Science Bite (3 min Oral Presentation) Lorne Infection and Immunity 2019

Characterisation of a family of novel secreted bacterial glycosyltransferases (#86)

Cristina Giogha 1 2 , Nichollas Scott 3 , Tania Wong Fok Lung 4 , Jaclyn S Pearson 2 , Elizabeth L Hartland 2 5
  1. Hudson Institute of Medical Research, Clayton, VIC, Australia
  2. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  3. University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  4. Department of Pediatrics, Columbia University at the Columbia University Medical Center, New York, USA
  5. Department of Molecular and Translational Science, Monash University, Clayton, Vic, Australia

Gut bacterial pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Salmonella enterica serovar Typhimurium are significant causes of diarrhoeal disease worldwide. During infection, the bacteria directly manipulate various aspects of host cell function by utilising type III secretion systems (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 of the host death-domain containing signaling proteins FADD, TRADD and RIPK1. NleB1 activity 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 (NleB2) and Salmonella Typhimurium (SseK1, SseK2, SseK3). Both SseK1 and SseK3 appear to function similarly to NleB1. However, using antibodies specific to the novel Arg-GlcNAc modification we found that NleB2 of EPEC and SseK2 of Salmonella Typhimurium do not catalyse Arg-GlcNAc modifications when expressed in mammalian cells. Using in vitro glycosylation assays combined with mass spectrometry we have determined the type of glycosylation catalysed by NleB2. In contrast to NleB1, NleB2 can utilise different sugar donors including UDP-GlcNAc, UDP-Glc and UDP-Gal. Sugar donor competition assays revealed that NleB2 prefers UDP-Glc, and peptide sequencing identified the modification site as an arginine residue. This is the first identification of a bacterial enzyme that can catalyse Arg-Glc modifications, which are rare and previously reported only in plants. We are currently developing tools to identify this unique type of biochemical modification which we will use to investigate 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.