Engineering Escherichia coli into a Protein Delivery System for Mammalian Cells

Abstract

Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

“…As previously described (9), HeLa cells were infected with the designated bacteria grown under conditions that induce T3SSs. At 1 h p.i., infected cells were fixed and stained with rabbit anti-E. coli 5.…”

“…coli Invade HeLa Cells at Levels Similar to WT Shigella. We recently reported the development of mT3 E. coli, laboratory strains of E. coli (i.e., DH10B) that express a functional Shigella T3SS (9). These strains carry a 31-kb region of the 220-kb S. flexneri VP, either on a plasmid (pmT3SS) or chromosomally integrated, plus a plasmid that encodes either the master Shigella T3SS transcriptional regulator, VirF, or its downstream target, VirB (10).…”

“…1A). Three of the effectors-IpaA, IpgB1, and IpgD-play roles in the invasion of Shigella into nonphagocytic epithelial cells (11), likely accounting for the ability of mT3 E. coli strains, like wild-type (WT) Shigella, to efficiently invade epithelial cells (9).…”

Sustainable therapies by engineered bacteria

“…Synthetic biology is providing modular parts and gene circuits that can be used to program the designed bacterial chassis to precisely control the expression and delivery of therapeutic proteins (Huh et al ., 2013; Reeves et al ., 2015; Ruano‐Gallego et al ., 2015), the adhesion of the engineered bacteria to target cells (Piñero‐Lambea et al ., 2015b) or modify their chemotactic behaviour (Hwang et al ., 2014). Bacteria have also been engineered with gene circuits that respond to disease signals in the gut (Riglar et al ., 2017).…”

Fabrication of nanocomposites and hybrid materials using microbial biotemplates

“…To perform this specific function, reengineering E. coli as a host cell was performed with a tunable type-3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. This reengineered system thus provided a highly flexible protein delivery platform with potential for future therapeutic applications [74]. Synthetic microbes, as a kind of cell therapy, have broad potential for future applications in human disease treatment.…”