A broad spectrum protein glycosylation system influences type II protein secretion and associated phenotypes in Vibrio cholerae

Protein secretion plays a crucial role for bacterial pathogens, exemplified by facultative human-pathoge

A broad spectrum protein glycosylation system influences type II protein secretion and associated phenotypes in Vibrio cholerae

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

Fic Proteins of Campylobacter fetus subsp. venerealis Form a Network of Functional Toxin–Antitoxin Systems

Enzymes containing the FIC (filamentation induced by cyclic AMP) domain catalyze post-translational modifications of target proteins. In bacteria the activity of some Fic proteins resembles classical toxin–antitoxin (TA) systems. An excess of toxin over neutralizing antitoxin can enable bacteria to survive some stress conditions by slowing metabolic processes and promoting dormancy. The cell can return to normal growth when sufficient antitoxin is present to block toxin activity. Fic genes of the human and animal pathogen Campylobacter fetus are significantly associated with just one subspecies, which is specifically adapted to the urogenital tract. Here, we demonstrate that the fic genes of virulent isolate C. fetus subsp. venerealis 84-112 form multiple TA systems. Expression of the toxins in Escherichia coli caused filamentation and growth inhibition phenotypes reversible by concomitant antitoxin expression. Key active site residues involved in adenylylation by Fic proteins are conserved in Fic1, Fic3 and Fic4, but degenerated in Fic2. We show that both Fic3 and the non-canonical Fic2 disrupt assembly and function of E. coli ribosomes when expressed independently of a trans-acting antitoxin. Toxicity of the Fic proteins is controlled by different mechanisms. The first involves intramolecular regulation by an inhibitory helix typical for Fic proteins. The second is an unusual neutralization by heterologous Fic–Fic protein interactions. Moreover, a small interacting antitoxin called Fic inhibitory protein 3, which appears unrelated to known Fic antitoxins, has the novel capacity to bind and neutralize Fic toxins encoded in cis and at distant sites. These findings reveal a remarkable system of functional crosstalk occurring between Fic proteins expressed from chromosomal and extrachromosomal modules. Conservation of fic genes in other bacteria that either inhabit or establish pathology in the urogenital tract of humans and animals underscores the significance of these factors for niche-specific adaptation and virulence