A new approach for analyzing an adhesive bacterial protein in the mouse gastrointestinal tract using optical tissue clearing

Keita Nishiyama,Nobuhiko Okada,Takao Mukai,Makoto Sugiyama,Hiroki Yamada,Kyoko Makino,Takashi Takaki,Sayaka Ishihara

doi:10.1038/s41598-019-41151-y

Keita Nishiyama, Nobuhiko Okada + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-41151-y

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Journal: Scientific Reports	Publication Date: Mar 18, 2019
Citations: 10	License type: open-access

Affiliation: Kitasato University, Showa University

Abstract

Several bacterial moonlighting proteins act as adhesion factors, which are important for bacterial colonization of the gastrointestinal (GI) tract. However, little is known about the adherence properties of moonlighting proteins in the GI tract. Here, we describe a new approach for visualizing the localization of moonlighting protein-coated fluorescent microbeads in the whole GI tract by using a tissue optical clearing method, using elongation factor Tu (EF-Tu) as an example. As a bacterial cell surface-localized protein mimic, recombinant EF-Tu from Lactobacillus reuteri was immobilized on microbeads. EF-Tu-coating promoted the interaction of the microbeads with a Caco-2 cell monolayer. Next, the microbeads were orally administered to mice. GI whole tissues were cleared in aqueous fructose solutions of increasing concentrations. At 1 h after administration, the microbeads were diffused from the stomach up to the cecum, and after 3 h, they were diffused throughout the intestinal tract. In the lower digestive tract, EF-Tu-beads were significantly more abundant than non-coated control beads, suggesting that EF-Tu plays an important role in the persistence of the microbeads in the GI tract. The new approach will help in evaluating how moonlighting proteins mediate bacterial colonization.

Highlights

Moonlighting refers to the ability of proteins or peptides to exert multiple biologically important functions in plants, animals, yeasts, and bacterial organisms[1,2,3]
The purified recombinant elongation factor Tu (EF-Tu) was chemically coupled to microbeads, and the complex mimicked a bacterial cell surface-localized protein (Fig. 1b)
Several bacterial moonlighting proteins function in adhesion, which has been considered the key process in bacterial colonization of the host mucosal surface[3,4,5]

Summary

Introduction

Moonlighting refers to the ability of proteins or peptides to exert multiple biologically important functions in plants, animals, yeasts, and bacterial organisms[1,2,3]. Beads modified with CD47, a member of the immunoglobulin superfamily, inhibit tumor development in mice and have shown promise in immunotherapeutic approaches[14], and beads modified with multivalent adhesion molecules prevent Pseudomonas aeruginosa infection in mice[15]. Based on these reports, we hypothesized that microbeads coated with a single moonlighting protein could be useful for evaluating its moonlighting function (in this case, as an intestinal adhesion factor). We previously found that the cell surface-associated EF-Tu from Lactobacillus reuteri binds to sulfated carbohydrate moieties of glycocalyx and mucins, suggesting that this protein is an adhesion factor in L. reuteri[10]

Methods

Results

Conclusion