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Lipopolysaccharide O-antigens—bacterial glycans made to measure

Open AccessPublished:May 18, 2020DOI:https://doi.org/10.1074/jbc.REV120.009402
      Lipopolysaccharides are critical components of bacterial outer membranes. The more conserved lipid A part of the lipopolysaccharide molecule is a major element in the permeability barrier imposed by the outer membrane and offers a pathogen-associated molecular pattern recognized by innate immune systems. In contrast, the long-chain O-antigen polysaccharide (O-PS) shows remarkable structural diversity and fulfills a range of functions, depending on bacterial lifestyles. O-PS production is vital for the success of clinically important Gram-negative pathogens. The biological properties and functions of O-PSs are mostly independent of specific structures, but the size distribution of O-PS chains is particularly important in many contexts. Despite the vast O-PS chemical diversity, most are produced in bacterial cells by two assembly strategies, and the different mechanisms employed in these pathways to regulate chain-length distribution are emerging. Here, we review our current understanding of the mechanisms involved in regulating O-PS chain-length distribution and discuss their impact on microbial cell biology.
      Lipopolysaccharides (LPSs) are a family of structurally related glycolipids found in the outer membranes of most Gram-negative bacteria (
      • Megrian D.
      • Taib N.
      • Witwinowski J.
      • Beloin C.
      • Gribaldo S.
      One or two membranes? Diderm Firmicutes challenge the Gram‐positive/Gram‐negative divide.
      ). A typical Escherichia coli cell possesses ∼2 × 106 LPS molecules, covering about three quarters of the cell surface, and it has been estimated that 70,000 molecules/min are exported to the outer membrane to sustain the growth rate of this organism (reviewed in Ref.
      • Whitfield C.
      • Trent M.S.
      Biosynthesis and export of bacterial lipopolysaccharides.
      ). These are important macromolecules with a substantial amount of cellular resources dedicated to their production.
      LPS molecules contain a mix of well-conserved and highly variable structural elements. All LPS molecules contain a lipid component (lipid A), whose archetypal structure is composed of a bisphosphorylated disaccharide backbone carrying 4-7 acyl chains (
      • Whitfield C.
      • Trent M.S.
      Biosynthesis and export of bacterial lipopolysaccharides.
      ). Lipid A is a principle structural component in the outer leaflet of the outer membrane and is essential for viability of almost all LPS producers. The few species (Neisseria meningitidis, Moraxella catarrhalis, and Acinetobacter baumannii) that can withstand disruption of the conserved lipid A-biosynthesis pathway (Raetz pathway) require major reconfiguration of outer membrane biogenesis and homeostasis, and this is accompanied by a fitness cost (reviewed in Ref.
      • Simpson B.W.
      • Trent M.S.
      Pushing the envelope: LPS modifications and their consequences.
      ). The unique structure of lipid A contributes to the barrier properties of the outer membrane and resistance to antimicrobial peptides and antibiotics like polymyxin and colistin. It also provides a pathogen-associated molecular pattern, recognized by complexes of Toll-like receptor 4 and myeloid differentiation factor 2 (TLR4:MD2), to activate a proinflammatory response (
      • Simpson B.W.
      • Trent M.S.
      Pushing the envelope: LPS modifications and their consequences.
      ). Regulated modifications to the base lipid A structure allow some pathogens to enhance resistance or evade immune detection, and much of our knowledge of lipid A structure results from efforts to understand the structural determinants of these key properties. However, an LPS composed solely of lipid A is so far confined to the intracellular pathogen, Francisella novicida (
      • Wang X.
      • Ribeiro A.A.
      • Guan Z.
      • McGrath S.C.
      • Cotter R.J.
      • Raetz C.R.H.
      Structure and biosynthesis of free lipid A molecules that replace lipopolysaccharide in Francisella tularensis subsp. novicida.
      ). In all other bacteria, lipid A is subject to further glycosylation, creating remarkable structural diversity.
      LPS molecules typically contain a short “core oligosaccharide” structure (core OS), which is attached to lipid A via an α-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. Whereas the core OS architecture is relatively conserved within a species, the structures can vary considerably between species in terms of the number and type of sugars, the extent of branching, and the presence of nonglycose substituents, such as phosphate (
      • Holst O.
      • Brade H.
      Chemical structure of the core region of lipopolysaccharides.
      ). The core OS may also contribute to outer membrane stability, but only the Kdo addition is essential for viability in vitro in the E. coli paradigm. This reflects the order of assembly where two Kdo residues are added to the nascent structure prior to completion of lipid A acylation; underacylated LPS is a poor substrate for the LPS export machinery, which cannot sustain growth without suppressor mutations (reviewed in Ref.
      • Whitfield C.
      • Trent M.S.
      Biosynthesis and export of bacterial lipopolysaccharides.
      ).
      In some bacteria (often mucosal pathogens), the core oligosaccharide can be modified by a phase-variable (on-off) extension of one or a few sugars in a form known as lipooliogsaccharide. However, the more prevalent (and classical) LPS format shows a tripartite structure where the core OS is further glycosylated by a long-chain repeat-unit polysaccharide. This is generally called the O-antigen (O-PS), with the term originating from the Kauffman–White serological classification system developed for Salmonella in the 1930s. LPS molecules carrying O-PS are called “smooth LPS” (S-LPS), whereas those lacking O-PS are termed “rough” (R)-LPS (Fig. 1A). Again, this follows Salmonella precedent, describing the colony morphologies of these bacteria grown on solid media. O-PSs are hypervariable repeat-unit polysaccharides with known structures compiled in the Carbohydrate Structure Database (RRID:SCR_018684) (
      • Toukach P.V.
