AdhE spirosome length in enterohaemorrhagic <i>Escherichia coli</i> is correlated with enzymatic directionality and is perturbed by salicylidene acylhydrazide binding
Bibliographic record
Abstract
ABSTRACT Antibiotics are contraindicated for the treatment of infection enterohemorrhagic Escherichia coli (EHEC), a human pathogen that causes diarrhea or hemorrhagic colitis in humans which can progress to hemolytic uremic syndrome (HUS). As an alternative to the use of antibiotics, previous studies developed the salicylidene acylhydrazides (SA), a family of anti-virulence compounds capable of blocking expression of the type three secretion system (T3SS), thereby reducing bacterial infections. Here we validate AdhE as the anti-virulence protein target of the SA compound ME0054. AdhE is a bidirectional enzyme able to catalyse the conversion of acetyl-CoA to ethanol and vice versa . AdhE oligomerises forming helicoidal filaments heterogeneous in length called spirosomes. In this work, we show that it is possible to partially fractionate AdhE spirosomes using size exclusion chromatography (SEC) and to characterise the spirosome oligomers present in each fraction with biophysical techniques such as small angle X-ray scattering (SAXS) and sedimentation velocity analytical ultracentrifugation (SV-AUC). Also, we observe that short spirosomes are more efficient in the reverse reaction whereas the spirosome length has no impact on the forward reaction. Therefore, for first time, we reveal that AdhE spirosome formation is necessary to regulate the direction of its enzymatic reactions. In addition, we show that ME0054 disrupts AdhE spirosomes, thereby enhancing the conversion of ethanol to acetyl-CoA. Importantly, SV-AUC data show that ME0054 binds to the AdhE filaments. Finally, time-resolved (TR) SAXS allowed us to follow the kinetics of spirosome disruption produced by ME0054, confirming its effectiveness at biologically relevant temperatures and timescales. SIGNIFICANCE STATEMENT There is an urgent need to develop alternative strategies to combat bacterial infections. Salicylidene acylhydrazides (SA) are able to reduce expression of the bacterial type three secretion system (T3SS), used by many pathogens to manipulate host eukaryotic cells, including our pathogen of interest: enterohaemorrhagic E. coli (EHEC). The mechanism underpinning these compounds is a mystery. Here we show how the SA compound ME0054, by disrupting AdhE spirosomes, enhances metabolic conversion of ethanol to acetyl-CoA. This finding is consistent with the phenotype observed in an EHEC AdhE mutant: alterations in acetate levels and changes in T3SS expression. Our work establishes a crucial mechanistic link between the binding of the SA compound to a key target protein and changes in bacterial metabolism.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".