Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation.

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.

Exogenous antigen targeted to FcgammaRI on myeloid cells is presented in association with MHC class I.

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.