Modification of Lipopolysaccharide with Colanic Acid (M-antigen) Repeats in Escherichia coli

Abstract

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide ( Enteric bacteria synthesize and display a complex array of various cell surface polysaccharides. There can be at least six distinct saccharide polymers simultaneously present within the glycocalyx of a typical strain of Escherichia coli. At present, the known components of the saccharide matrix include lipopolysaccharide (LPS)2 O-antigens (1), enterobacterial common antigen (ECA) (2), capsular polysaccharides (K-antigen) (3), poly ␤-1,6-N-acetyl-D-glucosamine (PNAG) (4), the ␤-1,4-glucan polymer bacterial cellulose (5), and colanic acid (CA or M-antigen) (6). The tremendous diversity within the serotype specific repeat units [ϳ170 O-antigens, ϳ80 K-antigens (7)], coupled with regulation of expression levels between polysaccharide classes, facilitates the expression of a multitude of glycocalyx compositions. For each strain and/or given growth environment, a number of cell coat polysaccharide states can be sampled to attain a balance that is suitable for a particular niche. The network of E. coli surface polysaccharides can be further subdivided into those that are tightly associated or covalently linked to the outer membrane (OM) (O/K-antigens, ECA) and those that are loosely associated, called exo-or slime polysaccharides. The exopolysaccharides (in particular CA and PNAG (8)) are integral components of biofilms, acting as the "cement," which holds together the various protein, lipid, and polysaccharide components (9). The vast majority of CA is secreted into this extracellular milieu, with no evidence existing of a lipid anchor tethering CA chains to the OM. Disruption of the putative acetyl transferase gene wcaF from the CA gene cluster in E. coli K-12 severely curtailed the maturation and development of the complex three-dimensional architecture typically associated with robust biofilms (10).CA is a polyanionic heteropolysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronic acid sugars that are nonstoichiometrically decorated with O-acetyl and pyruvate side chains (6, 11). The CA polysaccharide repeat is assembled on the membrane lipid undecaprenol pyrophosphate (Und-PP) by a series of glycosyl transferases on the cytoplasmic face of the inner membrane, after which the single repeat is flipped to the periplasmic side and polymerized by the Wzy-dependent pathway (reviewed in Refs. 1 and 3). Subsequently, the polymer is presumably cleaved from the Und-PP anchor, transported across the periplasm, and excreted into the extracellular space in a poorly understood process. The genetic determinant for CA biosynthesis resides ...

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli

“…Ligation of O-antigen to core by WaaL (RfaL) occurs at the periplasmic face of the IM (14). Despite the fact that E. coli K-12 lacks O-antigen (40), WaaL is present and can ligate colanic acid to the LPS core, and the resulting LPS migrates in gel electrophoresis as a ladder resembling those reported here (41). Moreover, colanic acid ligation to LPS occurs upon depletion of LptABDE (A. Polissi, personal communication).…”

Functional Characterization of UDP-Glucose:Undecaprenyl-Phosphate Glucose-1-Phosphate Transferases of Escherichia coli and Caulobacter crescentus

“…The chemical structure of the CA subunit ( Fig. 1) suggests that glucose is the initiating sugar for its synthesis (11,26). WcaJ is the only candidate protein encoded by the E. coli K-12 cps cluster that has features of a polyisoprenylphosphate hexose-1-phosphate transferase (44) and therefore could be the initiating enzyme for CA synthesis.…”

“…Unlike O antigen, which is attached to the cell surface via lipid A-core OS, cell surface CA has no identified lipid anchor and is loosely associated with the outer membrane. Under certain conditions, CA molecules can be attached covalently to lipid Acore OS (26), resulting in CA LPS . In this case, CA units are attached to a terminal heptose residue in the core OS (26), which is the normal attachment site recognized by the WaaL O-antigen ligase (10).…”

“…Under certain conditions, CA molecules can be attached covalently to lipid Acore OS (26), resulting in CA LPS . In this case, CA units are attached to a terminal heptose residue in the core OS (26), which is the normal attachment site recognized by the WaaL O-antigen ligase (10). Furthermore, CA LPS accumulates in E. coli strains with mutations in the LPS export pathway (42), and this process requires WaaL ligase function.…”

Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes

“…32,33 Likewise, S enterica sv Typhimurium LT2 does not produce a capsule (K. E. Sanderson, Salmonella Genetic Stock Center, Calgary, AB, e-mail, May 11, 2012). Whether expression of a capsule affects PF4 binding needs to be investigated in future studies.…”