Abstract
Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.
Highlights
Shigella spp. cause gastrointestinal disease, ranging from mild diarrheal episodes to complicated dysentery
We show that engineered Sf6TSP constructs can be applied in a rapid microtiter plate-based assay or as sensitive fluorescent probes for S. flexneri detection
The Sf6TSP binding site has been well characterized to interact with O-antigen fragments of serotype Y that lack branching glucose modifications
Summary
Shigella spp. cause gastrointestinal disease, ranging from mild diarrheal episodes to complicated dysentery. Thereby, most infections in developing countries are caused by Shigella flexneri (S. flexneri) strains, with serotype 2a as the most prominent [3]. [4], an increasing number of resistant Shigella strains are found [5], with livestock farming as a potential source [6]. Humans are the primary reservoir of Shigella spp. These emerging resistances against antimicrobial agents force the community to find new drugs and to keep outbreaks and contaminations to a minimum by fast and reliable diagnostic tools.
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