      • Egorova K.S.
      Carbohydrate structure database merged from bacterial, archaeal, plant and fungal parts.
      ). They can differ in glycose (i.e. sugar or sugar derivative) and nonglycose components, linkages, and topology of the repeat unit (Fig. 1C), generating distinct O-antigen epitopes. For example, there are 46 main O-serogroups in Salmonella, and some of these structures are also found among the >180 recognized O-serotypes in E. coli, reflecting the propensity for horizontal gene transfer (
      • Liu B.
      • Knirel Y.A.
      • Feng L.
      • Perepelov A.V.
      • Senchenkova S.N.
      • Reeves P.R.
      • Wang L.
      Structural diversity in Salmonella O antigens and its genetic basis.
      ,
      • Liu B.
      • Furevi A.
      • Perepelov A.V.
      • Guo X.
      • Cao H.
      • Wang Q.
      • Reeves P.R.
      • Knirel Y.A.
      • Wang L.
      • Widmalm G.
      Structure and genetics of Escherichia coli O antigens.
      ). The application of silver-stained SDS-PAGE offered the first insight into a critical LPS structural feature: heterogeneity in O-PS chain lengths within a population of bacteria (
      • Goldman R.C.
      • Leive L.
      Heterogeneity of antigenic-side-chain length in lipopolysaccharide from Escherichia coli O111 and Salmonella typhimurium LT2.
      ). O-PS chain-length distribution is characteristic for a particular isolate, and some examples are shown in Fig. 1B. An important feature of the SDS-PAGE profiles from WT isolates is the presence of “modal” clusters (marked by brackets in Fig. 1B) of stained molecules, where the O-PS chain-length distribution is skewed toward a particular size range. As will be described below, this is a regulated process and is important in LPS properties and functions, and the extent of the heterogeneity can offer insight into the O-PS assembly mechanism. The Salmonella enterica serovar Typhimurium pattern reveals a wide range of chain lengths, with the amount (and staining intensity) generally decreasing as chain lengths increase, with the exception of a higher-molecular-weight modal cluster of bands. In this species, the high-molecular-weight modal cluster is eliminated by deletion of wzz genes encoding chain length regulators (see the mutant profile). The examples shown from E. coli and Klebsiella pneumoniae lack similar chain length regulators and use a different mechanism to establish modality in chain length. In K. pneumoniae O2a, the pattern shows a relatively wide distribution of O-PS size. Capping of O2a chains with O1 antigen increases maximum chain length and adds an additional higher-molecular-weight cluster containing the O1 antigen. In contrast, E. coli O9a and K. pneumoniae O12 show much tighter modality with no appreciable amounts of shorter O-PS lengths. How are the distinctive chain-length patterns achieved, and what impact does this have on the biology of the bacteria? These questions are the subject of this review. We will describe our emerging understanding of the different molecular mechanisms involved in the biosynthesis of O-PS, highlighting relationships to systems involved in the production of other bacterial cell-surface polymers. Particular emphasis is placed on the regulation of O-PS polymerization machinery, to generate products with defined chain-length distributions, and the importance of this regulation to the success of bacterial pathogens.
      Figure thumbnail gr1
      Figure 1Structural overview of bacterial LPS molecules. A, cartoons of “smooth” O-antigen–containing LPS and “rough” LPS, which lacks the O-PS portion. B, examples of the size distribution of LPS molecules from different isolates, revealed by the profiles after separation on silver-stained SDS-polyacrylamide gels. C, representative O-PS repeat-unit structures. Specific features of these examples are discussed in the text.

      Biosynthetic bookends—shared processes in the initiation and termination of O-PS assembly

      All O-PSs are assembled as undecaprenyl diphosphate (Und-PP)-linked assembly intermediates (
      • Raetz C.R.H.
      • Whitfield C.
      Lipopolysaccharide endotoxins.
      ) in the cytoplasm and end with the ligation of Und-PP–linked O-PS glycans with defined chain-length distributions to lipid A core in the periplasm. Between these two conserved processes, the specific features of O-PS polymerization and export across the cytoplasmic membrane define three fundamentally different O-PS assembly strategies.
      Synthesis is initiated at the cytoplasmic face of the inner membrane by a phosphoglycosyltransferase (PGT) enzyme that transfers a hexose phosphate or acetamido sugar phosphate from the corresponding nucleotide diphosphoglycose donor to undecaprenyl phosphate (Und-P). PGT family members initiate all bacterial polysaccharides involving Und-PP–linked intermediates, including peptidoglycan, teichoic acids, N- and O-linked glycans in many glycosylated proteins, and some classes of capsular and exopolysaccharides. There have been important recent advances in understanding the molecular mechanisms of these enzymes (reviewed in Ref.
      • Allen K.N.
      • Imperiali B.
      Structural and mechanistic themes in glycoconjugate biosynthesis at membrane interfaces.
      ). PGT enzymes have been assigned to two superfamilies, which differ in membrane topology and mechanism (
      • Allen K.N.
      • Imperiali B.
      Structural and mechanistic themes in glycoconjugate biosynthesis at membrane interfaces.
      ). MraY from peptidoglycan biosynthesis is a prototypical “polytopic” PGT, with 10-11 transmembrane helices (TMHs) in its functional catalytic core and a proposed active site positioned within a cleft at the membrane surface created by the cytosolic loops (
      • Chung B.C.
