Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase

Abstract

Genetic editing has revolutionized biotechnology, but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here, we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. The most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118-amino-acid) delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implies that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications, ranging from studying T6SS effectors to genetic editing. IMPORTANCE Delivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here, we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.

Keywords: Aeromonas dhakensis; Cre recombinase; DNA recombination; Pseudomonas aeruginosa; T6SS; Vibrio cholerae; antimicrobial; bactericidal activity; biotechnology; effector; genetic editing; interspecies interactions; protein engineering; protein secretion; type six secretion system.

FIG 1

Active VgrG-Cre fusions are secreted and delivered to recipient cells in a T6SS-dependent manner. (A) Depiction (not to scale) showing fusions of Cre recombinase (yellow) and 3× V5 tag (orange) to VgrG1 and VgrG3 of V. cholerae V52. VgrG1 (gray) wild type includes a proline-rich region (PRR) and an actin cross-linking domain (red; ACD). VgrG3 (blue) wild type includes a lysozyme domain (red) with a linker region leading up to the peptidoglycan binding domain beginning at codon 718. nc, no Cre. (B) Western blot showing secretion of VgrG1 and VgrG3 fusions from wild-type V. cholerae V52 but not an equivalent tssM mutant. Sec (top) shows secreted fractions, and Cell (bottom) shows cell pellet lysates. In addition to α-V5, α-RpoB antibody was included as a cytoplasm control. Representative of three independent replicates. (C) Depiction of the floxed interruption in gentamicin resistance (FIGR) cassette. An ampicillin resistance cassette (purple), located in the reverse direction and flanked by loxP sites (red), interrupts the open reading frame of a gentamicin resistance gene (yellow striped). Exposure to Cre removes the block and allows expression of the gentamicin resistance gene (yellow). Promoters are shown as 90° arrows. Figure is not to scale. ATG, Gent^r start codon. (D) Recovery of V. cholerae Gent^r CFU (indicative of Cre-mediated FIGR cassette recombination) after delivery from wild-type (+) or ΔtssM (Δ) V. cholerae with the indicated Cre fusions to VgrG vectors. DL, detection limit. One-way analysis of variance (ANOVA) with Sidak’s multiple-comparison test comparing wild-type and ΔtssM donors with equivalent delivery fusions. Recovery from ΔtssM samples were not significantly above Cre only or no Cre (nc) controls. **, P < 0.01; ***, P < 0.001.

FIG 2

Improving Cre delivery using PAAR2 fusions, an effectorless donor strain, and removing 3V5 tags. (A) Depiction (not to scale) showing fusions of Cre recombinase (yellow) and 3× V5 tag (orange) to PAAR2 (green) of V. cholerae V52. Fusions include the full-length C-terminal tail (C-Tail) of PAAR2 (FL) or just 12 amino acids of it (C-); 12 amino acids (12aa) are also left over from the VgrG1₇₂₅-Cre_3V5 construct (codons 714 to 725), used as the template for PAAR2 insertion. (B) Recovery of Cre-recombined recipient bacteria (Gent^r CFU) after delivery from wild-type (+) or ΔtssM (Δ) V. cholerae with indicated Cre fusions to PAAR2 vectors. DL, detection limit. One-way ANOVA with Sidak’s multiple-comparison test. Recovery from ΔtssM samples was not significantly above the no-Cre (PAAR2 wild-type) control. ***, P < 0.001. (C) Cre fusion delivery from V. cholerae with catalytically inactivated antibacterial effectors (4eff_C) or ΔtssM (Δ). Data show recombination efficiency (Gent^r/Kan^r CFU recovered; recombined/total recipients) as a percentage. DL, approximate detection limit. One-way ANOVA with Dunnett’s multiple-comparison test comparing each sample to the ΔtssM donor strain. ***, P < 0.001; ns, not significant. (D) Recombination efficiency after delivery from 4eff_C (+) or ΔtssM (Δ) V. cholerae encoding PAAR₁₀₆-Cre with either a 3V5 or 6His tag. DL, approximate detection limit. One-way ANOVA with Tukey’s multiple-comparison test. ***, P < 0.001; ns, not significant. (E) Recombination efficiency after delivery from 4eff_C strain encoding indicated Cre fusions with the 3V5 tag present (+) or removed (−). DL, approximate detection limit. One-way ANOVA with Sidak’s multiple-comparison test comparing samples with and without 3V5 tags. *, P < 0.05; ns, not significant.

