The type III secretion system, or T3SS, is one of the most sophisticated weapons in the bacterial arsenal. It resembles a microscopic syringe, spanning the two bacterial membranes and protruding outward to puncture the membrane of a host cell. Through this conduit, disease-causing bacteria push effector proteins that hijack cellular machinery. In P. aeruginosa, a Gram-negative pathogen responsible for ventilator-associated pneumonia, bloodstream infections and devastating wound infections, the T3SS injects four known exoenzymes: ExoU, a potent phospholipase A2 cytotoxin; ExoS and ExoT, bifunctional Rho GTPase-activating proteins with ADP-ribosyltransferase activity; and ExoY, a promiscuous nucleotidyl cyclase. Together these toxins help make P. aeruginosa one of the six leading pathogens driving antimicrobial resistance-associated mortality, blamed for an estimated 559,000 deaths worldwide each year.

Yet the very machinery that makes the bacterium dangerous also makes it an attractive delivery vehicle. Crucially, proteins targeted for T3SS export require only a short N-terminal signal, roughly the first 50 to 60 amino acids, to be recognized by the secretion apparatus. That molecular simplicity means researchers can fuse this targeting sequence to almost any protein of interest and trick the bacterium into injecting it. Unlike viral vectors, which have earned a growing list of FDA approvals but carry risks of insertional mutagenesis, unregulated transgene expression and anti-vector immune responses, bacterial protein delivery leaves no foreign DNA behind in the host genome. The effect is transient, dose-controllable and potentially reversible, features that have fueled interest in microbial-based cancer therapy since the era of Busch and Coley more than a century ago.

To prove the concept in a living animal, the team, led by Amanda N. Tuckey and Robert W. Sobol, chose the Cre-loxP recombination system as a rigorous readout. Cre recombinase, derived from bacteriophage P1, recognizes 34 base-pair loxP sequences, each composed of two 13 base-pair palindromic repeats flanking an asymmetric eight base-pair core. When Cre finds two loxP sites oriented in the same direction, it excises the intervening DNA. This makes Cre an ideal test cargo: the protein must not only be secreted by the bacteria and translocated across the host cell membrane, but also imported into the nucleus and enzymatically active on chromosomal DNA before any detectable change occurs.

The researchers first validated the readout in human cells. They engineered U2OS osteosarcoma cells to carry a LoxP-DsRed-LoxP-EGFP reporter, originally developed by Jacco van Rheenen, in which cells glow red until Cre excises the DsRed cassette, switching fluorescence to green. Confocal microscopy and flow cytometry confirmed that EGFP appeared only when Cre was delivered, never in empty-vector controls. With the cellular assay established, the team turned to the bacterium itself.

They built an arabinose-inducible expression plasmid, pUCP18-Ara, by cloning the araC regulator and araBAD promoter from E. coli into a shuttle vector that replicates in both E. coli and Pseudomonas. Into this backbone they placed fusion genes linking the first 54 amino acids of each of the four exoenzymes to a nuclear localization sequence and Cre recombinase. Secretion tests, in which culture supernatants were concentrated by trichloroacetic acid precipitation and probed with an anti-Cre antibody, revealed that the ExoU targeting sequence drove the most reliable and abundant secretion of the fusion protein. The ExoU(54)-NLS-Cre strain therefore became the workhorse for the animal experiments.

A key safety consideration shaped the entire design. The team used strain PA103, a well-characterized P. aeruginosa isolate, but specifically an isogenic mutant, PA103ΔUT, lacking functional ExoU and ExoT. This deletion substantially attenuates virulence while preserving the intact T3SS needle. The bacteria were delivered by intratracheal instillation into the airways of Ai14 reporter mice, animals whose chromosomes carry a loxP-flanked transcriptional STOP cassette upstream of the red fluorescent protein tdTomato. Any cell that receives functional Cre in its nucleus will delete the STOP cassette and light up red, providing a permanent, cell-autonomous record of successful protein delivery. A low inoculum of 2 × 10⁴ colony-forming units was chosen to minimize lung pathology, and arabinose induction was maintained by intraperitoneal injection at the time of infection and on the two following days.