      • Zhao J.
      • Gillespie R.A.
      • Kwon D.-Y.
      • Guan Z.
      • Hong J.
      • Zhou P.
      • Lee S.-Y.
      Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.
      ). Homologs of MraY include WecA, which initiates O-PS biosynthesis in many bacteria E. coli, Shigella, and Salmonella by the transfer of 2-acetamido-2-deoxy-glucose-1-phosphate (GlcNAc-1-P) to Und-P (
      • Alexander D.C.
      • Valvano M.A.
      Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine.
      ). Some O-serotypes then perform an epimerization to convert Und-PP–GlcNAc to Und-PP–GalNAc (
      • Rush J.S.
      • Alaimo C.
      • Robbiani R.
      • Wacker M.
      • Waechter C.J.
      A novel epimerase that converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157.
      ) before further elaboration. PglC from the N-glycosylation pathway in Campylobacter provides the prototype for monotopic PGTs. The PglC catalytic core reveals a new protein fold. The catalytic domain is stabilized at the membrane interface by an unusual strategy, involving a reentrant helix (that penetrates one membrane leaflet), and additional helices that are oriented planar to the membrane surface (
      • Ray L.C.
      • Das D.
      • Entova S.
      • Lukose V.
      • Lynch A.J.
      • Imperiali B.
      • Allen K.N.
      Membrane association of monotopic phosphoglycosyl transferase underpins function.
      ). Some OP-S PGTs, including the founding galactose-1-P transferase (WbaP) from Salmonella, share the same catalytic core as PglC. Although they may possess additional TMHs, these are not essential for catalysis, allowing their inclusion in the “monotopic” family (
      • Patel K.B.
      • Furlong S.E.
      • Valvano M.A.
      Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica.
      ,
      • Patel K.B.
      • Ciepichal E.
      • Swiezewska E.
      • Valvano M.A.
      The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate.
      ).
      O-PS synthesis ends with the production of an Und-PP–linked glycan chain, now located in the periplasm, which provides a donor substrate for glycosylation of lipid A core. The reaction is performed by the O-antigen ligase, encoded by a gene typically designated waaL (
      • Raetz C.R.H.
      • Whitfield C.
      Lipopolysaccharide endotoxins.
      ). WaaL is an integral membrane protein, but no solved structures are available (
      • Ruan X.
      • Monjarás Feria J.M.
      • Hamad M.
      • Valvano M.A.
      Escherichia coli Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties.
      ). Although the ligation reaction has been demonstrated biochemically with partially or fully defined substrates (
      • Ruan X.
      • Loyola D.E.
      • Marolda C.L.
      • Perez-Donoso J.M.
      • Valvano M.A.
      The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases.
      ,
      • Han W.
      • Wu B.
      • Li L.
      • Zhao G.
      • Woodward R.
      • Pettit N.
      • Cai L.
      • Thon V.
      • Wang P.G.
      Defining function of lipopolysaccharide O-antigen ligase WaaL using chemoenzymatically synthesized substrates.
      ), its activity in most species is only inferred from the LPS phenotype of waaL mutants. The ligation reaction is analogous to protein glycosylation, and sequence similarities have been recognized between WaaL and some bacterial oligosaccharyltransferases (
      • Schulz B.L.
      • Jen F.E.C.
      • Power P.M.
      • Jones C.E.
      • Fox K.L.
      • Ku S.C.
      • Blanchfield J.T.
      • Jennings M.P.
      Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.
      ). After ligation, the completed LPS molecule is translocated to the outer membrane and inserted into the outer leaflet by a conserved LPS transport (Lpt) machine spanning the cell envelope. The components and functions of the Lpt complex have been reviewed elsewhere (
      • Okuda S.
      • Sherman D.J.
      • Silhavy T.J.
      • Ruiz N.
      • Kahne D.
      Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model.
      ,
      • Sperandeo P.
      • Martorana A.M.
      • Polissi A.
      The Lpt ABC transporter for lipopolysaccharide export to the cell surface.
      ), and structures of subcomplexes have been described (
      • Qiao S.
      • Luo Q.
      • Zhao Y.
      • Zhang X.C.
      • Huang Y.
      Structural basis for lipopolysaccharide insertion in the bacterial outer membrane.
      ,
      • Dong H.
      • Zhang Z.
      • Tang X.
      • Paterson N.G.
      • Dong C.
      Structural and functional insights into the lipopolysaccharide ABC transporter LptB2FG.
      ,
      • Li Y.
      • Orlando B.J.
      • Liao M.
      Structural basis of lipopolysaccharide extraction by the LptB2FGC complex.
      ,
      • Tang X.
      • Chang S.
      • Luo Q.
      • Zhang Z.
      • Qiao W.
      • Xu C.
      • Zhang C.
      • Niu Y.
      • Yang W.
      • Wang T.
      • Zhang Z.
      • Zhu X.
      • Wei X.
      • Dong C.
      • Zhang X.
      • et al.
      Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism.
      ).
      Various aspects of O-PS assembly have been reviewed previously (e.g. see Refs.
      • Raetz C.R.H.
      • Whitfield C.
      Lipopolysaccharide endotoxins.
      and
      • Valvano M.A.
      • Furlong S.E.
      • Patel K.B.
      ,
      • Kalynych S.
      • Morona R.
      • Cygler M.
      Progress in understanding the assembly process of bacterial O-antigen.
      ,
      • Islam S.T.
      • Lam J.S.
      Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway.
      ,
      • Tytgat H.L.P.
