In vitro activity of Neisseria meningitidis PglL O-oligosaccharyltransferase with diverse synthetic lipid donors and a UDP-activated sugar.

Oligosaccharyltransferases (OTases) are enzymes that catalyze the transfer of an oligosaccharide from a lipid carrier to an acceptor molecule, commonly a protein. OTases are classified as N-OTases and O-OTases, depending on the nature of the glycosylation reaction. The N-OTases catalyze the glycan transfer to amide groups in asparagines in a reaction named N-linked glycosylation. The O-OTases are responsible for protein O-linked glycosylation, which involves the attachment of glycans to hydroxyl groups of serine or threonine residues. These enzymes exhibit a relaxed specificity and are able to transfer a variety of glycan structures to different protein acceptors. This property confers OTases with great biotechnological potential as these enzymes can produce glycoconjugates relevant to the pharmaceutical industry. Furthermore, OTases are thought to be involved in pathogenesis mechanisms. Several aspects of the functionality of OTases are not fully understood. In this work, we developed a novel approach to perform kinetic studies on PglL, the O-OTase from Neisseria meningitidis. We investigated the importance of the acyl moiety of the lipid glycan donor substrate on the functionality of PglL by testing the efficiency of glycosylation reactions using synthetic substrates carrying the same glycan structure but different acyl moieties. We found that PglL can function with many lipids as glycan donors, although the length and the conformation of the lipid moiety significantly influenced the catalytic efficiency. Interestingly, PglL was also able to transfer a monosaccharide employing its nucleotide-activated form, acting as a Leloir glycosyltransferase. These results provide new insights on the function and the evolution of oligosaccharyltransferases.

Enterobacterial common antigen integrity is a checkpoint for flagellar biogenesis in Serratia marcescens.

Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.