Microbes and Beneficial Microbes

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 eff ectors are enzymes that modify host proteins to subvert their normal functions. NleB

is a glycosyltransferase that modifi es host proteins with N-acetyl-D-glucosamine to inhibit antibacterial and infl ammatory host

responses. NleB is conserved among the attaching/eff acing pathogens enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli

(EPEC), and Citrobacter rodentium. Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3.

However, there are confl icting reports regarding the activities and host protein targets among the NleB/SseK orthologs. We performed

in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specifi cities of these eff ectors. SseK1,

SseK3, EHEC NleB1, EPEC NleB1, and C. rodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and

NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host glyceraldehyde 3- phosphate dehydrogenase (GAPDH).

C. rodentium NleB, EHEC NleB1, EPEC NleB1,and SseK2 glycosylated the Fas-associated death domain protein (FADD). SseK3

and NleB2 were not active against either substrate. EHEC NleB1 glycosylates two GAPDH arginine residues, R197 and R200. Th ese

two residues are essential for GAPDHmediated activation of tumor necrosis factor (TNF) Receptor-Associated Factor 2 (TRAF2)

ubiquitination. Th ese results provide evidence that members of this highly conserved family of bacterial virulence eff ectors target

diff erent host protein substrates and exhibit distinct cellular modes of action to suppress host responses.