      • Lebeer S.
      The sweet tooth of bacteria: common themes in bacterial glycoconjugates.
      ), and here we focus on recent advances in understanding chain length regulation and its impact on cellular functions.

      Regulating a polymerase—the Wzx/Wzy-dependent pathway

      The most prevalent O-PS biosynthesis process is the Wzx/Wzy-dependent pathway; as an example, it is used for 165 of 176 currently identified E. coli O-serotypes (
      • Liu B.
      • Furevi A.
      • Perepelov A.V.
      • Guo X.
      • Cao H.
      • Wang Q.
      • Reeves P.R.
      • Knirel Y.A.
      • Wang L.
      • Widmalm G.
      Structure and genetics of Escherichia coli O antigens.
      ). Studies involving E. coli, Shigella, Pseudomonas, and Salmonella provide the framework of biochemical knowledge gathered on this O-PS pathway, and many of the central steps have parallels in the biosynthesis of capsular and extracellular polysaccharides in Gram-positive and Gram-negative bacteria (
      • Whitfield C.
      • Wear S.S.
      • Sande C.
      Assembly of bacterial capsular polysaccharides and exopolysaccharides.
      ).
      After PGT-mediated initiation of O-PS biosynthesis, a series of reactions catalyzed by classical sugar nucleotide–dependent glycosyltransferases (GTs) completes the carbohydrate structure of the Und-PP–linked repeat unit at the cytoplasmic face of the inner membrane (Fig. 2A). Nonglycose components are sometimes added at this stage. The pathway is defined by the export of Und-PP–linked repeat units to the periplasm, where they are polymerized in a blockwise process by transfer of the growing O-PS from its lipid carrier to the nonreducing terminus of the incoming Und-PP repeat unit. These steps are performed by the Wzx exporter and Wzy polymerase, respectively. Modality of O-PS chain length distribution is established in the polymerization stage, prior to ligation (
      • Daniels C.
      • Griffiths C.
      • Cowles B.
      • Lam J.S.
      Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core.
      ,
      • Feldman M.F.
      • Wacker M.
      • Hernandez M.
      • Hitchen P.G.
      • Marolda C.L.
      • Kowarik M.
      • Morris H.R.
      • Dell A.
      • Valvano M.A.
      • Aebi M.
      Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.
      ), by proteins belonging to the Wzz family.
      Figure thumbnail gr2
      Figure 2O-PS biosynthesis in the Wzx/Wzy-dependent pathway. A, cartoon representation of the pathway identifying the major protein classes, using S. Typhimurium O:4 as the example (note that this O-PS can be further modified post-polymerization). The cartoon is intended to depict the components, and there is currently no understanding of their stoichiometry or relative positioning. As indicated in the text, the nascent lipid-linked O-PS may be associated with the surface of the Wzz oligomer, or polymerization may occur within its lumen. B, model of the S. Typhimurium WzzST oligomer derived from the cryo-EM structure (
      • Collins R.F.
      • Kargas V.
      • Clarke B.R.
      • Siebert C.A.
      • Clare D.K.
      • Bond P.J.
      • Whitfield C.
      • Ford R.C.
      Full-length, oligomeric structure of Wzz determined by cryoelectron microscopy reveals insights into membrane-bound states.
      ) with a ribbon depiction of the polypeptide chains (courtesy of R. C. Ford). The N- and C-terminal cytoplasmic segments are in green, the TMH segments in red, and the large periplasmic barrel–forming segments in blue. The transmembrane helices are embedded in a simulated lipid bilayer composed of POPC molecules (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) shown in stick representation, with the hydrocarbon chains in cyan, the glycerophosphate groups in red, and the choline groups in blue.
      Wzx is a subfamily of the MOP (multidrug/oligosaccharidyl-lipid/polysaccharide) flippase family of transporters. These are diverse integral membrane proteins that exert substrate preference for the structure of the O-PS repeat unit. The principles of substrate recognition are complicated and poorly understood, but some structural determinants appear to be particularly important for high efficiency flipping (reviewed in Refs.
      • Hong Y.
      • Liu M.A.
      • Reeves P.R.
      Progress in our understanding of Wzx flippase for translocation of bacterial membrane lipid-linked oligosaccharide.
      and
      • Liu M.A.
      • Morris P.
      • Reeves P.R.
      Wzx flippases exhibiting complex O-unit preferences require a new model for Wzx-substrate interactions.
      ). Export of nonnative O-PS can occur under conditions of Wzx overexpression. This suggests that the flippase can operate independently of a presumed assembly complex possessing fixed stoichiometry of components, and the physiological basis requires more investigation. Although the structure of Wzx has not been solved, structural and mechanistic insight is provided by a related flippase (MurJ) that exports Und-PP–linked repeat units of peptidoglycan. An overexpressed Wzx exporter from exopolysaccharide assembly can substitute for MurJ, reinforcing the possibility of a conserved mechanism in this class of exporters (
      • Sham L.-T.
      • Zheng S.
      • Yakhnina A.A.
      • Kruse A.C.
      • Bernhardt T.G.
      Loss of specificity variants of WzxC suggest that substrate recognition is coupled with transporter opening in MOP‐family flippases.
      ). The initial structures of two MurJ homologs in inward-facing conformations revealed a two-lobed structure with a predominantly cationic lumen that may be accessed via a lateral gate (
      • Kuk A.C.Y.
      • Mashalidis E.H.
      • Lee S.-Y.
      Crystal structure of the MOP flippase MurJ in an inward-facing conformation.
