Development and Application of a Cellular, Gain-of-Signal, Bioluminescent Reporter Screen for Inhibitors of Type II Secretion in Pseudomonas aeruginosa and Burkholderia pseudomallei
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Notice bibliographique
Résumé
The type II secretion (T2S) system in gram-negative bacteria comprises the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, the authors used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive upregulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the isopropyl-β-D-thiogalactopyranoside concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73 000) were detected as primary hits due to stimulation of luminescence with a z score ≥5. Direct secretion assays of the nine most potent hits, representing five chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm but do inhibit T2S-mediated extracellular secretion of elastase with IC50 values from 5 to 25 µM. In addition, seven of the nine compounds also inhibited the T2S-mediated extracellular secretion of phospholipase C by P. aeruginosa and protease activity by Burkholderia pseudomallei. The type II secretion (T2S) system in gram-negative bacteria comprises the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, the authors used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive upregulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the isopropyl-β-D-thiogalactopyranoside concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73 000) were detected as primary hits due to stimulation of luminescence with a z score ≥5. Direct secretion assays of the nine most potent hits, representing five chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm but do inhibit T2S-mediated extracellular secretion of elastase with IC50 values from 5 to 25 µM. In addition, seven of the nine compounds also inhibited the T2S-mediated extracellular secretion of phospholipase C by P. aeruginosa and protease activity by Burkholderia pseudomallei.
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Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découle