Exploiting Prophage-Mediated Lysis for Biotherapeutic Release by Lactobacillus reuteri

Abstract

Lactobacillus reuteri has the potential to be developed as a microbial therapeutic delivery platform because of an established safety profile, health-promoting properties, and available genome editing tools. Here, we show that L. reuteri VPL1014 exhibits a low mutation rate compared to other Gram-positive bacteria, which we expect will contribute to the stability of genetically modified strains. VPL1014 encodes two biologically active prophages, which are induced during gastrointestinal transit. We hypothesized that intracellularly accumulated recombinant protein can be released following bacteriophage-mediated lysis. To test this, we engineered VPL1014 to accumulate leptin, our model protein, inside the cell. In vitro prophage induction of recombinant VPL1014 released leptin into the extracellular milieu, which corresponded to bacteriophage production. We also employed a plasmid system that does not require antibiotic in the growth medium for plasmid maintenance. Collectively, these data provide new avenues to exploit native prophages to deliver therapeutic molecules. IMPORTANCE Lactic acid bacteria (LAB) have been explored as potential biotherapeutic vehicles for the past 20 years. To secrete a therapeutic in the extracellular milieu, one typically relies on the bacterial secretion pathway, i.e., the Sec pathway. Overexpression of a secreted protein can overload the secretory pathway and impact the organism’s fitness, and optimization of the signal peptide is also required to maximize the efficiency of the release of mature protein. Here, we describe a previously unexplored approach to release therapeutics from the probiotic Lactobacillus reuteri. We demonstrate that an intracellularly accumulated recombinant protein is released following prophage activation. Since we recently demonstrated that prophages are activated during gastrointestinal transit, we propose that this method will provide a straightforward and efficient approach to deliver therapeutics in vivo.

Second-generation Probiotics Producing IL-22 Increase Survival of Mice After Total Body Irradiation

“…It presents evidence of successful radiation mitigation with two different platforms for enteric delivery of IL-22, with one involving cytokine secretion by Escherichia coli, the other involving the lysis of bacteria and release of cytokine in L. reuteri. It is known that bacteriophage lyse L. reuteri during gastrointestinal transit resulting in release of IL-22 (31). L. reuteri is known to target the jejunum and ilium, of the small intestine (40).…”

“…By singlestranded DNA recombineering, we inactivated thyA in a rifampicinresistant derivative of L. reuteri VPL1014 [LR:rpoB(H488R)] to yield LRΔthyA (Rif ® ), as described previously (30,31) (Tables I and II). The gene providing chloramphenicol resistance in the vector pVPL31126 was replaced with the thyA gene derived from L. reuteri VPL1014 via blunt-end ligation (T4 DNA ligase: Thermo Fisher Scientific) and transformed into LR* by electroporation to construct LR*/pIL-22-thyA, as described previously (31)(32)(33). LR* harboring the previously constructed vector pCtl-thyA served as an empty vector control (31).…”

“…The gene providing chloramphenicol resistance in the vector pVPL31126 was replaced with the thyA gene derived from L. reuteri VPL1014 via blunt-end ligation (T4 DNA ligase: Thermo Fisher Scientific) and transformed into LR* by electroporation to construct LR*/pIL-22-thyA, as described previously (31)(32)(33). LR* harboring the previously constructed vector pCtl-thyA served as an empty vector control (31). E. coli EC1000 was used as an intermediate cloning host.…”

Secretion of Recombinant Interleukin-22 by Engineered Lactobacillus reuteri Reduces Fatty Liver Disease in a Mouse Model of Diet-Induced Obesity

“…As is evident from our Western blot analysis, only part of the recombinant protein was processed correctly by the bacterial cell secretion machinery. We made a similar observation with leptin (14). While incomplete cleavage may be overcome by testing different leader sequences or by optimizing the combination of the leader sequence and the N-terminal sequence of the mature protein, these are not trivial tasks (19).…”

“…The organism pertinent to this study, Lactobacillus reuteri, has proven to be functional as a therapeutic delivery platform in animal disease models (11). Advantages of using L. reuteri are the availability of high-throughput genome editing tools (12,13) and its ability to thrive in the gut ecosystem, and L. reuteri has-compared to other lactobacilli and L. lactis-an extraordinarily low mutation rate (14), which is expected to improve the genetic stability of the recombinant organism. Lastly, L. reuteri ATCC PTA 6475, the strain we used in this study, has probiotic features, including amelioration of obesity (15) (Fig.…”