      ,
      • Zheng S.
      • Sham L.-T.
      • Rubino F.A.
      • Brock K.P.
      • Robins W.P.
      • Mekalanos J.J.
      • Marks D.S.
      • Bernhardt T.G.
      • Kruse A.C.
      Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.
      ). Mutagenesis, trapping of intermediates, and structures of different conformations (
      • Kuk A.C.Y.
      • Mashalidis E.H.
      • Lee S.-Y.
      Crystal structure of the MOP flippase MurJ in an inward-facing conformation.
      ,
      • Zheng S.
      • Sham L.-T.
      • Rubino F.A.
      • Brock K.P.
      • Robins W.P.
      • Mekalanos J.J.
      • Marks D.S.
      • Bernhardt T.G.
      • Kruse A.C.
      Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.
      ,
      • Kumar S.
      • Rubino F.A.
      • Mendoza A.G.
      • Ruiz N.
      The bacterial lipid II flippase MurJ functions by an alternating-access mechanism.
      ,
      • Kuk A.C.Y.
      • Hao A.
      • Guan Z.
      • Lee S.-Y.
      Visualizing conformation transitions of the Lipid II flippase MurJ.
      ,
      • Rubino F.A.
      • Mollo A.
      • Kumar S.
      • Butler E.K.
      • Ruiz N.
      • Walker S.
      • Kahne D.E.
      Detection of transport intermediates in the peptidoglycan flippase MurJ identifies residues essential for conformational cycling.
      ) support an alternating access model, where substrate binds at the lateral entry gate and is captured in the lumen in the inward-facing conformation. The substrate is then proposed to be flipped by a “rocker switch” mechanism in transition to the outward-facing conformation, and its release is encouraged by lumen shrinkage.
      Wzy polymerases transfer the growing O-PS onto the nonreducing end of the newly delivered repeat unit (
      • Bray D.
      • Robbins P.W.
      The direction of chain growth in Salmonella anatum O-antigen biosynthesis.
      ). These proteins are integral membrane proteins with a conserved membrane topology (
      • Islam S.T.
      • Lam J.S.
      Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway.
      ). Wzy proteins share a periplasmic Wzy_C motif with O-antigen ligases and some oligosaccharyltransferases for O-linked protein glycosylation, which also use Und-PP oligosaccharide substrates (
      • Schulz B.L.
      • Jen F.E.C.
      • Power P.M.
      • Jones C.E.
      • Fox K.L.
      • Ku S.C.
      • Blanchfield J.T.
      • Jennings M.P.
      Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.
      ). In Salmonella, Wzy proteins have complex specificities involving the glycose composition of the donor and acceptor oligosaccharides (
      • Reeves P.R.
      • Cunneen M.M.
      • Liu B.
      • Wang L.
      Genetics and evolution of the Salmonella galactose-initiated set of O antigens.
      ), but some homologs seem more promiscuous (
      • Merino S.
      • Gonzalez V.
      • Tomás J.M.
      The first sugar of the repeat units is essential for the Wzy polymerase activity and elongation of the O-antigen lipopolysaccharide.
      ). The Wzy mechanism is still speculative, and its structure and function are expected to be complex, based on precedent from other enzymes that use lipid-linked sugar or oligosaccharide donors, including bacterial N-linked oligosaccharyltransferase (PglB) (
      • Napiórkowska M.
      • Boilevin J.
      • Sovdat T.
      • Darbre T.
      • Reymond J.-L.
      • Aebi M.
      • Locher K.P.
      Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase.
      ) and GT-C-fold GT enzymes such as ALG6 GT from eukaryotic N-linked glycan assembly (
      • Bloch J.S.
      • Pesciullesi G.
      • Boilevin J.
      • Nosol K.
      • Irobalieva R.N.
      • Darbre T.
      • Aebi M.
      • Kossiakoff A.A.
      • Reymond J.-L.
      • Locher K.P.
      Structure and mechanism of the ER-based glucosyltransferase ALG6.
      ) (see below). In vitro demonstrations of Wzy activity lead to the conclusion that Wzy operates in a “distributive” mechanism (
      • Woodward R.
      • Yi W.
      • Li L.
      • Zhao G.
      • Eguchi H.
      • Sridhar P.R.
      • Guo H.
      • Song J.K.
      • Motari E.
      • Cai L.
      • Kelleher P.
      • Liu X.
      • Han W.
      • Zhang W.
      • Ding Y.
      • et al.
      In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.
      ,
      • Zhao G.
      • Wu B.
      • Li L.
      • Wang P.G.
      O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis.
      ). In contrast to processive enzymes that retain the growing glycan chain throughout chain extension, distributive enzymes release the glycan between additions, creating a broad profile of product sizes (
      • Yakovlieva L.
      • Walvoort M.T.C.
      Processivity in bacterial glycosyltransferases.
      ). This type of activity cannot explain the modal clusters seen in bacteria such as E. coli and Salmonella (Fig. 1B) and highlights the necessity for the final component of the assembly machinery, chain-length regulatory Wzz proteins belonging to the polysaccharide copolymerase family 1 (PCP-1, formerly MPA-1) (
      • Morona R.
      • Van Den Bosch L.
      • Daniels C.
      Evaluation of Wzz/MPA1/MPA2 proteins based on the presence of coiled-coil regions.
      ,
      • Morona R.
      • Purins L.
      • Tocilj A.
      • Matte A.
      • Cygler M.
      Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins.
      ).
