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

Darren E. Higgins, Nilabh Shastri, Daniel A. Portnoy

Research output: Contribution to journalArticlepeer-review

70 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1631-1641
Number of pages11
JournalMolecular Microbiology
Issue number6
StatePublished - 1999
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology


Dive into the research topics of 'Delivery of protein to the cytosol of macrophages using Escherichia coli K-12'. Together they form a unique fingerprint.

Cite this