NleB1 (Non-LEE-encoded type III effector B1) is a protein-arginine N-acetylglucosaminyltransferase that is secreted by the type III secretion system (T3SS) into host cells during EHEC infection. The enzyme catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to the guanidino nitrogen of arginine residues on host target proteins, primarily death domain-containing proteins such as FADD (Arg117), TRADD (Arg235), and RIPK1 (Arg603). This unusual post-translational modification disrupts homotypic and heterotypic death domain interactions required for death receptor signaling complex assembly, thereby inhibiting TNF/TRAIL/Fas-mediated apoptosis, necroptosis, and NF-kappaB activation. NleB1 also glycosylates GAPDH at Arg197/Arg200, preventing GAPDH-TRAF2 interaction. The enzyme requires Mn2+ as a cofactor and contains a GT-A fold with a characteristic DXD motif and HEN catalytic triad. NleB1 undergoes auto-GlcNAcylation which is required for activity toward death domain-containing substrates. Importantly, NleB1 is a signaling modulator/effector that manipulates host cell processes rather than a classical toxin that directly kills cells (nleB1-deep-research-falcon.md).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: NleB1 is secreted via the type III secretion system into the extracellular milieu and then translocated directly into host cells. The annotation reflects that the protein leaves the bacterial cytoplasm and enters the extracellular space transiently before entering host cells (PMID:30619781, PMID:28522607). Reason: This annotation is correct as NleB1 is a T3SS effector that is secreted from the bacterium. UniProt confirms the subcellular location as "Secreted" with subsequent translocation to "Host cytoplasm" via T3SS. The IEA annotation based on UniProt subcellular location vocabulary mapping is appropriate. Supporting Evidence: PMID:30619781 The enteropathogenic and enterohemorrhagic Escherichia coli NleB proteins as well as the Salmonella enterica SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | ACCEPT | Summary: NleB1 is a glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to arginine residues on protein substrates. This broad transferase activity annotation is correct but less specific than GO:0106362 (protein-arginine N-acetylglucosaminyltransferase activity) which is also annotated. Reason: This is a correct parent term annotation. While more specific annotations exist (GO:0016757, GO:0106362), having parent terms from IEA mappings is acceptable and does not conflict with more specific annotations. The UniProt record explicitly lists EC=2.4.1.- confirming transferase activity. Supporting Evidence: PMID:30619781 The kinetic parameters of NleB1 (150 nM at 30Β°C) were calculated as follows: Vmax: 2,975.3 Β± 125 RLU/min/ΞΌg protein; Km: 379 Β± 43 ΞΌM; Kcat (s-1): 50, Kcat/Km (s-1, M-1): 130,703.4 |
| GO:0016757 glycosyltransferase activity | IEA GO_REF:0000043 | ACCEPT | Summary: NleB1 catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to arginine residues on host proteins. Multiple publications confirm glycosyltransferase activity (PMID:28522607, PMID:30619781). The protein belongs to the glycosyltransferase NleB family and has a GT-A fold. Reason: This annotation is experimentally validated. PMID:28522607 states "NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine". PMID:30619781 further characterizes the kinetic parameters. This is an appropriate intermediate-level MF annotation. Supporting Evidence: PMID:28522607 NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses PMID:30619781 The NleB1/NleB2 (enteropathogenic and enterohemorrhagic E. coli; EPEC and EHEC) and SseK1/SseK2/SseK3 (Salmonella enterica) T3SS effectors are glycosyltransferases that modify protein substrates on arginine residues |
| GO:0030430 host cell cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: NleB1 is translocated into host cells via the T3SS where it functions in the host cell cytoplasm to modify death domain-containing proteins. This localization is essential for its function in disrupting host signaling pathways. Reason: Correct localization annotation. The deep research report confirms "NleB1 is secreted by the T3SS into host cells, where it acts in the host cytosol to modify death-domain proteins." UniProt also lists "Host cytoplasm" as the subcellular location with evidence by similarity to related effectors. Supporting Evidence: PMID:28522607 Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | ACCEPT | Summary: NleB1 requires manganese (Mn2+) as a cofactor for its glycosyltransferase activity. The DXD motif (residues 221-223) coordinates the metal ion. Mutation of the DXD motif to AAA abolishes activity (PMID:30619781). Reason: Correct annotation supported by experimental evidence. While GO:0030145 (manganese ion binding) is more specific and also annotated with IDA evidence, this broader term is acceptable as an IEA annotation. The DXD motif essential for Mn2+ coordination is confirmed in the UniProt feature table. Supporting Evidence: PMID:30619781 an inactive form of NleB1 (NleB1-AAA) in which the aspartic acid residues required for Mn2+ stabilization were mutated to alanines |
