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.
Highlights
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
The mutation rate of L. reuteri VPL1014 was 3.4-fold lower than that of the strain with the second-lowest mutation rate, Lactobacillus salivarius (29.7 ϫ 10Ϫ10 mutations/cell/generation), with the mutation rate in the latter being comparable to the mutation rates of the seven remaining lactobacillus strains, ranging from 42.9 ϫ 10Ϫ10 (Lactobacillus gasseri ATCC 33323) to 60.6 ϫ 10Ϫ10 (Lactobacillus plantarum BAA-793) mutations/cell/generation
We recently demonstrated that L. reuteri ATCC PTA 6475, a precursor of VPL1014, encodes two biologically active prophages, which are bacterial viruses that are integrated in the bacterial genome [55]
Summary
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. The organism has evolved to thrive in the intestine, and select strains exhibit probiotic features, including modulation of inflammation [32,33,34,35], prevention of bone loss in menopausal females [36], and production of reuterin, an antimicrobial molecule that has activity against Escherichia coli O157:H7 and Listeria monocytogenes, for example [37, 38] Genetic tools, such as single-stranded DNA recombineering [39], CRISPR-Cas genome editing [40], and a counterselection marker [41], have been developed for L. reuteri and provide the species the potential to be developed as a therapeutic delivery vehicle
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