agr Expression precedes escape of internalized Staphylococcus aureus from the host endosome

Staphylococcus aureus is a versatile pathogen capable of causing life-threa tening infections. Many of its cell wall and exoproduct virulence determina nts are controlled via the accessory gene regulator (agr). Although conside red primarily as an extracellular pathogen, it is now recognized that S. au reus can be internalized by epithelial and endothelial cells. Traditional e xperimental approaches to investigate bacterial internalization are extreme ly time-consuming and notoriously irreproducible. We present here a new rep orter gene method to assess intracellular growth of S. aureus in MAC-T cell s that utilizes a gfp-luxABCDE, reporter operon under the control of the Ba cillus megaterium xylA promoter, which in S. aureus is expressed in a growt h-dependent manner. This facilitates assessment of the growth of internaliz ed bacteria in a nondestructive assay. The dual gfp-lux reporter cassette w as also evaluated as a reporter of agr expression and used to monitor the t emporal induction of agr during the MAC-T internalization process. The data obtained suggest that agr induction occurs prior to endosomal lysis and th at agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.