Engineering Cyborg Pathogens through Intracellular Hydrogelation

Synthetic biology primarily focuses on two kinds of cell chassis: living cells and nonliving systems. Living cells are autoreplicating systems that have active metabolism. Nonliving systems, including artificial cells and nanoparticles, are nonreplicating systems typically lacking active metabolism. In recent work, Cyborg bacteria that are nonreplicating-but-metabolically active have been engineered through intracellular hydrogelation. Intracellular hydrogelation is conducted by infusing gel monomers and photoactivators into cells, followed by the activation of polymerization of the gel monomers inside the cells. However, the previous work investigated only <i>Escherichia coli</i> cells. Extending the Cyborg-Cell method to pathogenic bacteria could enable the exploitation of their pathogenic properties in biomedical applications. Here, we focus on different strains of <i>Pseudomonas aeruginosa</i>, <i>Staphylococcus aureus</i>, and <i>Klebsiella pneumoniae</i>. To synthesize the Cyborg pathogens, we first reveal the impact of different hydrogel concentrations on the metabolism, replication, and intracellular gelation of Cyborg pathogens. Next, we demonstrate that the Cyborg pathogens are taken up by macrophages in a similar magnitude as wild-type pathogens through confocal microscopy and real-time PCR. Finally, we show that the macrophage that takes up the Cyborg pathogen exhibits a similar phenotypic response to the wild-type pathogen. Our work generalizes the intracellular hydrogelation approach from lab strains of <i>E. coli</i> to bacterial pathogens. The new Cyborg pathogens could be applied in biomedical applications ranging from drug delivery to immunotherapy.