      Initially, wzz genes were called rol (regulator of O-chain length) or cld (chain-length determinant) based on the loss of modal clusters of O-PS in the corresponding mutants (
      • Batchelor R.A.
      • Alifano P.
      • Biffali E.
      • Hull S.I.
      • Hull R.A.
      Nucleotide sequences of the genes regulating O-polysaccharide chain length (rol) from Escherichia coli Salmonella typhimurium: protein homology and functional complementation.
      ,
      • Bastin D.A.
      • Stevenson G.
      • Brown P.K.
      • Haase A.
      • Reeves P.R.
      Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.
      ) (Fig. 1B). A specific Wzz protein imparts a characteristic modality in O-chain lengths, but some organisms, including Salmonella (
      • Murray G.L.
      • Attridge S.R.
      • Morona R.
      Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz.
      ), Shigella (
      • Stevenson G.
      • Kessler A.
      • Reeves P.R.
      A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants.
      ), and P. aeruginosa (
      • Kintz E.
      • Scarff J.M.
      • DiGiandomenico A.
      • Goldberg J.B.
      Lipopolysaccharide O-antigen chain length regulation in Pseudomonas aeruginosa serogroup O11 strain PA103.
      ,
      • Huszczynski S.M.
      • Coumoundouros C.
      • Pham P.
      • Lam J.S.
      • Khursigara C.M.
      • Brun Y.V.
      Unique regions of the polysaccharide copolymerase Wzz2 from Pseudomonas aeruginosa are essential for O-specific antigen chain length control.
      ), express two wzz genes, imparting different modalities on the same O-PS. For example, some Salmonella isolates produce three ranges of O-PS lengths: “short” (<16 repeat units), “long” (
      • Patel K.B.
      • Furlong S.E.
      • Valvano M.A.
      Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica.
      ,
      • Patel K.B.
      • Ciepichal E.
      • Swiezewska E.
      • Valvano M.A.
      The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate.
      ,
      • Ruan X.
      • Monjarás Feria J.M.
      • Hamad M.
      • Valvano M.A.
      Escherichia coli Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties.
      ,
      • Ruan X.
      • Loyola D.E.
      • Marolda C.L.
      • Perez-Donoso J.M.
      • Valvano M.A.
      The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases.
      ,
      • Han W.
      • Wu B.
      • Li L.
      • Zhao G.
      • Woodward R.
      • Pettit N.
      • Cai L.
      • Thon V.
      • Wang P.G.
      Defining function of lipopolysaccharide O-antigen ligase WaaL using chemoenzymatically synthesized substrates.
      ,
      • Schulz B.L.
      • Jen F.E.C.
      • Power P.M.
      • Jones C.E.
      • Fox K.L.
      • Ku S.C.
      • Blanchfield J.T.
      • Jennings M.P.
      Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.
      ,
      • Okuda S.
      • Sherman D.J.
      • Silhavy T.J.
      • Ruiz N.
      • Kahne D.
      Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model.
      ,
      • Sperandeo P.
      • Martorana A.M.
      • Polissi A.
      The Lpt ABC transporter for lipopolysaccharide export to the cell surface.
      ,
      • Qiao S.
      • Luo Q.
      • Zhao Y.
      • Zhang X.C.
      • Huang Y.
      Structural basis for lipopolysaccharide insertion in the bacterial outer membrane.
      ,
      • Dong H.
      • Zhang Z.
      • Tang X.
      • Paterson N.G.
      • Dong C.
      Structural and functional insights into the lipopolysaccharide ABC transporter LptB2FG.
      ,
      • Li Y.
      • Orlando B.J.
      • Liao M.
      Structural basis of lipopolysaccharide extraction by the LptB2FGC complex.
      ,
      • Tang X.
      • Chang S.
      • Luo Q.
      • Zhang Z.
      • Qiao W.
      • Xu C.
      • Zhang C.
      • Niu Y.
      • Yang W.
      • Wang T.
      • Zhang Z.
      • Zhu X.
      • Wei X.
      • Dong C.
      • Zhang X.
      • et al.
      Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism.
      ,
      • Valvano M.A.
      • Furlong S.E.
      • Patel K.B.
      ,
      • Kalynych S.
      • Morona R.
      • Cygler M.
      Progress in understanding the assembly process of bacterial O-antigen.
      ,
      • Islam S.T.
      • Lam J.S.
      Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway.
      ,
      • Tytgat H.L.P.
      • Lebeer S.
      The sweet tooth of bacteria: common themes in bacterial glycoconjugates.
      ,
      • Whitfield C.
      • Wear S.S.
      • Sande C.
      Assembly of bacterial capsular polysaccharides and exopolysaccharides.
      ,
      • Daniels C.
      • Griffiths C.
      • Cowles B.
      • Lam J.S.
      Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core.
      ,
      • Feldman M.F.
      • Wacker M.
      • Hernandez M.
      • Hitchen P.G.
      • Marolda C.L.
      • Kowarik M.
      • Morris H.R.
      • Dell A.
      • Valvano M.A.
      • Aebi M.
      Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.
      ,
      • Hong Y.
      • Liu M.A.
      • Reeves P.R.
      Progress in our understanding of Wzx flippase for translocation of bacterial membrane lipid-linked oligosaccharide.
      ), and very long (>100) (
      • Hölzer S.U.
      • Schlumberger M.C.
      • Jäckel D.
      • Hensel M.
      Effect of the O-antigen length of lipopolysaccharide on the functions of type III secretion systems in Salmonella enterica.
      ). The Wzz proteins compete for the same pool of Und-PP–linked intermediates, and high levels of Wzy are required to support the very long modality (
      • Carter J.A.
