Development of a Limosilactobacillus reuteri therapeutic delivery platform with reduced colonization potential.

Autor:	Alexander LM; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Khalid S; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Gallego-Lopez GM; Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA.; Morgridge Institute for Research, Madison, Wisconsin, USA., Astmann TJ; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Oh J-H; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Heggen M; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Huss P; Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA., Fisher R; Department of Radiation Oncology, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA., Mukherjee A; Department of Radiation Oncology, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA., Raman S; Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA., Choi IY; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Smith MN; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA., Rogers CJ; ChromoLogic, LLC, Monrovia, California, USA., Epperly MW; Department of Radiation Oncology, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA., Knoll LJ; Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA., Greenberger JS; Department of Radiation Oncology, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA., van Pijkeren J-P; Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Jazyk:	angličtina
Zdroj:	Applied and environmental microbiology [Appl Environ Microbiol] 2024 Nov 20; Vol. 90 (11), pp. e0031224. Date of Electronic Publication: 2024 Oct 31.
DOI:	10.1128/aem.00312-24
Abstrakt:	Bacterial biotherapeutic delivery vehicles have the potential to treat a variety of diseases. This approach obviates the need to purify the recombinant effector molecule, allows delivery of therapeutics in situ via oral or intranasal administration, and protects the effector molecule during gastrointestinal transit. Lactic acid bacteria have been broadly developed as therapeutic delivery vehicles though risks associated with the colonization of a genetically modified microorganism have so-far not been addressed. Here, we present an engineered Limosilactobacillus reuteri strain with reduced colonization potential. We applied a dual-recombineering scheme for efficient barcoding and generated mutants in genes encoding five previously characterized and four uncharacterized putative adhesins. Compared with the wild type, none of the mutants were reduced in their ability to survive gastrointestinal transit in mice. CmbA was identified as a key protein in L. reuteri adhesion to HT-29 and enteroid cells. The nonuple mutant, a single strain with all nine genes encoding adhesins inactivated, had reduced capacity to adhere to enteroid monolayers. The nonuple mutant producing murine IFN-β was equally effective as its wild-type counterpart in mitigating radiation toxicity in mice. Thus, this work established a novel therapeutic delivery platform that lays a foundation for its application in other microbial therapeutic delivery candidates and furthers the progress of the L. reuteri delivery system towards human use.IMPORTANCEOne major advantage to leverage gut microbes that have co-evolved with the vertebrate host is that evolution already has taken care of the difficult task to optimize survival within a complex ecosystem. The availability of the ecological niche will support colonization. However, long-term colonization of a recombinant microbe may not be desirable. Therefore, strategies need to be developed to overcome this potential safety concern. In this work, we developed a single strain in which we inactivated the encoding sortase, and eight genes encoding characterized/putative adhesins. Each individual mutant was characterized for growth and adhesion to epithelial cells. On enteroid cells, the nonuple mutant has a reduced adhesion potential compared with the wild-type strain. In a model of total-body irradiation, the nonuple strain engineered to release murine interferon-β performed comparable to a derivative of the wild-type strain that releases interferon-β. This work is an important step toward the application of recombinant L. reuteri in humans. Competing Interests: J.-P.V.P received unrestricted funds from BioGaia AB, a probiotic-producing company. J.-P.V.P is the founder of the consulting company Next-Gen Probiotics, LLC. J.-P.V.P. and L.M.A. are inventors on a patent application describing the development of microbes with reduced adhesion characteristics
Databáze:	MEDLINE
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