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

Samir El Qaidi,Kangming Chen,Adnan Halim,Lina Siukstaite,Christian Rueter,Ramon Hurtado-Guerrero,Henrik Clausen,Philip R Hardwidge

doi:10.1074/jbc.m117.790675

Abstract

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.

Highlights

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells
We found that Escherichia coli (EHEC) NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptorassociated factor 2 ubiquitination
We found that EHEC NleB1 and C. rodentium NleB glycosylate both GAPDH and FADD, whereas SseK1 only glycosylates GAPDH, and SseK2 and enteropathogenic E. coli (EPEC) NleB1 are only active with FADD

Summary

Results

NleB is a glycosyltransferase that glycosylates several host proteins with GlcNAcs [5,6,7]. EHEC NleB1 glycosylated WT GAPDH but did not glycosylate Arg-free GAPDH (Fig. 3A) We subjected these protein samples to mass spectrometry analysis and observed mass increments corresponding to one and two single GlcNAc modifications on the LWRDGRGALQNIPASTGAAK peptide of GAPDH. His-GAPDH mutants were individually incubated with each EHEC NleB1 and subjected to GST pulldown assays using glutathione-Sepharose beads (GE Healthcare). To determine the functional importance of the GAPDH Arg197 and Arg200 residues in the context of TRAF2 activity, we mutated Arg197 and/or Arg200 to either alanine or lysine for use in transfection experiments in a stable GAPDH knockdown cell line (Fig. 4A) We transfected these cells with TRAF2-FLAG and with different Myc-GAPDH mutants. We had predicted that the GAPDH Arg197/Arg200 mutants would be resistant to EHEC NleB1-mediated inhibition in these cell culture experiments, our finding that the GAPDH mutants failed to interact with TRAF2 precluded us from testing this hypothesis directly

Discussion

55 GAPDH-Myc

Experimental procedures