Delivery of protein to the cytosol of macrophages using Escherichia coli K-12

Listeriolysin O (LLO) is an essential determinant of pathogenicity whose na tural biological role is to mediate lysis of Listeria monocytogenes contain ing phagosomes. In this study, we report that Escherichia coli expressing c ytoplasmic recombinant LLO can efficiently deliver co-expressed proteins to the cytosol of macrophages, We propose a model in which subsequent or conc omitant to phagocytosis the E. coli are killed and degraded within phagosom es causing the release of LLO and target proteins from the bacteria. LLO ac ts by forming large pores in the phagosomal membrane, thus releasing the ta rget protein into the cytosol. Delivery was shown to be rapid, within minut es after phagocytosis. Using this method, a large enzymatically active prot ein was delivered to the cytosol, Furthermore, we demonstrated that the E. coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the ma jor histocompatibility (MHC) class I pathway for antigen processing and pre sentation, greater than 4 logs compared with E. coli expressing OVA alone, Moreover, the time required for processing and presentation of an OVA-deriv ed peptide was similar to that previously reported when purified OVA was in troduced directly into the cytosol by other methods. Using this system, pot entially large amounts of any protein that can be expressed in E. coli can be delivered to the cytosol without protein purification. The potential use of this system for the delivery of antigenic protein in vivo and the deliv ery of DNA are discussed.