title: Bacterial delivery of nuclear proteins into pluripotent and differentiated cells.
abstract: Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming.
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Candace
authors.initials: C
authors.lastname: Bichsel
authors.forename: Dennis K
authors.initials: DK
authors.lastname: Neeld
authors.forename: Takashi
authors.initials: T
authors.lastname: Hamazaki
authors.forename: Donghai
authors.initials: D
authors.lastname: Wu
authors.forename: Lung-Ji
authors.initials: LJ
authors.lastname: Chang
authors.forename: Lijun
authors.initials: L
authors.lastname: Yang
authors.forename: Naohiro
authors.initials: N
authors.lastname: Terada
authors.forename: Shouguang
authors.initials: S
authors.lastname: Jin
mesh_terms: D036002:ADP Ribose Transferases / Q000008:administration & dosage / Q000493:pharmacokinetics; D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D001427:Bacterial Toxins / Q000008:administration & dosage / Q000493:pharmacokinetics; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D016503:Drug Delivery Systems / Q000379:methods*; D006801:Humans; D019426:Integrases; D051379:Mice; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000008:administration & dosage* / Q000493:pharmacokinetics; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000008:administration & dosage* / Q000493:pharmacokinetics
chemical_list: D058947:Bacterial Secretion Systems; D001427:Bacterial Toxins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D014157:Transcription Factors; D036002:ADP Ribose Transferases; C444820:exoenzyme S; C045073:Cre recombinase; D019426:Integrases