Progress in understanding the assembly process of bacterial O-antigen

Abstract

The discovery that the surfaces of Gram-negative bacteria often carry unique polysaccharide signatures pre-dates most seminal discoveries of molecular biology and biochemistry of the 20th century. The O-antigen component of the lipopolysaccharide has been one of the most intensely studied bacterial polysaccharide surface structures for over 80 years. Yet, many questions about the mechanism of biosynthesis of the O-antigen and its transport to the cell surface remain unanswered. In this review we provide an overview of how the molecular basis of the O-antigen assembly and trafficking were unraveled in a historical context. We pay particular attention to the emergence of novel technological approaches and how they fueled the elucidation of the O-antigen maturation process. Moreover, we provide a brief perspective on the biosynthesis of enterobacterial common antigen and underline the similarities and differences between the pathways used to assemble these two surface polysaccharides. Finally, we highlight key discoveries that led to the understanding of the mechanistic basis of bacteriophage-induced O-antigen modifications. We place special emphasis on the regulation of the length of O-antigen polymers and provide a detailed overview of the models explaining the O-antigen length determination. Finally, we highlight outstanding questions that need to be addressed both structurally and functionally to advance our understanding of the O-antigen assembly, trafficking and export within cellular and molecular contexts.

Making a membrane on the other side of the wall

“…The tripartite structure of LPS consists of lipid A component, a core oligosaccharide component, and an O-antigen (OAg) polysaccharide chain that extends into the extracellular milieu [17] (Figure 1). The Oag component is not synthesized in the model organism E. coli K-12 [18], but it plays an important role in protecting commensal strains in their host environment and is also a crucial virulence determinant in pathogenic strains [19]. Lipid A is a diglucosamine phosphate-based lipid (Figure 3A) that is a notoriously potent activator of the innate immune system and is commonly referred to as ‘endotoxin’ [20].…”

“…The lipid A component is synthesized via an eponymous pathway largely characterized by Chris Raetz and his colleagues [17,25] . Sugars of the core polysaccharide are sequentially attached to lipid A; the Oag component is synthesized and translocated separately [19]. Nascent LPS must be translocated across the IM bilayer.…”

“…The ABC transporter MsbA has been convincingly shown to be responsible for LPS flipping activity [26-28]. Now, in the periplasmic leaflet of the IM, nascent LPS can be modified by the glycosyltransferase WaaL with Oag polysaccharide [19]. The LPS is then efficiently transported to the OM.…”

Wzx flippases exhibiting complex O‐unit preferences require a new model for Wzx–substrate interactions

“…The vast majority of heteropolymeric O antigens are synthesized via the Wzx/Wzy pathway (Kalynych, Morona, & Cygler, ; Reeves & Cunneen, ), with most of the responsible genes grouped together in a gene cluster at a conserved chromosomal location. O‐unit synthesis is initiated at the cytoplasmic face of the inner membrane (IM), where a phosphoglycosyl transferase attaches the first sugar phosphate (typically N ‐acetylglucosamine phosphate (GlcNAc‐P) in the Enterobacteriaceae) onto the polyisoprenoid lipid carrier molecule undecaprenyl phosphate (UndP), generating a pyrophosphate (PP) linkage (Price & Momany, ).…”

Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units

“…Most O antigens are synthesized by the Wzx/ Wzy-dependent pathway. The several steps of this pathway have been reviewed many times since its discovery, and readers are referred to some recent reviews (Islam & Lam, 2014;Kalynych et al, 2014;Reeves & Cunneen, 2009) for details of the pathway outlined below. Synthesis begins with assembly of O units on the lipid carrier undecaprenyl phosphate (Und-P), at the cytoplasmic face of the inner membrane.…”