| GO:0090729 toxin activity | IEA GO_REF:0000043 | REMOVE | Summary: This annotation derives from the UniProt keyword "Toxin" (KW-0800) mapping to GO. However, the GO definition of toxin activity requires "Interacting selectively with one or more biological molecules in another (target) organism, initiating pathogenesis (leading to an abnormal, generally detrimental state)." While NleB1 is a virulence factor, it is fundamentally different from classical toxins. NleB1 is a signaling modulator that manipulates host cell processes by enzymatically modifying host proteins - it does NOT directly kill or damage cells like true toxins (e.g., Shiga toxin which inhibits protein synthesis, hemolysin which lyses cells by pore formation). NleB1 actually INHIBITS cell death (apoptosis, necroptosis) rather than causing it (nleB1-deep-research-falcon.md). Reason: This is a clear case of SPKW over-annotation. The UniProt keyword "Toxin" is applied broadly to bacterial virulence factors, but the GO term "toxin activity" has a specific definition that does not fit NleB1. Key distinctions: 1) True toxins (Shiga toxin, diphtheria toxin, hemolysins) directly damage/kill cells 2) NleB1 is a T3SS effector that MODULATES host signaling without direct cytotoxicity 3) NleB1 actually SUPPRESSES host cell death pathways (apoptosis, necroptosis) 4) The mechanism is enzymatic modification of host proteins to subvert signaling, not direct cellular damage 5) PMID:30619781 explicitly notes that inhibitors of NleB1 "were not significantly toxic to mammalian cells" and did not cause "significant macrophage death" The appropriate GO terms for NleB1's function are process terms like GO:0052041 (symbiont-mediated suppression of host programmed cell death), GO:0085034 (symbiont-mediated suppression of host NF-kappaB cascade), and GO:0140403 (effector-mediated suppression of host innate immune response). Supporting Evidence: PMID:28522607 NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses PMID:30619781 We also failed to observe significant macrophage death in our studies |
| GO:0106362 protein-arginine N-acetylglucosaminyltransferase activity | IEA GO_REF:0000002 | ACCEPT | Summary: This is the most precise molecular function annotation for NleB1. The enzyme specifically catalyzes the transfer of GlcNAc to the guanidino nitrogen of arginine residues on protein substrates. This activity is directly demonstrated in PMID:28522607 and PMID:30619781. Reason: Correct and appropriate annotation. This GO term precisely describes NleB1's enzymatic activity. UniProt lists this exact term with IDA evidence from PMID:28522607 and PMID:30619781. The IEA annotation from InterPro is consistent with experimental evidence. Supporting Evidence: PMID:28522607 EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200 PMID:30619781 This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles |
| GO:0005515 protein binding | IPI PMID:27018634 Quantitative Mass Spectrometry Identifies Novel Host Binding... | MODIFY | Summary: PMID:27018634 identified ensconsin (MAP7) as a binding partner for NleB1 using quantitative mass spectrometry. The study confirmed that NleB1 interacted with ensconsin in a region corresponding to its microtubule binding domain. However, "protein binding" is an uninformative annotation per GO curation guidelines. Reason: While the interaction with ensconsin/MAP7 is experimentally validated, the GO term "protein binding" (GO:0005515) is considered too vague to be informative. Per GO curation guidelines, more specific binding terms should be used. The interaction with MAP7 led to disruption of intracellular trafficking (see GO:0052038 annotation). A more informative annotation would describe the functional consequence or use a specific binding term if available. Proposed replacements: effector-mediated perturbation of host process by symbiont Supporting Evidence: PMID:27018634 we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain |
| GO:0030145 manganese ion binding | IDA PMID:30619781 High-Throughput Screening for Bacterial Glycosyltransferase ... | ACCEPT | Summary: NleB1 requires Mn2+ as a cofactor for its glycosyltransferase activity. The DXD motif (residues 221-223) is essential for Mn2+ coordination. PMID:30619781 demonstrated that mutation of the DXD motif to AAA (NleB1-AAA) abolished enzymatic activity, confirming the requirement for metal coordination. Reason: This is a well-supported annotation with direct experimental evidence. The kinetic assays in PMID:30619781 used 25 mM MnCl2 and showed that the DXD->AAA mutant lacked activity. UniProt features annotate residues 223, 320, and 322 as Mn2+ binding sites. Supporting Evidence: PMID:30619781 UDP-Glo luminescence assays were performed in 96 well-microplates, containing 250 nM NleB1 in 125 mM Tris pH 7.4, 25 mM MnCl2, 2.5 mM DTT, and 100 ΞΌM UDP-GlcNAc PMID:30619781 an inactive form of NleB1 (NleB1-AAA) in which the aspartic acid residues required for Mn2+ stabilization were mutated to alanines |
