Cyborg Bacteria as Living Organelles for Mammalian Cells Augmentation

Introduction: : Mitochondrial dysfunction underlies a spectrum of debilitating disorders, from inherited mutations and age-related decline to progressive neurodegeneration and cancer-driven metabolic hijacking, yet effective strategies for restoring lost organelle function remain elusive. Synthetic-biology efforts have attempted de novo organelle replacement with protein cages, biomolecular condensates, and other artificial compartments, but these constructs cannot match the intricate metabolism, signaling, and adaptability of healthy mitochondria. To bridge this gap, we introduce Cyborg Bacterial Cells: metabolically active, replication-incompetent Salmonella engineered to operate as living synthetic organelles inside mammalian hosts. Rendered non-proliferative through intracellular hydrogelation, these Cyborg Cells invade cells without cytotoxicity and carry organelle-level roles, maintaining cellular functions during chemical uncoupling, sensing extracellular signals through programmable gene circuits, and executing localized enzyme therapies. By uniting the systems-level sophistication of living microbes with the safety and programmability of engineered intracellular hydrogels, Cyborg Bacteria establish a new paradigm for organelle replacement therapy and broader intracellular disease intervention.

Materials and

Methods: : Cyborg E. coli and Salmonella were generated by permeating bacterial cells with PEG-based monomers followed by UV-induced intracellular crosslinking to arrest proliferation while preserving metabolic activity. HEK293FT and SKBR-3 cells were co-incubated with Cyborg Cells at multiplicities of infection (MOI) ranging from 1×10⁶ to 1×10⁸. Confocal microscopy confirmed intracellular invasion, and 1×10⁸ MOI per 1×10⁵ mammalian cells was identified as optimal for high invasion without cytotoxicity. Viability assays confirmed host cell survival post-invasion. For therapeutic functionality, Cyborg Cells were engineered to express cytosine deaminase on their outer membrane, and prodrug activation (5-FC to 5-FU) was assessed via imaging-based viability assays. Programmable functionality was tested using small molecule-inducible circuits with fluorescence reporters, evaluated through microscopy and plate reader assays. Protection of host mitochondria was tested by FCCP-induced uncoupling followed by TMRE staining to assess mitochondrial membrane potential. Differential gene expression from RNA sequencing provided insight into underlying host-Cyborg interactions.

Results, Conclusions, and Discussions:: We demonstrated Cyborg Bacterial Cells as a novel class of synthetic organelles that are metabolically active yet replication-incompetent, capable of persistent, non-cytotoxic integration into mammalian host cells. Following intracellular hydrogelation, Cyborg Salmonella retained viability and biosynthetic function while losing the ability to divide, for >3 weeks. Upon co-incubation with HEK293FT and SKBR-3 cells, these engineered bacteria efficiently invaded host cytoplasm at an optimized MOI of 1×10⁸ per 1×10⁵ cells, as confirmed by confocal microscopy, without compromising host cell viability. We demonstrated three core functionalities of Cyborg Bacterial Organelles. First, as therapeutic biofactories, Cyborg Bacterial Organelles expressing surface-anchored cytosine deaminase converted the prodrug 5-fluorocytosine into cytotoxic 5-fluorouracil, reducing host cell viability approximately sixfold relative to untreated controls. This result confirms the feasibility of spatially confined, cell-internalized enzyme-based therapy. Second, we established that Cyborg Cyborg Bacterial Organelles could operate as intracellular biosensors. Engineered with small molecule-inducible gene circuits, they retained responsiveness to seven distinct extracellular cues from inside mammalian cells, enabling real-time sensing and actuation within the intracellular space. Lastly, we investigated the role of Cyborg Bacterial Organelles in rescuing mitochondrial function under chemical stress. Specifically, across FCCP titration (0.032–100 µM), the ΔTMRE curve, defined as TMRECyborg invaded – TMREnon-invaded stayed positive. The percent protection rose from 50–80 % at sub-micromolar doses to 196 % at 10 µM and peaked at 433 % at 100 µM. This mitochondrial protection effect followed a sigmoidal trend, indicating robust functional crosstalk between Cyborg Bacterial Organelles and host metabolism. RNA sequencing revealed upregulation of genes involved in ion transport, membrane remodeling, and glucuronate metabolism, suggesting the protective effects may result from metabolic buffering or membrane stabilization rather than direct transcriptional repair. Overall, this work positions Cyborg Bacterial Organelles as a multifunctional platform for intra-mammalian cell augmentation and as living therapeutics that blend the cross-taxa complexity with the precision of engineered control. Their ability to persist, sense, and perform localized therapeutic functions while avoiding replication makes them ideal candidates for organelle replacement therapy, cancer treatment, and future synthetic endosymbiosis-based biomedical strategies.

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