An unnatural amino acid dependent, conditional *Pseudomonas* vaccine prevents bacterial infection

The study addresses the challenge of developing safe and effective live bacterial vaccines that avoid reversion or escape to pathogenic forms. Live-attenuated vaccines can induce robust immunity but carry risks, particularly in immunocompromised individuals, due to potential reversion and bacterial adaptability. An alternative is to engineer auxotrophic bacteria whose replication depends on a metabolite absent from the host, ideally a non-natural molecule, thereby minimizing genetic changes relative to the wild-type pathogen and reducing escape risk. Unnatural amino acids (UAAs) enable creation of stringent auxotrophies by site-specific incorporation into essential proteins via orthogonal aminoacyl-tRNA synthetase/tRNA pairs, but prior strategies using multiple nonsense codons reduced protein expression and growth. Building on earlier work that created a BzF-dependent dimer interface in the essential DNA replication protein DnaN (β-sliding clamp) in E. coli with very low escape frequencies, the current study aims to extend this approach to the human pathogen Pseudomonas aeruginosa. The purpose is to generate a live, conditional vaccine strain that is strictly dependent on the UAA p-benzoyl-L-phenylalanine (BzF) for replication, ensuring an excellent safety profile while eliciting protective immunity, and to establish a generalizable platform for other bacterial pathogens given the conservation of DnaN.

The paper situates its work within prior vaccine strategies and genetic biocontainment literature. Live-attenuated bacterial vaccines (e.g., for cholera, tuberculosis, typhoid) are effective but pose safety risks due to escape. Traditional attenuation targets virulence/cytotoxicity genes but can be overcome by bacterial evolution. UAA-based auxotrophies have been explored by introducing orthogonal aaRS/tRNA pairs to incorporate UAAs at nonsense codons in essential genes. Early approaches used multiple nonsense codons across one or several essential genes to suppress escape, but this compromised protein function and growth. The authors’ previous research engineered UAA-dependent active sites, metal coordination sites, and orthogonal dimer interfaces in essential proteins, highlighting the advantage of targeting non-metabolic core life processes. Notably, they evolved the E. coli DnaN homodimer interface to oligomerize only in the presence of BzF, yielding an auxotroph with exceptionally low escape frequencies (<2×10⁻¹⁰). Given DnaN’s conservation and the clinical burden of multidrug-resistant P. aeruginosa (a leading cause of mortality in cystic fibrosis and significant nosocomial pathogen), this strategy is proposed as a promising vaccination platform.

• UAA incorporation system in P. aeruginosa: Tested BzF incorporation using orthogonal pairs. A pyrrolysyl-based MbPylRS/tRNA pair (codon-optimized and non-optimized) failed to suppress an amber codon in a Prolyl-tRNA synthetase (ProS) reporter at site Y174X, even with dipeptide Ala-BzF. A Methanocaldococcus jannaschii TyrRS/tRNA variant evolved for BzF successfully suppressed amber in P. aeruginosa, confirmed by western blot.
• Strain engineering: Due to DnaN conservation (56% identity between E. coli and P. aeruginosa; similar dimer interface), mutations from previously evolved E. coli DnaN (M3, M5, M8) were overlaid onto P. aeruginosa DnaN. A temperature-sensitive (TS) complement system was created: wild-type dnaN expressed from a TS-origin plasmid allowed deletion of genomic dnaN in PAO1, yielding a TS ΔdnaN strain (validated by PCR and temperature-dependent growth). P. aeruginosa variants carrying E. coli-derived mutations did not complement growth at 42 °C.
• Directed evolution via E. coli two-hybrid: An adenylate cyclase-based bacterial two-hybrid in E. coli BTH101 was used to evolve BzF-dependent P. aeruginosa DnaN dimerization. Libraries: (1) NNK saturation at position I273 on Pa DnaN-T18 (adjacent to BzF274 helix end); (2) 218 bp error-prone library spanning the BzF pocket on T25-Pa DnaN (homologous to Pa M3 variant). Libraries were transformed into BTH101 bearing pUltra-BzFRS (MjTyrRS/tRNA), selected on maltose minimal media ±1 mM BzF and IPTG. Twenty-six BzF-dependent clones formed four sequence clusters (e.g., 1273P; others with P65S/Q95H, L111I/E120V, K80BzF/N114S). Dimerization was assessed by UV-induced photocrosslinking of BzF-containing DnaN variants on western blots.
• Vaccine plasmid optimization and construction: To enhance expression/dimerization, combinations of strong promoters (psbA, tac, 14g, 77) and origins (mSF, RSF1010) were screened; tac promoter with RSF1010 ori gave maximal expression and crosslinking. The final plasmid, dnaN.L274BzF.M2, encoded Pa DnaN M2 with mutations V70A, I78F, L97I, F106V, L108G, I273P, and L274BzF, plus the Mj BzFRS/tRNA pair. This plasmid complemented the TS ΔdnaN strain at non-permissive temperature; curing of the TS complement plasmid was confirmed. The resulting BzF-dependent auxotrophic strain is termed Pa Vaccine.
• Growth and escape assays: Pa Vaccine growth was measured at 37 °C in LB with varying BzF concentrations (0–1 mM) using a plate reader. Washout experiments evaluated residual replication after removing BzF. Escape frequency was assayed by plating 10⁷ cells on LB agar without BzF (7-day incubation) and CFU quantitation from serial dilutions on BzF-containing plates. Functional indispensability of BzF at position 274 was tested by replacing L274BzF with each canonical amino acid and assessing growth in the ΔdnaN background at 42 °C.
• Mouse vaccination and challenge: Female BALB/c mice (5–6 weeks) were immunized intranasally (IN) or intraperitoneally (IP) with 6×10⁷ CFU of Pa Vaccine (grown with 1 mM BzF, washed) or formalin-inactivated PAO1 (FI PAO1) on days 0, 14, 21, and 28. Safety assessed via dose-escalation; body weight monitored. IgG and IgA responses were measured by whole-cell ELISA longitudinally (days 0, 14, 21, 28, 35, 42) against PAO1 and PA14; cross-reactivity tested at day 42 against a panel of pathogenic P. aeruginosa strains spanning multiple O-antigen serogroups. On day 43, lethal challenges were performed: IN groups received 8×10⁶ CFU PA14 IN; IP groups received 3×10⁷ CFU PA14 IP. Survival monitored for 7 days. Statistics used Welch’s two-tailed t-tests; doubling times obtained from exponential growth fits.