| GO:0106362 protein-arginine N-acetylglucosaminyltransferase activity | IDA PMID:28522607 NleB/SseK effectors from Citrobacter rodentium, Escherichia ... | ACCEPT | Summary: PMID:28522607 directly demonstrated that EHEC NleB1 glycosylates host proteins GAPDH (at Arg197 and Arg200) and FADD on arginine residues using in vitro glycosylation assays and mass spectrometry. Reason: This is the primary experimental evidence for NleB1's enzymatic activity. The study used in vitro assays and cell culture experiments to demonstrate arginine-specific N-acetylglucosaminyltransferase activity. The specific modification sites were identified by mass spectrometry. Supporting Evidence: PMID:28522607 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 PMID:28522607 C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein) |
| GO:0106362 protein-arginine N-acetylglucosaminyltransferase activity | IDA PMID:30619781 High-Throughput Screening for Bacterial Glycosyltransferase ... | ACCEPT | Summary: PMID:30619781 characterized the kinetic parameters of NleB1's glycosyltransferase activity (Km = 379 uM for UDP-GlcNAc, kcat = 50 s-1) and developed high-throughput screening assays to identify inhibitors. The study used purified recombinant NleB1 and demonstrated activity against GAPDH and TRADD substrates. Reason: This provides additional IDA evidence for the same molecular function. The study provides quantitative kinetic characterization of the enzyme. Having multiple IDA annotations from different publications strengthens the evidence base. Supporting Evidence: PMID:30619781 The kinetic parameters of NleB1 (150 nM at 30Β°C) were calculated as follows: Vmax: 2,975.3 Β± 125 RLU/min/ΞΌg protein; Km: 379 Β± 43 ΞΌM; Kcat (s-1): 50, Kcat/Km (s-1, M-1): 130,703.4 |
| GO:0052038 symbiont-mediated perturbation of host intracellular transport | IMP PMID:27018634 Quantitative Mass Spectrometry Identifies Novel Host Binding... | ACCEPT | Summary: PMID:27018634 demonstrated that NleB1 and EspL interact with ensconsin (MAP7), an essential cofactor of kinesin-1 required for intracellular trafficking. The study showed that intracellular trafficking was severely disrupted during wild-type EPEC infections but not during infections with ΞnleB1 or ΞespL mutants. Reason: This is a well-supported annotation with genetic evidence (IMP). The phenotype was observed in wild-type but not mutant infections, providing direct evidence that NleB1 contributes to perturbation of host intracellular transport through its interaction with the microtubule-associated protein ensconsin. Supporting Evidence: PMID:27018634 Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΞnleB1 or ΞespL mutants |
| GO:0085034 symbiont-mediated suppression of host NF-kappaB cascade | IDA PMID:28522607 NleB/SseK effectors from Citrobacter rodentium, Escherichia ... | NEW | Summary: NleB1 blocks TNF-mediated NF-kappaB pathway activation by glycosylating death domain proteins FADD, TRADD, and RIPK1, preventing their assembly into signaling complexes. This is a core function of NleB1. PMID:28522607 directly demonstrated that EHEC NleB1 blocked TNF-mediated NF-kappaB pathway activation. Reason: This annotation is strongly supported by the literature and represents a core biological function of NleB1. The mechanism (arginine glycosylation of death domain proteins preventing signaling complex assembly) is well characterized. Supporting Evidence: PMID:28522607 SseK1, SseK3, EHEC NleB1, EPEC NleB1, and Crodentium NleB blocked TNF-mediated NF-ΞΊB pathway activation PMID:30619781 This modification is biologically important because the glycosylation of arginines on protein substrates disrupts the normal functioning of the innate immune system |
| GO:0033668 symbiont-mediated suppression of host apoptosis | IDA PMID:28522607 NleB/SseK effectors from Citrobacter rodentium, Escherichia ... | NEW | Summary: NleB1 inhibits host apoptosis by glycosylating death domain proteins (FADD, TRADD, RIPK1) which prevents death receptor signaling complex assembly. This blocks TNF/TRAIL/Fas-mediated apoptotic signaling pathways (nleB1-deep-research-falcon.md). Reason: This is a well-documented function of NleB1. The enzyme specifically targets death domain-containing proteins that are essential for death receptor-mediated apoptosis. By glycosylating these proteins, NleB1 prevents their interaction and blocks apoptotic signaling. Supporting Evidence: PMID:28522607 NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses PMID:30619781 Several death domain-containing proteins have been described as substrates of some of the NleB/SseK orthologs, including the Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), and the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) |
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Download this section (compressed HTML)Q: Should UniProt keyword "Toxin" be applied to T3SS effectors that modulate host signaling without direct cytotoxic activity? The current mapping leads to inappropriate GO annotations.
Q: What is the relative importance of different NleB1 substrates (FADD, TRADD, RIPK1, GAPDH) for virulence during natural infection?
Experiment: Comparative analysis of NleB1 activity on different death domain proteins to establish substrate hierarchy and physiological relevance during infection.
Hypothesis: FADD may be the primary physiological target based on structural and kinetic data
Experiment: Structural characterization of NleB1-substrate complexes to understand substrate recognition and guide inhibitor development.
Hypothesis: Substrate recognition involves specific interactions with death domain fold
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