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

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 -acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic (EHEC), enteropathogenic (EPEC), and Moreover, 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 glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. 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, Arg and Arg, 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.

Salmonellaeffectors SseK1 and SseK3 target death domain proteins in the TNF and TRAIL signaling pathways

“…Although previous reports had suggested both TRADD and FADD were substrates of SseK1 [8, 9, 11], our data suggested that TRADD is the preferred substrate of SseK1, as we could not detect modification of FADD during S. Typhimurium infection. TRADD plays a key role in the activation of canonical NF-κB signaling leading to pro-inflammatory cytokine secretion (reviewed in [17, 18]) and programmed cell death via TNF induced apoptosis or necroptosis [19].…”

“…A number of reports have used in vitro experiments to identify possible substrates of SseK1 [8, 9, 11]. While these studies have demonstrated the ability of SseK1 to GlcNAcylate a given target, the approach is substrate specific and there requires knowledge of the potential targets.…”

“…Elucidating the biochemical activity of bacterial effector proteins, their preferred host substrates, and contribution to virulence, remains a major priority of host-pathogen research to help elucidate basic processes that are manipulated by highly evolved pathogens, and to provide for new opportunities to disrupt disease causing processes. A number of studies have described putative substrates of the Salmonella Typhimurium effectors SseK1 and SseK3 [8, 9, 11, 12] but these studies have relied substantially on in vitro experiments using recombinant proteins or overexpression of the effectors in mammalian cells. Here, we identified the substrates of SseK1 and SseK3 when expressed at native levels during S. Typhimurium infection of RAW264.7 cells.…”

“…One report suggested that recombinant SseK1 modifies recombinant TRADD in vitro [8], whereas a subsequent report suggested SseK1 modifies both TRADD and FADD when co-expressed ectopically in mammalian cell lines [9]. Yet another report using in vitro glycosylation assays suggested that recombinant SseK1 glycosylates GAPDH but not FADD [11]. SseK3 on the other hand was reported to bind but not modify the E3-ubiquitin ligase TRIM32 [12], and was shown to weakly modify TRADD but not FADD [9].…”

Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

“…Shortly after this report, another function of NleB in interfering with TNF RSC assembly was uncovered in two reports that showed that NleB transfers GlcNac on arginine residues in death domains in TRADD (Arg 235), FADD (Arg 117), and RIPK1 (Arg 603, based on homology) (40,41). The GlcNac moiety interferes with death domain oligomerization, preventing the assembly of the TNF death receptor complex as well as NF-B activation by TNF (40)(41)(42). This unique posttranslational modification prevents activation of necroptosis and NF-B-mediated transcription of downstream cytokines and chemokines (43).…”