title: Delivery of protein to the cytosol of macrophages using Escherichia coli K-12.
abstract: Listeriolysin O (LLO) is an essential determinant of pathogenicity whose natural biological role is to mediate lysis of Listeria monocytogenes containing phagosomes. In this study, we report that Escherichia coli expressing cytoplasmic recombinant LLO can efficiently deliver co-expressed proteins to the cytosol of macrophages. We propose a model in which subsequent or concomitant to phagocytosis the E. coli are killed and degraded within phagosomes causing the release of LLO and target proteins from the bacteria. LLO acts by forming large pores in the phagosomal membrane, thus releasing the target protein into the cytosol. Delivery was shown to be rapid, within minutes after phagocytosis. Using this method, a large enzymatically active protein was delivered to the cytosol. Furthermore, we demonstrated that the E. coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the major histocompatibility (MHC) class I pathway for antigen processing and presentation, greater than 4 logs compared with E. coli expressing OVA alone. Moreover, the time required for processing and presentation of an OVA-derived peptide was similar to that previously reported when purified OVA was introduced directly into the cytosol by other methods. Using this system, potentially 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 delivery of DNA are discussed.
authors: Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
authors: D E
authors: DE
authors: Higgins
authors: N
authors: N
authors: Shastri
authors: D A
authors: DA
authors: Portnoy
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D001427:Bacterial Toxins; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D003600:Cytosol / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005805:Genes, MHC Class I / Q000502:physiology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D010047:Ovalbumin / Q000378:metabolism; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D010047:Ovalbumin; D001616:beta-Galactosidase; C038705:hlyA protein, Listeria monocytogenes