FIG 3

Active Cre can be delivered as effector fusions and by A. dhakensis. (A) Depiction (not to scale) showing fusions of Cre recombinase (yellow) and 3× V5 tag (orange) to V. cholerae T6SS effectors (red) or to A. dhakensis PAAR proteins (dark green) and VgrG proteins with (dark blue) or without (dark gray) a flexible linker inserted. (B) Recombination efficiency after delivery from V. cholerae 4eff_C (+) or ΔtssM (Δ) strains encoding Cre fusions to indicated effector proteins. DL, approximate detection limit. One-way ANOVA with Sidak’s multiple-comparison test comparing 4eff_C and ΔtssM donors with equivalent delivery fusions. ***, P < 0.001; ns, not significant. (C) Recombination efficiency after delivery of Cre fusions from A. dhakensis with catalytically inactivated antibacterial effectors (3eff_C) or ΔtssM (Δ). DL, approximate detection limit. One-way ANOVA with Dunnett’s multiple-comparison test comparing each sample to the ΔtssM donor. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 4

Fusion of PAAR2₁₀₆ to the A. dhakensis effector, TseC, empowers V. cholerae to kill P. aeruginosa. (A) Prey P. aeruginosa recovery after incubation with A. dhakensis killer cells. Prey P. aeruginosa has ΔhsiB (ΔtssB) mutations in all three T6SS. Killer strains have T6SS-null mutation (ΔtssM) or a wild-type T6SS with all effectors intact (wt), all antibacterial effectors catalytically inactivated (3eff_C), or a single active effector (indicated), while the other effectors are inactivated. One-way ANOVA with Dunnett’s multiple-comparison test compared all samples to the 3eff_C killer strain. **, P < 0.01; ***, P < 0.001; ns, not significant. (B) Prey V. cholerae (ΔtssM) recovery after incubation with V. cholerae killer cells encoding PAAR2₁₀₆ fusions to A. dhakensis TseC (TseC_Ad) or Cre as a control. Killer strains have ΔtssM (Δ) or a wild-type T6SS with all effectors intact (wt) or all native antibacterial effectors catalytically inactivated (4eff_C). DL, detection limit. One-way ANOVA with Sidak’s multiple-comparison test. ***, P < 0.001; ns, not significant. (C) Prey P. aeruginosa (ΔhsiB mutations in all three T6SS) recovery after incubation with V. cholerae killer cells with no fusion construct (n/a) or encoding PAAR2₁₀₆ fusions to A. dhakensis TseC (TseC_Ad) or Cre as a control. Killer strains have ΔtssM (Δ) or a wild-type T6SS with all effectors intact (wt) or all native antibacterial effectors catalytically inactivated (4eff_C). One-way ANOVA with Sidak’s multiple-comparison test. **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 5

Model of engineered T6SS-mediated delivery of Cre recombinase and TseC_Ad. (A) Engineered T6SSs of V. cholerae or A. dhakensis donor cells deliver active Cre recombinase protein. Delivered Cre instigates recombination of the floxed interruption in the gentamicin resistance (FIGR) cassette in recipient cells. (B) P. aeruginosa exhibits natural resistance (by unknown mechanisms) to all four antibacterial effectors of wild-type V. cholerae. (C) V. cholerae engineered to deliver the A. dhakensis effector, TseC_Ad, gains the ability to kill P. aeruginosa.