• Successful creation of a strictly UAA-dependent P. aeruginosa vaccine strain (Pa Vaccine) by engineering BzF-dependent dimerization of the essential replication factor DnaN at position L274.
• Auxotrophic containment: Undetectable escape in plate assays. Plating 10⁷ cells without BzF showed no colonies after 7 days, implying an escape frequency below the detection limit (reported as <10⁻⁶ in the assay; abstract notes <10⁻¹¹ overall). All 20 canonical amino acid substitutions at position 274 failed to complement growth, confirming BzF essentiality for function.
• Growth characteristics: In LB with 1 mM BzF at 37 °C, Pa Vaccine doubled in ~100 min versus 35 min for wild-type PAO1. Minimal growth at 0.125 mM BzF and no growth without BzF. After BzF removal, Pa Vaccine completed approximately 2.6 additional replication cycles before arrest, consistent with depletion of intracellular BzF.
• Safety: No deaths up to 6×10⁸ CFU for Pa Vaccine when administered IP; at 2×10⁹ CFU, deaths were attributed to strong immune reactions (e.g., LPS), not productive infection. FI PAO1 showed no deaths up to 2×10⁹ CFU.
• Immunogenicity: Both Pa Vaccine and FI PAO1 elicited robust anti-PAO1 serum IgG via both IN and IP routes. Notably, IN Pa Vaccine induced significantly higher IgG titers against heterologous pathogenic PA14 than FI PAO1, evident after a single boost and sustained to day 42. IN vaccination produced a transient increase in anti-PA14 IgA after the second dose; IP did not.
• Protection: Complete protection against lethal PA14 challenge in both IN and IP models; all vaccinated mice (Pa Vaccine or FI PAO1) survived (N=8 per group) following matched-route challenges.
• Cross-reactivity: Day 42 sera from Pa Vaccine-immunized mice exhibited similar or higher IgG titers across a panel of pathogenic P. aeruginosa strains from multiple O-antigen serogroups (e.g., O2/5, O6, O11, O12, O19) compared to FI PAO1. IN Pa Vaccine often yielded higher titers than IP, suggesting superior mucosal immunization benefits.

The work demonstrates that a highly conserved essential protein, DnaN, can be engineered to be strictly dependent on a non-natural amino acid (BzF) for function in P. aeruginosa, achieving stringent biocontainment and maintaining strong vaccine efficacy. The E. coli two-hybrid platform, leveraging structural homology, enabled rapid directed evolution of a BzF-dependent dimer interface in the P. aeruginosa DnaN, overcoming transformation limitations in P. aeruginosa. Pa Vaccine’s undetectable escape frequency and inability to revert via canonical substitutions at position 274 underscore stringent auxotrophy. As a live conditional vaccine, Pa Vaccine elicited robust systemic and mucosal immune responses; notably, IN administration produced higher cross-reactive IgG titers against heterologous strains (e.g., PA14) than formalin-inactivated bacteria, and both vaccination routes provided full protection from lethal challenge. The authors attribute enhanced mucosal responses and cross-reactivity to transient intrahost replication (2–3 divisions) before BzF depletion, preserving intact, immunogenic surface structures (e.g., LPS O-antigen) compared to chemically fixed cells. The platform is generalizable to other pathogens due to DnaN conservation. For clinical translation, genomic integration of the engineered dnaN and the BzF aaRS/tRNA system, removal of antibiotic markers, and vigilant monitoring for potential reversion mechanisms (e.g., horizontal gene transfer of functional dnaN) are emphasized, especially in immunocompromised populations.

The study establishes a generalizable strategy to create a live, conditional P. aeruginosa vaccine strain (Pa Vaccine) that is strictly dependent on the unnatural amino acid BzF, ensuring stringent biocontainment and excellent safety. Pa Vaccine induces robust and cross-reactive immune responses via intranasal or intraperitoneal routes and confers complete protection against lethal PA14 challenge in mice. The approach, grounded in engineering a UAA-dependent interface within an essential replication protein (DnaN), provides a platform for developing safe, effective live vaccines against diverse bacterial pathogens. Future work should focus on genomic integration of the engineered components to eliminate plasmid antibiotic markers, optimization of expression for clinical use, expanded evaluation across serogroups and clinical isolates, and rigorous surveillance for potential escape via horizontal gene transfer or other mechanisms.

• Reported escape frequency is assay-limited; while no escape was detected after plating 10⁷ cells without BzF (implying <10⁻⁷), the abstract cites <10⁻¹¹, and the discussion notes <10⁻⁷; more sensitive, high-throughput assays could refine the true escape rate.
• Plasmid-based expression with antibiotic resistance markers is not directly suitable for clinical use; genomic integration and marker removal are required.
• Potential for reversion/escape via horizontal gene transfer of a functional dnaN remains a concern, particularly in immunocompromised hosts.
• Growth of the vaccine strain is slower than wild-type (doubling time ~100 min vs 35 min), which may affect antigen load kinetics.
• Some experiments were performed once unless otherwise indicated, which may limit reproducibility and statistical power for certain findings.