title: Engineering Escherichia coli into a protein delivery system for mammalian cells.
abstract: Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.
authors: †Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, United States.|‡Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02138, United States.
authors: Analise Z
authors: AZ
authors: Reeves
authors: †Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, United States.
authors: William E
authors: WE
authors: Spears
authors: †Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, United States.|‡Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02138, United States.
authors: Juan
authors: J
authors: Du
authors: §Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, United States.|∥Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, United States.|⊥Joslin Diabetes Center, Boston, Massachusetts 02215, United States.
authors: Kah Yong
authors: KY
authors: Tan
authors: §Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, United States.|∥Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, United States.|⊥Joslin Diabetes Center, Boston, Massachusetts 02215, United States.
authors: Amy J
authors: AJ
authors: Wagers
authors: †Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, United States.|‡Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02138, United States.|∥Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, United States.
authors: Cammie F
authors: CF
authors: Lesser
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008322:Mammals / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D012763:Shigella flexneri / Q000235:genetics; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems
keywords: bacterial engineering; protein delivery; synthetic biology; type 3 secretion system