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.

Keywords: Lactobacillus reuteri; bacteriophage; leptin; microbial delivery; probiotic; prophage; therapeutic.

FIG 1

Assessment of potential biotherapeutic delivery vehicles. (a) Mutation rates of selected lactic acid bacteria determined by the FALCOR method (6). L. reuteri (Lre) exhibits a low mutation rate relative to other lactic acid bacteria, L. salivarius (Ls), L. gasseri (Lg), L. fermentum (Lf), L. jensenii (Lj), L. casei (Lc), L. rhamnosus (Lrh), L. acidophilus (La), L. plantarum (Lp), and L. lactis (Ll). *, P < 0.05; **, P < 0.01 (relative to L. reuteri). The results shown are averages from three independent experiments ± standard error. (b) LAB survival following GI transit in a mouse. L. reuteri [LR::rpoB(H488R)], L. rhamnosus, and L. plantarum survived GI transit at least 10-fold better than L. lactis (P < 0.001, Tukey’s HSD). Each dot represents a single mouse. Different letters indicate statistical differences between the respective treatment groups.

FIG 2

L. reuteri-mediated leptin production. (a) Western blotting results for intracellularly accumulated leptin indicated that leptin-3×FLAG is produced at the expected size, while the majority of secreted leptin is incorrectly cleaved. Lane I, LR/pLeptin-3×FLAG (19 kDa); lane SP, secreted leptin LR/pSP-Leptin-3×FLAG (23 kDa). (b) ELISA confirmed leptin production by LR/pLeptin. Ctl, LR/pCtl; nd, not detected. The results shown are averages from three independent experiments ± standard error, normalized per 1 mg of cell pellet dry weight.

FIG 3

Leptin release from recombinant VPL1014 following mitomycin C treatment. (a) Numbers of PFU derived from leptin-producing VPL1014 culture. No PFU were produced by induced or uninduced LRΔΦ1ΔΦ2/pLeptin. The results shown are averages from three independent experiments ± standard error. (b) ELISA data showing the percentage of total leptin (from the supernatant [SN] plus the cell lysate) released into the extracellular milieu. The results shown are averages from three independent experiments ± standard error. (c) Growth curves of uninduced LR/pLeptin, uninduced LRΔΦ1ΔΦ2/pLeptin, induced LR/pLeptin, and induced LRΔΦ1ΔΦ2/pLeptin are shown. Asterisks indicate statistical differences between respective induced and uninduced groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Tukey’s HSD). The results shown are averages of three independent experiments ± standard error.

FIG 4

Plasmid stability of the pLeptin-ThyA construct in LRΔthyA::rpoB(H488R). Plasmid stability is represented by the percentage of cells from plain MRS broth that retained pLeptin-ThyA, pCtl-ThyA, or pLeptin over the course of ∼100 generations without antibiotic in the medium. (Inset) Plasmid stability of pLeptin-ThyA, pCtl-ThyA, or pLeptin from mMRS without thymidine (no beef extract) (P < 0.01, Tukey’s HSD). The results shown are averages from three independent experiments ± standard error. Different letters indicate statistical differences between the respective treatment groups.