Seventy-two hours after infection, the mice were euthanized and their lungs perfused, fixed and cut into 300-micrometer serial sections with a vibratome. Confocal microscopy of five fields per animal revealed a striking difference: mice infected with the empty-vector control strain showed minimal red fluorescence background, while those receiving the ExoU-NLS-Cre bacteria displayed significantly more tdTomato-positive cells throughout the lung slices. Quantification of red fluorescence normalized to DAPI-stained tissue area confirmed the difference was statistically significant, with a two-tailed Welch’s t-test yielding p = 0.035 and a large effect size. Every animal survived, with no weight loss or overt signs of stress, demonstrating that functional protein delivery to the lung cell nucleus is feasible in a living mammal without causing measurable harm.

The long-term ambition is to engineer this platform, sometimes called a Type III Protein Delivery System, to treat lung cancer, which accounts for roughly 14 percent of all new cancers and is projected by the American Cancer Society to claim more than 150,000 lives annually in the United States. Because the T3SS bypasses endosomal degradation and deposits proteins directly into the cytosol, it could deliver tumor suppressors, pro-apoptotic factors or synthetic lethal enzymes to cancer cells while sidestepping the genotoxic risks of gene transfer. Related systems built in Yersinia enterocolitica and Salmonella enterica have already been explored for anticancer payloads, and the Pseudomonas T3SS has previously been used in cell culture to inject transcription factors for cellular reprogramming and even antigens for SARS-CoV-2 vaccine development.

Significant engineering challenges remain before the platform approaches the clinic. Although PA103ΔUT is attenuated, the researchers found that residual cytotoxicity, likely from Exotoxin A, the Type II-secreted ADP-ribosyltransferase that inactivates host elongation factor 2, caused cultured lung cells to round up and detach within 48 hours of exposure, even though the effect was not apparent in the intact mouse. Future strains will need additional mutations, such as a disrupted toxA gene, and possibly auxotrophies that force the bacteria to die outside the controlled infection setting, ensuring biocontainment without relying on antibiotics. The team also acknowledges that Cre itself can cause off-target genomic effects, and plans to compare additional T3SS targeting sequences in animal models to maximize efficiency and specificity. Still, the demonstration that a disarmed pathogen can rewrite the genome of lung cells purely through protein injection marks a compelling proof of principle for protein-based therapeutics that never touch DNA.

Subject of Research: Type III secretion system-mediated protein delivery causing genomic recombination in murine lungs

Article Title: Exploiting the bacterial type III secretion system of Pseudomonas aeruginosa to impact genomic recombination in murine lungs

Article References: N.Tuckey, A., Clark, J., Q.Al-Rahahleh, R., H.Siddiqui, A., A.DeFreitas, S., P.Roos, W., T.Lin, M., Audia, J. P., & W.Sobol, R. (2026). Exploiting the bacterial type III secretion system of Pseudomonas aeruginosa to impact genomic recombination in murine lungs. Current Research in Biotechnology, Article 100420. https://doi.org/10.1016/j.crbiot.2026.100420

Image Credits: AI Generated

DOI: 10.1016/j.crbiot.2026.100420

Keywords: Pseudomonas aeruginosa, type III secretion system, Cre-loxP recombination, protein delivery, bacterial therapeutics, lung cancer, tdTomato reporter mice, ExoU, microbial cancer therapy, gene recombination, PA103ΔUT, in vivo delivery

Cell engineering has become one of the most promising frontiers in biomedicine, underpinning therapies ranging from CAR-T cancer treatments to engineered probiotics. Yet the dominant tool for endowing cells with new abilities—genetic manipulation—carries inherent constraints. Editing multiple genes simultaneously is technically complex, and the tools that work in one species often fail to translate to another. As a result, building cells with sophisticated, multi-part functionalities through genetic means remains slow, costly and sometimes infeasible.

The cell surface offers a compelling alternative target. Because it mediates virtually every interaction a cell has with its environment, the surface is where therapeutic behavior can be most directly tuned. Surface biomolecules present a rich array of functional groups—amines, thiols and other reactive chemistry—that serve as natural anchoring points for chemical modification. Wang and Liu exploited this chemical accessibility by harnessing dopamine, a small molecule that self-polymerizes under mild, alkaline conditions into polydopamine, a adhesive layer first described in a landmark 2007 Science paper on mussel-inspired surface chemistry.

