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

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.

The crystal structure of CbpD clarifies substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…A number of these post-transitionally modified residues are also found in the AA10-GbpA2 domain interface of CbpD, likely encouraging a more elongated overall conformation, similar to the observed solution scattering conformation. Additionally, a recent bioinformatics analysis of 27 000 LPMOs found that most of the eight LPMO AA families are enriched in Ser and Thr residues in C-terminal regions that are prime candidates for PTM, specifically O-glycosylation (Tamburrini et al, 2021), which may affect secretion (Vorkapic et al, 2019). Of note, the Tma12 structure has an N-glycosylation at Asn158, structurally equivalent to Ser136 in CbpD, which has been identified as phosphorylated when CbpD is expressed and secreted by P. aeruginosa (Ouidir et al, 2014).…”

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses

“…Previous comparative genomic as well as ex vivo studies have identified a number of potential bacterial glycosylation systems, for which the native substrates and glycans are yet to be defined. For example, in Vibrio cholerae,a functional O-linked glycosylation system has been identified and shown to be required for cholera toxin secretion, yet the glycoproteins of this system are still unknown. Because of the diversity of glycans used for bacterial protein glycosylation, ,,,,,,,,, it is not surprising that a single approach such as ZIC-HILIC enrichment is inapplicable to all glycosylation systems or substrates within a single glycoproteome.…”

CbpD crystal structure adds intrigue to substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…A number of these post-transitionally modified residues are also in the AA10:GbpA2 domain interface of CbpD, likely encouraging a more elongated overall conformation, similar to the observed solution scattering conformation. Additionally, a recent bioinformatics analysis of 27,000 LPMOs found that most of the eight LPMO AA families are enriched for Ser and Thr residues in C-terminal regions that are prime candidates for PTM, specifically O - glycosylation (Tamburrini et al ., 2021), which may affect secretion (Vorkapic et al ., 2019). Of note, the Tma12 structure has an N-glycosylation at Asn158, structurally equivalent to Ser136 in CbpD, which has been identified as phosphorylated when CbpD is expressed and secreted by P. aeruginosa (Ouidir et al ., 2014).…”