title: agr expression precedes escape of internalized Staphylococcus aureus from the host endosome.
abstract: Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfp-lux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest that agr induction occurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.
authors.affiliation: School of Biosciences, University of Nottingham, Loughborough, Leicestershire LE12 5RD, United Kingdom.
authors.forename: S N
authors.initials: SN
authors.lastname: Qazi
authors.forename: E
authors.initials: E
authors.lastname: Counil
authors.forename: J
authors.initials: J
authors.lastname: Morrissey
authors.forename: C E
authors.initials: CE
authors.lastname: Rees
authors.forename: A
authors.initials: A
authors.lastname: Cockayne
authors.forename: K
authors.initials: K
authors.lastname: Winzer
authors.forename: W C
authors.initials: WC
authors.lastname: Chan
authors.forename: P
authors.initials: P
authors.lastname: Williams
authors.forename: P J
authors.initials: PJ
authors.lastname: Hill
mesh_terms: D000217:Acyltransferases; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002417:Cattle; D002460:Cell Line; D004705:Endocytosis / Q000276:immunology*; D011992:Endosomes / Q000276:immunology / Q000382:microbiology*; D004847:Epithelial Cells / Q000166:cytology; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D015394:Molecular Structure; D009876:Operon; D010088:Oxidoreductases; D013211:Staphylococcus aureus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*
chemical_list: C095698:Agr protein, Staphylococcus aureus; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; C055198:LuxC protein, Bacteria; D000217:Acyltransferases; C065928:luxD protein, Bacteria