      • Jiménez J.C.
      • Zaldívar M.
      • Álvarez S.A.
      • Marolda C.L.
      • Valvano M.A.
      • Contreras I.
      The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a.
      ), but the precise cellular concentration of Wzy and its stoichiometry with other essential components of the assembly pathway remain to be determined. The longer O-PS modalities require WzzST and WzzFepE, respectively, whereas short chains are Wzz-independent and generate an SDS-PAGE pattern where the probability of further O-PS extension decreases as the chains get longer (Fig. 1B). Synthesis of the shorter chains requires Wzy and two proteins of currently unknown function, PbgE2 and PbgE3 (
      • Farizano J.V.
      • Pescaretti M.d. L.
      • López F.E.
      • Hsu F.-F.
      • Delgado M.A.
      The PmrAB system-inducing conditions control both lipid A remodeling and O-antigen length distribution, influencing the Salmonella Typhimurium-host interactions.
      ). Both are integral membrane proteins, but they share no compelling structural similarity with Wzz proteins, and their mechanism(s) of action requires investigation.
      Wzz proteins form oligomers located in the inner membrane, and each protomer possesses two TMHs separated by a large periplasmic domain (
      • Morona R.
      • Purins L.
      • Tocilj A.
      • Matte A.
      • Cygler M.
      Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins.
      ). Crystal structures of periplasmic domains from several Wzz homologs from E. coli and Salmonella reveal a conserved fold with an α/β membrane-proximal base and an extended α-helical hairpin extending more than 100 Å into the periplasm (
      • Tocilj A.
      • Munger C.
      • Proteau A.
      • Morona R.
      • Purins L.
      • Ajamian E.
      • Wagner J.
      • Papadopoulos M.
      • Van Den Bosch L.
      • Rubinstein J.L.
      • Féthière J.
      • Matte A.
      • Cygler M.
      Bacterial polysaccharide co-polymerases share a common framework for control of polymer length.
      ). The protomers interact at their bases to create oligomers varying in size from trimers to nonamers in crystals. In contrast, EM images of negatively stained full-length proteins appear more uniform and reveal octamers (
      • Kalynych S.
      • Cherney M.
      • Bostina M.
      • Rouiller I.
      • Cygler M.
      Quaternary structure of WzzB and WzzE polysaccharide copolymerases.
      ). Reconstitution of three different Wzz proteins in proteoliposomes also generated a homogeneous population interpreted as hexamers (
      • Larue K.
      • Kimber M.S.
      • Ford R.
      • Whitfield C.
      Biochemical and structural analysis of bacterial O-antigen chain length regulator proteins reveals a conserved quaternary structure.
      ), but the data could potentially fit an octamer (
      • Kalynych S.
      • Cherney M.
      • Bostina M.
      • Rouiller I.
      • Cygler M.
      Quaternary structure of WzzB and WzzE polysaccharide copolymerases.
      ). Subsequently, a cryo-EM structure of full-length detergent-solubilized Wzz was solved (
      • Collins R.F.
      • Kargas V.
      • Clarke B.R.
      • Siebert C.A.
      • Clare D.K.
      • Bond P.J.
      • Whitfield C.
      • Ford R.C.
      Full-length, oligomeric structure of Wzz determined by cryoelectron microscopy reveals insights into membrane-bound states.
      ) (Fig. 2B). The packing of the protomers was consistent with the crystallographic structures, but they formed a dodecameric structure. Modeling and coarse-grained simulation in a lipid environment offered further structural insights: dodecamers are unstable in the membrane; the importance of the α/β domain in oligomerization was reinforced; the periplasmic α-helical domain has flexibility; and the TMHs are unlikely to contact one another within the lipid bilayer. Extensive mutagenesis has been performed on several Wzz homologs, and chimeric proteins have been constructed based on sequence and structural data. In general, surface-exposed residues in the periplasmic domains appear most likely to influence O-PS chain length, and regions distinguishing different modalities conferred by two Wzz proteins from P. aeruginosa O13 map to the periplasmic domain of those proteins (e.g. Refs.
      • Huszczynski S.M.
      • Coumoundouros C.
      • Pham P.
      • Lam J.S.
      • Khursigara C.M.
      • Brun Y.V.
      Unique regions of the polysaccharide copolymerase Wzz2 from Pseudomonas aeruginosa are essential for O-specific antigen chain length control.
      and
      • Kalynych S.
      • Ruan X.
      • Valvano M.A.
      • Cygler M.
      Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control.
      ). However, some residues in the base and oligomer lumen are also important. It has also been reported that mutations that shift oligomer stability also affect O-PS length, reinforcing the functional importance of an oligomeric structure (e.g. Refs.
      • Kalynych S.
      • Ruan X.
      • Valvano M.A.
      • Cygler M.
      Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control.
      and
      • Kintz E.N.
      • Goldberg J.B.
      Site-directed mutagenesis reveals key residue for O antigen chain length regulation and protein stability in Pseudomonas aeruginosa Wzz2.
      ). However, it remains to be established whether the differences in the numbers of protomers in oligomers reported in different studies are simply a result of the varied preparation approaches or if they reflect a mechanism where a dynamic oligomeric complex is integral to its function.
      How does this all translate into the function as an O-PS chain-length regulator? Despite the amount of available data, a compelling structure-function interpretation is elusive (
      • Kalynych S.
      • Valvano M.A.
      • Cygler M.
