Characterization of Heterologously Expressed Putative Cytolysins from “ <i>Brachyspira hampsonii “</i>
Notice bibliographique
Résumé
Cytolysins produced by Brachyspira hyodysenteriae and “ Brachyspira hampsonii” strain 30446 are believed to be important virulence factors in the pathology of swine dysentery. To date four putative cytolysin genes termed tlyA, tlyB, tlyC, and hlyA have been identified, all of which are present in both Brachyspira hyodysenteriae and “ Brachyspira hampsonii” . While the contributions of tlyB, tlyC, and hlyA to the pathogenesis of swine dysentery are not yet known, tlyA knockout strains of Brachyspira hyodysenteriae have shown decreased virulence in mouse models. In addition, homologues of tlyA are believed to be important virulence factors for human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis , functioning as both cytolysins and contributing to antibiotic resistance through ribosomal RNA methylation. While active hlyA protein has been purified from Brachyspira hyodysenteriae broth cultures, studies of tlyA, tlyB, and tlyC to date have focused on their activity when introduced into Escherichia coli via plasmid. These results were cast into doubt several years later upon the discovery that many lab strains of Escherichia coli exhibit a hemolytic phenotype when foreign genes were introduced. To examine the effects of these putative cytolysins the tlyA and tlyC genes were amplified from “ Brachyspira hampsonii” genomic DNA and ligated into expression vectors. The resulting protein products were overexpressed in Escherichia coli and purified by Ni 2+ and amylose affinity chromatography. Cytolytic activity of purified recombinant tlyA and tlyC on a sheep erythrocyte suspension was examined, and no cytolysis greater than a phosphate buffered saline control was observed from tlyA at concentrations ranging from 0 to 88 μg/mL or from tlyC at concentrations ranging from 0–34 μg/mL. At the time of writing, attempts are being made to express and purify the α‐toxin from Staphylococcus aureus utilizing the same methodology in order to rule out inactivation of tlyA and tlyC by the purification process. While it is also possible that higher concentrations of tlyA and tlyC are required for hemolysis or that full activity of these proteins require unidentified cofactors or post‐translational modifications not possible in our current Escherichia coli based expression system, the possibility that tlyA and tlyC have been misannotated as cytolysins cannot be ruled out. Support or Funding Information Natural Sciences and Engineering Research Council of Canada (NSERC) 371364 /2010 to MEL & Alberta Livestock and Meat Association project 2013R054R to JCH, MEL & JEH
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Comment cette classification a été obtenuedéplier
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 | 0,001 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 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 découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».