A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis.

Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.

Challenges and Pitfalls in the Engineering of Human Interleukin 22 (hIL-22) Secreting Lactobacillus reuteri.

Engineered microbes for the delivery of intestinally directed therapeutics is a promising avenue for the treatment of various intestinal diseases including inflammatory bowel disease (IBD) and intestinal graft vs. host disease (GVHD). This modality of treatment would allow for the targeted delivery of therapeutics to the site of inflammation or disease while minimizing the systemic side effects that often accompany treatment of these pathologies. Here, we show the challenges encountered and overcome in successfully engineering Lactobacillus reuteri to secrete high levels of biologically active human interleukin 22 (hIL-22). Initial hIL-22 constructs secreted high levels of hIL-22, however we found the majority of hIL-22 was cleaved and not biologically active. Several strategies were explored to improve the production of intact hIL-22, with the optimization of the signal sequence for peptide secretion having the most impact of production of intact hIL-22. This resulted in L. reuteri secreting high concentrations (up to 700 ng/mL) of hIL-22. Bioactivity of hIL-22 was confirmed by the secretion of interleukin 10 (IL-10) from the colon cancer derived epithelial cell line Colo205 and the secretion of Regenerating islet-derived protein 3 alpha (Reg3α) from human jejunal enteroids. The secretion of bioactive hIL-22 imposed a significant cost for L. reuteri as bacterial growth was significantly impaired upon induction. Future challenges and optimization strategies for the delivery of hIL-22 to the human intestinal tract are discussed.

Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri.

BACKGROUND: Lactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency. RESULTS: Strains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA. CONCLUSIONS: Our findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.

Genetic Tools for the Enhancement of Probiotic Properties.

The Lactobacillus genus is a diverse group of microorganisms, many of which are of industrial and medical relevance. Several Lactobacillus species have been used as probiotics, organisms that when present in sufficient quantities confer a health benefit to the host. A significant limitation to the mechanistic understanding of how these microbes provide health benefits to their hosts and how they can be used as therapeutic delivery systems has been the lack of genetic strategies to efficiently manipulate their genomes. This article will review the development and employment of traditional genetic tools in lactobacilli and highlight the latest methodologies that are allowing for precision genome engineering of these probiotic organisms. The application of these tools will be key in providing mechanistic insights into probiotics as well as maximizing the value of lactobacilli as either a traditional probiotic or as a platform for the delivery of therapeutic proteins. Finally, we will discuss concepts that we consider relevant for the delivery of engineered therapeutics to the human gut.

Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation.

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two-component systems from marine Shewanella species, and validate them in laboratory Escherichia coli Then, we port these sensors into a gut-adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.