Salt-Induced Stress Stimulates a Lipoteichoic Acid-Specific Three-Component Glycosylation System in Staphylococcus aureus

Abstract

Lipoteichoic acid (LTA) in Staphylococcus aureus is a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, including d-alanine and glycosyl groups, to alter cellular function. While the genetic determinants of d-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation in S. aureus We demonstrate here that S. aureus utilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und) N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-type S. aureus LTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenous d-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCE The role of glycosylation in the structure and function of Staphylococcus aureus lipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role of N-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As the dlt and glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreased d-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.

Keywords: SigB; cell envelope; cell envelope stress; glycosylation; lipoteichoic acid; teichoic acids.