A Broad Spectrum Protein Glycosylation System Influences Type II Protein Secretion and Associated Phenotypes in Vibrio cholerae.

Dina Vorkapic,Deborah R Leitner,Franz G Zingl,Joachim Reidl,Fabian Mitterer,Adina Schulze,Torben Kuehnast,Stefan Schild,Lisa-Maria Mauerhofer,Mario F Feldman,Michael Koomey,Laura Liesinger,Ruth Birner-Gruenberger,Jan Haug Anonsen,Katharina Pressler,Manuela Toeglhofer

doi:10.3389/fmicb.2019.02780

Abstract

Protein secretion plays a crucial role for bacterial pathogens, exemplified by facultative human-pathogen Vibrio cholerae, which secretes various proteinaceous effectors at different stages of its lifecycle. Accordingly, the identification of factors impacting on protein secretion is important to understand the bacterial pathophysiology. PglLVc, a predicted oligosaccharyltransferase of V. cholerae, has been recently shown to exhibit O-glycosylation activity with relaxed glycan specificity in an engineered Escherichia coli system. By engineering V. cholerae strains to express a defined, undecaprenyl diphosphate-linked glycoform precursor, we confirmed functional O-linked protein glycosylation activity of PglLVc in V. cholerae. We demonstrate that PglLVc is required for the glycosylation of multiple V. cholerae proteins, including periplasmic chaperones such as DegP, that are required for efficient type II-dependent secretion. Moreover, defined deletion mutants and complementation strains provided first insights into the physiological role of O-linked protein glycosylation in V. cholerae. RbmD, a protein with structural similarities to PglLVc and other established oligosaccharyltransferases (OTases), was also included in this phenotypical characterization. Remarkably, presence or absence of PglLVc and RbmD impacts the secretion of proteins via the type II secretion system (T2SS). This is highlighted by altered cholera toxin (CT) secretion, chitin utilization and biofilm formation observed in ΔpglLVc and ΔrbmD single or double mutants. This work thus establishes a unique connection between broad spectrum O-linked protein glycosylation and the efficacy of type II-dependent protein secretion critical to the pathogen’s lifecycle.

Highlights

The Gram-negative pathogen Vibrio cholerae, the causative agent of the severe secretory diarrheal disease cholera, transits between two dissimilar habitats along its life cycle
We establish a clear association between O-linked protein glycosylation mediated by the oligosaccharyltransferase PglLVc and the efficacy of type II protein secretion-dependent processes in the human pathogen V. cholerae
Perhaps the simplest explanation would be that one or more of the glycoproteins targeted by PglLVc influences type II protein secretion proficiency and that the activity or functionality of that target protein is influenced by its glycosylation status

Summary

Introduction

The Gram-negative pathogen Vibrio cholerae, the causative agent of the severe secretory diarrheal disease cholera, transits between two dissimilar habitats along its life cycle. A hallmark of environmental survival and transmission of V. cholerae is the ability to form biofilms on chitinous surfaces in the aquatic reservoirs. V. cholerae passes the acidic barrier in the stomach and reaches the small intestine, the primary site of colonization. During this passage, V. cholerae activates a complex regulatory cascade to induce the expression of virulence factors and achieve full colonization fitness (Childers and Klose, 2007). The pathology of cholera is mainly due to the activity of the secreted cholera toxin (CT), which induces a massive efflux of chloride ions and water into the intestinal lumen resulting in a secretory diarrhea (Childers and Klose, 2007)

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