Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Détails

ID Serval
serval:BIB_034F966F2E0E
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.
Périodique
Infection and Immunity
Auteur(s)
Que Y.A., Haefliger J.A., Francioli P., Moreillon P.
ISSN
0019-9567 (Print)
ISSN-L
0019-9567
Statut éditorial
Publié
Date de publication
2000
Volume
68
Numéro
6
Pages
3516-3522
Langue
anglais
Résumé
Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.
Mots-clé
Bacterial Adhesion, Coagulase/biosynthesis, Coagulase/genetics, Fibrinogen, Genes, Bacterial, Genetic Vectors, Lactococcus lactis/genetics, Recombinant Proteins/biosynthesis, Staphylococcus aureus/enzymology, Staphylococcus aureus/genetics
Pubmed
Web of science
Open Access
Oui
Création de la notice
07/04/2008 7:46
Dernière modification de la notice
20/08/2019 12:25
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