Science Bite - 3 min Oral Presentation (Students and ECRs only) Lorne Infection and Immunity 2020

A novel glycosyltransferase effector from enteropathogenic Escherichia coli that targets innate immune signalling proteins (#73)

Cristina Giogha 1 , Tania Wong Fok Lung 2 , Jaclyn S Pearson 1 3 , Nichollas Scott 4 , Elizabeth L Hartland 1 3
  1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  2. Department of Pediatrics, Columbia University at the Columbia University Medical Center, New York, NY, USA
  3. Department of Molecular and Translational Science, Monash University, Clayton, Vic, Australia
  4. University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, VIC, Australia

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

 

In recent years, 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 by preventing formation of the death-inducing signaling complex (DISC) in response FasL stimulation.

 

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 when expressed in mammalian cells. 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. Sugar donor competition assays revealed that NleB2 prefers UDP-Glucose, and peptide sequencing identified the modification site as an arginine residue. Thus, this is the first identification of a bacterial enzyme that can catalyse Arginine-Glucose modifications, which are rare and previously reported only in plants. We have identified residues that dictate this unique sugar preference, and have used this knowledge and the Arg-GlcNAc-specific antibody to identify targets of NleB2 in transfected cells. We are currently investigating these targets to determine the role of NleB2 during infection.

  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.