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Staphylococcus aureus colonizes the lungs of cystic fibrosis patients and treatment with antibiotics usually results in recurrent and relapsing infections. We have shown that S. aureus can invade and replicate within a cystic fibrosis epithelial cell line (CFT-1), and that these internalized bacteria subsequently escape from the endocytic vesicle. The accessory gene regulator, agr, in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence. Here we show that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Trafficking analysis of live S. aureus which did not express alpha-haemolysin, a specific agr regulated toxin, revealed a defect in vesicular escape that was undistinguishable from the trafficking defect exhibited by the agr mutant. Furthermore, overexpression of alpha-haemolysin under an inducible promoter in an agr mutant of S. aureus partially restored the phagosome-escaping phenotype of an agr mutant. These results demonstrate that the expression of agr is required for vesicular escape, and that biologically active alpha-haemolysin is required for S. aureus escape from the endocytic vesicle into the cytosol of CFT-1 cells.

Staphylococcus aureus is frequently the initial bacterium isolated from young cystic fibrosis (CF) patients, and yet its role in CF disease progression has not been determined. Recent data from our lab demonstrates that S. aureus can invade and replicate within the CF tracheal epithelial cell line (CFT-1). Here we describe the finding that the fate of internalized S. aureus in CFT-1 cells differs from its complemented counterpart (LCFSN). S. aureus strain RN6390 was able to replicate within the mutant CFT-1 cells after invasion but not in the complemented LCFSN cells. At 1 h postinvasion, S. aureus containing vesicles within both cell lines acquired vacuolar-ATPase, lysosomal markers LAMP 1 and 2, and the lysomotrophic dye LysoTracker to a similar degree. However, at 4 h postinvasion, the percentage of S. aureus within CFT-1 cells associated with these markers decreased significantly compared to LCFSN, where the association approached 100%. Transmission electron microscopic analysis revealed that the majority of bacteria within CFT-1 cells were free in the cytosol at 4 h after invasion, whereas most S. aureus bacteria internalized by LCFSN cells remained within vesicles. These results demonstrate a fundamental difference in the fate of live S. aureus after invasion of CFT-1 versus LCFSN cell lines and may explain the propensity of S. aureus to cause chronic lung infection in CF patients.

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible P(BAD) promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp(uvr) fusion carried on a shuttle plasmid, we demonstrated that dose-dependent tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

Vaccine therapy is attractive for prostate cancer patients because the tumor is slow growing (allowing time to augment host responses) and occurs in an older population less likely to tolerate more toxic treatments. We have constructed an expression vector based on a monoclonal antibody (mAb) that targets the high affinity receptor for IgG (FcgammaRI, CD64) which is exclusively expressed on myeloid cells including dendritic cells (DC). The heavy chain of mAb H22 CH2 and CH3 domains were removed and replaced with the gene for prostate specific antigen (PSA). Using that vector, we have constructed and purified FPH22.PSA, a fusion protein that targets PSA to FcgammaRI on antigen presenting cells (APC). This fusion protein has an apparent molecular mass of 80-83 kDa, binds to FcgammaRI with high affinity and expresses PSA. We demonstrate that FPH22.PSA targeted PSA was internalized and processed by the human myeloid THP-1 cell line resulting in presentation of MHC class I-associated PSA peptides and lysis of THP-1 by PSA-specific human CTL. Moreover, pretreatment of THP-1 cells with antibodies to block either FcgammaRI or MHC class I, blocked lysis indicating that targeting to FcgammaRI results in presentation of exogenous antigen on MHC class I molecules. These data demonstrate that FPH22.PSA was processed in such a manner by the myeloid cell line to allow for presentation of immunodominant peptides in MHC class I molecules and suggests that uptake of antigen via FcgammaRI results in cross-priming.