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.

Figure 1. Protein delivery by type III secretion system of P. aeruginosa.

(A) Comparison of ExoS secretion by standard laboratory strains of PAO1, PAK and a hyper secreting strain PAK-J. Strains were grown under type III secretion inducing conditions and culture supernatants were subjected to Western blot analysis using anti-ExoS antibody which recognizes both ExoS and ExoT. (B) Immunohistochemistry of MEF cells following infection by PAK-J (60 min at MOI 20). Cells were stained with anti-ExoS antibody followed by FITC labeled secondary antibody; (i) visualization of ExoS; (ii) visualization of nuclei with Propidium Iodine stain; (iii) compilation of (i) & (ii). (C) Comparison of MEF viability after 3 hour infection with PAK-J or PAK-JΔSTY at indicated MOIs.

Figure 2. Injection of indicated ExoS-Cre fusions.

(A) Cre recombinase was fused in frame with various N-terminal portions of ExoS, with a nuclear localization sequence (NLS) in the fusion junction. (B) Detection of the five ExS54-Cre fusion proteins in PAK- JΔSTY harboring the corresponding fusion constructs. Bacterial cells were grown under type III inducing condition and cell lysates were subjected to Western blot using anti-Cre antibody. (C) TE26 cells were infected with PAK-JΔSTY containing the aforementioned constructs, lysed, and subject to Western Blot with anti-Cre antibody. N, no infection control; V, vector control, PAK-JΔSTY/pUCP20; P, T3SS mutant control, PAK-JΔpopD/pExoS54-Cre; Lanes1-5 are Cre fusions to ExoS17, ExoS54, ExoS96, ExoS234 and ExoS full length, respectively.

Figure 3. Assessment of Cre recombinase activity.

Cre function was assessed by LacZ positive TE26 cells which contain a floxed terminator preventing lacZ expression (A). TE26 cells were infected for various times and MOIs and subsequently stained with X-gal for β-galactosidase (B) to determine the optimal infection conditions as indicated by percentage LacZ positive cells (C). TE26 cells were infected at an MOI of 50 for 3 hours before or after cell cycle synchronization and X-gal stained for β-galactosidase activity (D).

Figure 4. Protein injection into mouse embryonic stem cells (mESC).

(A) mESC were infected with PAK-J strains at a MOI of 50 for 2.5 hours, lysed and assayed for injected ExoS-Flag by anti-Flag Western Blot. (B) mESC were infected with PAK-JΔSTY(pExoS-Flag) for 2.5 hours and subsequently fixed and immunostained with anti-Flag to illuminate translocated ExoS-Flag protein. (C) R26R-EYFP mESC cells contain a floxed terminator preventing downstream EYFP expression. (D) R26R-EYFP were infected with PAK-JΔSTY(pExoS54-Cre) at various MOIs for 2.5 hours. Cells were collected for FACS analysis 48 hours post infection. (E) EYFP-positive mESC were plated after infection to assess EYFP expression by confocal fluorescence microscopy.

Figure 5. ExoS54-Cre mediated excision of an iPSC nuclear reprogramming cassette.

TNGim05 iPSC contain a floxed reprogramming cassette (A). ExoS54-Cre mediated recombination results in loss of mOrange expression, which can be observed by fluorescence confocal microscopy (B) and flow cytometry (C). Reprogramming cassette excision is confirmed by PCR analysis (D). Lanes 1 and 3, IRES primer set, lanes 2 and 4, CAG primer set.