Design Principles of Phase-Separated Compartmentalization for Engineering Enzymatic Reactions in a Living Cell

Shiga toxin-producing E. coli (STEC) is a leading cause of foodborne illnesses that currently lacks treatment options. We devised a novel therapeutic approach, MAGICaST, for in situ genetic manipulation of STEC that reduces virulence and promotes clearance. MAGICaST utilizes a donor strain to deliver a self-transmissible vector carrying a CRISPR transposon system for precise integration of genetic payloads into target species. Using a mouse model infected with human STEC strain ATCC 43895, we assessed whether disrupting shiga toxin genes, Stx1b or Stx2b, with integration of a non-coding payload improved host outcomes. Mice administered with wildtype or Stx1b-integrated STEC died within five days. However, mice who received Stx2b-integrated STEC survived and were asymptomatic over 20 days. Consequently, Stx2b was chosen as the target for testing MAGICaST’s ability to inactivate virulence in situ. Three days after administration of the MAGICaST donor, the vector infiltrated 90% of the pathogenic population and inactivated 80% of the virulent Stx2b gene, although survival remained unchanged. To promote survival, we used MAGICaST to deliver a genetic payload expressing a secreted TD4 nanobody that inhibits epithelial adhesion by binding the translocated intimin receptor. With integration of the TD4 nanobody into Stx2b, survival doubled to ten days. Our results show that MAGICaST is an effective targeted approach to attenuate virulence genes and substitute them with functional payloads to enhance pathogen clearance in situ.