SPKW E. coli O157:H7 (ECO57) Subproject

Parent project: SPKW.md

Overview

E. coli O157:H7 is a pathogenic bacterium with well-characterized virulence factors including:
- Shiga toxins (Stx1, Stx2) - True toxins that kill host cells
- Type III secretion system (T3SS) effectors - Signaling modulators that manipulate host cells

This provides an ideal test case for distinguishing between:
1. True toxins - Proteins that directly damage/kill host cells (LEGITIMATE toxin annotations)
2. Effectors - Proteins that modulate signaling without direct cytotoxicity (potential OVER-ANNOTATIONS)

Statistics (2026-01-31)

Status

• [x] Initial exploration complete (2026-01-31)
• [x] Deep research for 2 genes (2026-01-31)
• [x] Annotation review complete (2026-01-31)
• [x] Write-up complete (2026-01-31)
• [ ] Additional T3SS effectors (nleB2, nleE, etc.) - future work

Case Studies (2 genes reviewed)

Case 1: nleB1 - Type III Effector (OVER-ANNOTATED)

Gene: nleB1 (Q8XBX8) - Protein-arginine N-acetylglucosaminyltransferase

Review file: genes/ECO57/[nleB1](../../genes/ECO57/nleB1/nleB1-ai-review.html)/nleB1-ai-review.yaml

SPKW annotation: GO:0090729 (toxin activity)

Review decision: REMOVE

Analysis: NleB1 is a T3SS effector that modulates host signaling, NOT a toxin:

True Toxin (e.g., Stx2A):
┌─────────────────────────────────────────────────────────────────────────────┐
│ Enters cell → Inactivates ribosomes → Halts translation → CELL DEATH       │
│                                                                             │
│ Direct cytotoxicity = TRUE TOXIN                                            │
└─────────────────────────────────────────────────────────────────────────────┘

NleB1 (Type III Effector):
┌─────────────────────────────────────────────────────────────────────────────┐
│ Injected via T3SS → GlcNAcylates death domain proteins → Blocks apoptosis  │
│                                                                             │
│ Modulates signaling = EFFECTOR, NOT TOXIN                                   │
│                                                                             │
│ Key difference: NleB1 PREVENTS cell death, not causes it!                   │
└─────────────────────────────────────────────────────────────────────────────┘

NleB1's actual function:
1. Glycosyltransferase that adds GlcNAc to arginine residues
2. Targets death domain proteins (FADD, TRADD, RIPK1)
3. Blocks death receptor signaling complex assembly
4. SUPPRESSES apoptosis and necroptosis - opposite of toxin!

Literature evidence (PMID:30619781):

"NleB1 inhibitors were not significantly toxic to mammalian cells"
"Did not cause significant macrophage death"

Core function: GO:0106362 (protein-arginine N-acetylglucosaminyltransferase activity)

Case 2: stx2A - Shiga Toxin A Subunit (LEGITIMATE)

Gene: stx2A (A0A9Q6Z964) - rRNA N-glycosylase

Review file: genes/ECO57/[stx2A](../../genes/ECO57/stx2A/stx2A-ai-review.html)/stx2A-ai-review.yaml

SPKW annotation: GO:0090729 (toxin activity)

Review decision: ACCEPT (Legitimate annotation)

Analysis: Stx2A is a TRUE TOXIN with direct cytotoxic activity:

Shiga Toxin Mechanism:
┌─────────────────────────────────────────────────────────────────────────────┐
│ 1. B pentamer binds Gb3 receptor → Endocytosis                              │
│ 2. A chain cleaved by furin → A1 (catalytic) released to cytosol            │
│ 3. A1 depurinates A4324 in 28S rRNA sarcin-ricin loop                       │
│ 4. Ribosomes inactivated → Translation halted → CELL DEATH                  │
│                                                                             │
│ Direct ribosome inactivation = TRUE TOXIN ACTIVITY                          │
└─────────────────────────────────────────────────────────────────────────────┘

Stx2A's actual function:
1. rRNA N-glycosylase (EC 3.2.2.22)
2. Depurinates specific adenine in 28S rRNA sarcin-ricin loop
3. Inactivates ribosomes (ribosome-inactivating protein, RIP)
4. Causes hemolytic uremic syndrome (HUS) in humans

This is EXACTLY what "toxin activity" (GO:0090729) describes:

"initiating pathogenesis (leading to an abnormal, generally detrimental state)"

Key Finding: Distinguishing Toxins from Effectors

Over-Annotation Pattern: "Toxin" Applied to Effectors

The UniProt "Toxin" keyword is being applied too broadly to T3SS effectors:

Genes correctly annotated as toxins:
- stx1A, stx1B, stx2A, stx2B (Shiga toxins) - TRUE TOXINS
- hlyA, hlyE (hemolysins) - TRUE TOXINS (pore-forming)

Genes incorrectly annotated as toxins:
- nleB1, nleB2 (glycosyltransferases) - EFFECTORS
- nleE (cysteine methyltransferase) - EFFECTOR

Comparison: O157:H7 vs Other Organisms

Recommendations for Bacterial Pathogen SPKW Curation

1. Distinguish toxins from effectors: Toxins directly kill; effectors modulate signaling

2. Check mechanism of action: Does the protein directly damage cells or manipulate them?

3. Consider the biological outcome: If a protein PREVENTS cell death (like NleB1), it's not a toxin

4. Validate Shiga toxin family: These are legitimate toxin annotations

5. Review T3SS effector annotations: Many may have incorrect "toxin" annotations

Review Files Location

genes/ECO57/
├── [nleB1](../../genes/ECO57/nleB1/nleB1-ai-review.html)/nleB1-ai-review.yaml   (REMOVE: toxin - is effector that blocks apoptosis)
└── [stx2A](../../genes/ECO57/stx2A/stx2A-ai-review.html)/stx2A-ai-review.yaml   (ACCEPT: toxin - true ribosome-inactivating protein)