NleB/SseK effectors from Citrobacter rodentium, Escherichia coli, and Salmonella enterica display distinct differences in host substrate specificity

J Biol Chem. 2017 Jul 7;292(27):11423-11430. doi: 10.1074/jbc.M117.790675. Epub 2017 May 18.


Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and Crodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.

Keywords: bacterial pathogenesis; glycosyltransferase; innate immunity; signal transduction; virulence factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Citrobacter rodentium / enzymology*
  • Citrobacter rodentium / genetics
  • Citrobacter rodentium / pathogenicity
  • Enterohemorrhagic Escherichia coli / enzymology*
  • Enterohemorrhagic Escherichia coli / genetics
  • Enterohemorrhagic Escherichia coli / pathogenicity
  • Enteropathogenic Escherichia coli / enzymology*
  • Enteropathogenic Escherichia coli / genetics
  • Enteropathogenic Escherichia coli / pathogenicity
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Fas-Associated Death Domain Protein / genetics
  • Fas-Associated Death Domain Protein / metabolism
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Glycosylation
  • Mice
  • RAW 264.7 Cells
  • Receptors, Tumor Necrosis Factor, Type II / genetics
  • Receptors, Tumor Necrosis Factor, Type II / metabolism
  • Salmonella enterica / enzymology*
  • Salmonella enterica / genetics
  • Salmonella enterica / pathogenicity
  • TNF Receptor-Associated Factor 2 / genetics
  • TNF Receptor-Associated Factor 2 / metabolism
  • Ubiquitination
  • Virulence Factors / genetics
  • Virulence Factors / metabolism*


  • Bacterial Proteins
  • Escherichia coli Proteins
  • Fadd protein, mouse
  • Fas-Associated Death Domain Protein
  • NleB protein, E coli
  • Receptors, Tumor Necrosis Factor, Type II
  • SseK1 protein, Salmonella enterica
  • SseK2 protein, Salmonella enterica
  • TNF Receptor-Associated Factor 2
  • Virulence Factors
  • Glyceraldehyde-3-Phosphate Dehydrogenases