Emerging strategies for engineering Escherichia coli Nissle 1917-based therapeutics

Abstract

Engineered microbes are rapidly being developed for the delivery of therapeutic modalities to sites of disease. Escherichia coli Nissle 1917 (EcN), a genetically tractable probiotic with a well-established human safety record, is emerging as a favored chassis. Here, we summarize the latest progress in rationally engineered variants of EcN for the treatment of infectious diseases, metabolic disorders, and inflammatory bowel diseases (IBDs) when administered orally, as well as cancers when injected directly into tumors or the systemic circulation. We also discuss emerging studies that raise potential safety concerns regarding these EcN-based strains as therapeutics due to their secretion of a genotoxic colibactin that can promote the formation of DNA double-stranded breaks in mammalian DNA.

Keywords: Escherichia coli Nissle 1917; bioengineering; colibactin; microbial therapeutics; synthetic biology.

Figure 1.. Variants of EcN engineered with enhanced antimicrobial properties.

(a) EcN J25 is engineered to constitutively secrete the microcin MccJ25. The expression of McjA, the MccJ25 precursor, and ProTeOn, are controlled via a ProTeOn-dependent promoter, thus generating a positive feedback loop. Genes encoding the proteins needed for J25 biosynthesis and transport are in an operon present on the same plasmid under the control of a constitutive promoter maintained via antibiotic selection. (b) EcN pttrMcH47 is engineered to secrete the microcin Mcc47 upon sensing tetrathionate, a reporter of inflammation. Genes encoding the proteins needed for tetrathionate sensing are positioned upstream of those involved in MccH47 biosynthesis and export. (c) EcN BHA is engineered to constitutively secrete Enterocins A and B, and Hiracin, each fused to a MccV N-terminal secretion tag. The expression of all three bacteriocins, the proteins that mediate their secretion, and ProTeOn are controlled via the expression of a ProTeOn-dependent promoter on a plasmid maintained via antibiotic selection. (d) Sense-Kill-EcN SED is engineered to produce molecules that eradicate Pseudomonas aeruginosa. Sense-Kill-EcN SED constitutively expresses LasR, a transcription factor activated upon binding a quorum-sensing (QS) molecule produced by P. aeruginosa. Activated LasR promotes the transcription of pyocin S5, E7 lysis protein, and Dispersin B. E7 proceeds to lyse the cells, releasing pyocin S5 and Dispersin B. The genes encoding the ‘sense-kill’ circuit are encoded on a plasmid that is maintained via auxotrophic selection.

Figure 2.. Engineered EcN variants for the treatment of inflammatory bowel disease.

(a) PBP8 CsgA-TFF3 is engineered to display TFF3 on its surface. TFF3 is fused to CsgA such that it is assembled and exposed on curli fibers attached to EcN. The expression of the plasmid-encoded CsgA-TFF3, and other components of the curli operon, including the assembly/transport component CsgG, is expressed in an arabinose-dependent manner from a plasmid. (b) EGF-EcN is engineered to constitutively express a lipase ABC transporter. EGF is fused to a C-terminal LARD (Lipase ABC transporter Recognition Domain). The genes encoding the transporter and EGF are integrated into the EcN chromosome. (c) Lresb pDGAT is engineered with a blue-light responsive circuit that controls expression of Ag43, an adhesin (‘bio-glue’) that promotes EcN biofilm formation. Since blue-light does not penetrate the intestines, upconversion molecules (UCMs) that convert the NIR light to blue light are co-administered with the bacteria. Lresb pDGAT secrete TGF-ß1 fused to an OmpA secretion signal peptide. The genes encoding this system are encoded on a plasmid maintained via antibiotic selection.

Figure 3.. EcN variants developed for the treatment of cancer.

(a) SYNB1891 engineered to express Listeria monocytogenes DacA, which promotes the biosynthesis of cyclic di-AMP. dacA is encoded in the chromosome under the control P_fnrS, a hypoxia-induced promoter. For biocontainment, SYNB1891 lacks thyA and dapA. (b) SLIC variants of EcN each contain a chromosomally encoded lysis circuit, whereby the expression of the QS molecule by LuxI promotes activation of LuxR, which promotes the expression of additional LuxI and the E7 lysis protein. When the bacteria reach a certain density, they coordinately lyse releasing therapeutic payloads. The payload proteins are constitutively expressed and encoded on a plasmid maintained via a toxin-antitoxin system. Strains expressing different payloads can be used in combination, as exemplified by the coordinated release of anti-PD-L1 and anti-CTLA-4 nanobodies and recombinant GM-CSF for the treatment of cancer. (c) Eda-l1-HlpA secretes myrosinase, an enzyme that catalyzes the conversion of glucosinolate to sulphoraphane, and HlpA, a protein that promotes binding to cancer cells. Myrosinase is targeted for secretion via the Sec pathway and OMP porins via fusion to a YebF secretion tag. HlpA is exposed on the other surface of the E. coli via fusion to INP (ice nuclease protein). Both proteins are constitutively expressed from a plasmid maintained via auxotrophic selection. (d) L-Arg EcN is engineered to convert ammonia to L-arginine when grown under hypoxic conditions. L-Arg no longer encodes argR, a repressor of the L-argnine biosynthesis, and carries a feedback-resistant mutant of ArgA, the first enzyme in this biosynthesis pathway, under the control of the P_fnrS promoter.