Vic, Australia
  3. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  4. Department of Pediatrics, Columbia University at the Columbia University Medical Center, New York, NY, USA
  5. Department of Microbiology, Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University,, Clayton, Victoria, Australia
  6. ACRF Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are significant causes of diarrhoeal disease worldwide. During infection, the bacteria manipulate various aspects of host cell function by utilising a type III secretion system (T3SS) to translocate effector proteins directly into host cells. These effectors are essential for the bacteria to survive, replicate and cause disease.

We have identified several T3SS effectors with highly novel enzymatic activities, including the glycosyltransferase NleB1 of EPEC (1). Unlike other glycosyltransferases which add sugars to serine, threonine or asparagine residues, NleB1 transfers a single N-acetylglucosamine (GlcNAc) sugar to arginine residues, mediating Arg-GlcNAc modifications. NleB1 specifically glycosylates the death-domain of the adapter protein FADD and blocks host cell death during infection.

Homologues of NleB1 with conserved glycosyltransferase motifs are found within EPEC (termed NleB2) and Salmonella Typhimurium. Those from Salmonella appear to have similar enzymatic activities to NleB1. However, using Arg-GlcNAc-specific antibodies we found that NleB2 of EPEC does not catalyse this type of glycosylation during infection. In vitro glycosylation assays combined with mass spectrometry identified that in contrast to NleB1, NleB2 can utilise different sugar donors including UDP-GlcNAc, UDP-glucose and UDP-galactose to glycosylate the death domain of human RIPK1. Sugar donor competition assays revealed that NleB2 prefers UDP-glucose, and peptide sequencing identified the modification site within RIPK1 as an arginine residue, indicating that NleB2 catalyses arginine-glucose modifications.

We identified the residue in NleB1 and NleB2 that dictates this unique catalytic activity, using site-directed mutagenesis. Although these mutations switch sugar donor preference, we found that they do not affect the ability of these enzymes to inhibit inflammatory or cell death signaling during transfection or EPEC infection. Thus, this is the first identification of a bacterial enzyme that catalyses arginine-glucose modifications, which are rare and previously reported only in plants. The switch in sugar donor preference that has arisen in NleB2 may allow for adaption to changes in sugar donor availability within the host cell cytoplasm.

  1. 1. Pearson JS, Giogha C, Ong SY, et al. (2013). A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature 501:247- 251.