The beauty of the method lies in its simplicity and universality. When dopamine is added to a suspension of living bacteria under the right conditions, it polymerizes directly on the cell surface, forming a thin reactive coating that can simultaneously capture a second component of choice. In the protocol’s first procedure, that component is polyethylene glycol, or PEG, a hydrophilic polymer long known to help nanoparticles slip through mucus. The result is a PEGylated bacterium capable of penetrating the intestinal mucus layer and reinforcing the mucosal barrier—a potential preventive strategy against colitis. Excluding bacterial culture, preparing these mucus-penetrating bacteria takes roughly three hours.

The second procedure goes a step further with dual-functionalization. Here, polydopamine serves as the bridge between two distinct bioactive molecules on a single bacterium: an anti-PD1 antibody, a celebrated immune checkpoint inhibitor, and the S1 subunit of the SARS-CoV-2 spike protein, a viral antigen. The resulting synergy-immunoactivation bacteria are designed to simultaneously provoke anticancer immunity and antiviral immunity, offering a two-in-one platform for treating tumors while guarding against infection. This dual-functionalization procedure requires only about one hour of hands-on preparation time beyond bacterial culture.

Compared with conventional genetic manipulation and existing physicochemical surface modification techniques, the protocol’s versatility stands out. The polydopamine intermediate accepts both natural biological macromolecules, such as proteins and antibodies, and synthetic materials, and it tolerates the attachment of multiple diverse components in sequence. Because the chemistry does not depend on species-specific genetic machinery, it can in principle be applied to a wide range of cell types, from probiotic Escherichia coli Nissle 1917 to potentially other living cells, making it a genuinely cross-species platform.

The scientific foundations of the work draw on more than a decade of polydopamine research. Catecholic chemistry, first systematized for surface modification by Messersmith, Lee and colleagues, has been applied to countless materials, but extending it to living cells required careful optimization to ensure the polymerization conditions do not compromise cell viability. The Wang and Liu groups had previously demonstrated polymerization-mediated multifunctionalization of living cells in Advanced Materials in 2021, and their primary research papers—published in Nature Biomedical Engineering in 2024 on mucus-penetrating PEGylated bacteria and in Advanced Materials in 2023 on hybrid immunoactive nanosurface bacteria—provide the proof-of-concept data that underpin this protocol.

The therapeutic implications are substantial. For inflammatory bowel disease, the mucus-penetrating bacteria represent a novel way to shore up a failing intestinal barrier rather than simply delivering drugs. For oncology, bacteria coated with checkpoint inhibitors concentrate immunotherapy at tumor sites, potentially reducing the systemic toxicity associated with injected antibodies while leveraging the natural tumor-homing behavior of certain bacterial strains. And because the coating chemistry is modular, the same scaffold could in principle carry different antigen–antibody pairs to address other cancers or emerging viral threats.

The authors also confront the practical challenges facing any live biotherapeutic. Regulatory frameworks for live biotherapeutic products, as outlined by the US Food and Drug Administration, demand rigorous characterization of manufacturing and control, and a surface-engineered bacterium must demonstrate stability, safety and reproducibility at every step. The protocol addresses these concerns by providing detailed characterization procedures, including assessments of coating thickness, surface composition, bacterial viability, mucus penetration, and the immunogenicity and intratumoral distribution of the dual-functionalized bacteria. Jinyao Liu has additionally filed a patent related to the technology, signaling commercial interest alongside the academic contribution.

By transforming cell surface engineering from a specialized genetic exercise into an accessible chemical operation, the protocol lowers the barrier to entry for laboratories seeking to develop next-generation living therapeutics. The authors anticipate that the platform will offer valuable guidance for engineering living cells with designable, tailorable functionalities for innovative cell-based therapy. If the approach translates from the bench to the clinic as hoped, the humble chemistry of mussel glue may soon help dress living cells for battle against some of medicine’s most stubborn diseases.

Subject of Research: Dopamine polymerization-mediated chemical surface functionalization of living cells for cell-based therapy

Article Title: Dopamine polymerization-mediated surface functionalization of living cells for advanced therapeutic applications

Article References: Dopamine polymerization-mediated surface functionalization of living cells for advanced therapeutic applications. (n.d.). https://doi.org/10.1038/s41596-026-01422-1

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01422-1

Keywords: dopamine polymerization, polydopamine, surface functionalization, living cells, bacterial therapeutics, mucus-penetrating bacteria, PEGylation, immune checkpoint inhibitor, anticancer immunity, antiviral immunity, cell engineering, colitis