      Polysaccharide co-polymerases: the enigmatic conductors of the O-antigen assembly orchestra.
      ), but the role of Wzz does appear to be structural rather than catalytic. Over the years, several possible models have been proposed. In one, O-antigen length is modulated by the transient nature of the interaction between Wzy and Wzz, eventually allowing transfer of the growing glycan to WaaL for ligation to lipid A core (
      • Bastin D.A.
      • Stevenson G.
      • Brown P.K.
      • Haase A.
      • Reeves P.R.
      Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.
      ). In another proposed model, Wzz would facilitate interactions between Wzy and WaaL (
      • Morona R.
      • van den Bosch L.
      • Manning P.A.
      Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.
      ), but we have since learned that modality is established in the absence of WaaL (
      • Daniels C.
      • Griffiths C.
      • Cowles B.
      • Lam J.S.
      Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core.
      ,
      • Feldman M.F.
      • Wacker M.
      • Hernandez M.
      • Hitchen P.G.
      • Marolda C.L.
      • Kowarik M.
      • Morris H.R.
      • Dell A.
      • Valvano M.A.
      • Aebi M.
      Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.
      ). The initial Wzz structures led to a new hypothesis that Wzz and Wzy interact and different oligomer sizes influence modality (
      • Tocilj A.
      • Munger C.
      • Proteau A.
      • Morona R.
      • Purins L.
      • Ajamian E.
      • Wagner J.
      • Papadopoulos M.
      • Van Den Bosch L.
      • Rubinstein J.L.
      • Féthière J.
      • Matte A.
      • Cygler M.
      Bacterial polysaccharide co-polymerases share a common framework for control of polymer length.
      ). In support of interaction, Shigella Wzz seems to play a role in Wzy stability (
      • Nath P.
      • Morona R.
      Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity.
      ), and high levels of Wzy are needed to sustain synthesis of longer chains (
      • Carter J.A.
      • Jiménez J.C.
      • Zaldívar M.
      • Álvarez S.A.
      • Marolda C.L.
      • Valvano M.A.
      • Contreras I.
      The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a.
      ), but attempts to directly demonstrate Wzy:Wzz interactions have met with mixed results; interactions were detected in one study (
      • Nath P.
      • Morona R.
      Detection of Wzy/Wzz interaction in Shigella flexneri.
      ) but not in another (
      • Carter J.A.
      • Jiménez J.C.
      • Zaldívar M.
      • Álvarez S.A.
      • Marolda C.L.
      • Valvano M.A.
      • Contreras I.
      The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a.
      ). A more recent (hybrid) model proposes that polymerization continues until the binding capacity or lumen volume of a Wzz oligomer is met or Wzy:Wzz interactions cease (
      • Collins R.F.
      • Kargas V.
      • Clarke B.R.
      • Siebert C.A.
      • Clare D.K.
      • Bond P.J.
      • Whitfield C.
      • Ford R.C.
      Full-length, oligomeric structure of Wzz determined by cryoelectron microscopy reveals insights into membrane-bound states.
      ). This invokes measurement of chain length against some structural element of the Wzz oligomer, and O-PS:Wzz interaction has been reported (
      • Purins L.
      • Van Den Bosch L.
      • Richardson V.
      • Morona R.
      Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2.
      ). However, further elaboration of models requiring Wzy:Wzz interaction must accommodate the low levels of Wzy expression relative to Wzz and are limited by not knowing the site of interaction with the polymerase (i.e. is Wzy on the exterior surface of the Wzz oligomer or within the lumen?). Genetic studies have led to the hypothesis that Wzx, Wzy, and Wzz all interact to form a membrane-associated polymerization complex (
      • Marolda C.L.
      • Tatar L.D.
      • Alaimo C.
      • Aebi M.
      • Valvano M.A.
      Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide O antigen.
      ), and it is conceivable that this complex may also include the PGT and GT enzymes to coordinate synthesis, export, and polymerization of Und-PP–linked repeat units, which would afford other regulatory opportunities. For example, the flow of building blocks into the system may influence chain-length distributions, and a role for Wzz in modulating the activities of the PGT and Wzx proteins could also underpin chain-length regulation. Notably, an altered chain length phenotype has been reported for derivatives of the Salmonella PGT (WbaP) with a deletion in a periplasmic loop (
      • Saldias M.S.
      • Patel K.
      • Marolda C.L.
      • Bittner M.
      • Contreras I.
      • Valvano M.A.
      Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit.
      ). Genetic studies have also led to an interesting hypothesis where WaaL controls the level of Und-PP–linked intermediates available in the periplasm, potentially by regulating the PGT enzyme (
      • Hong Y.
      • Reeves P.R.
      Model for the controlled synthesis of O-antigen repeat units involving the WaaL ligase.
      ). In summary, there are many possible mechanistic explanations for regulating O-PS chain length distribution in Wzx/Wzy-dependent systems. Ultimately, a solution to the question is likely to require definitive structural insight into the identities and interactions of components in (multi)enzyme complexes to drive biochemical experiments.

      Molecular rulers and transport coupling—ABC transporter-dependent processes

      Another widespread O-PS assembly strategy is characterized by polymerization of the Und-PP–linked glycan at the cytoplasmic face of the membrane, prior to export to the periplasm by a member of the ABC transporter superfamily (Fig. 3A). In these systems, the PGT enzyme acts once per O-PS (providing the reducing terminal acetamido sugar), rather than once per repeat unit like Wzx/Wzy systems. Any acetamido sugars found in the repeat unit are added by additional GTs. O-PS chain-length distribution is still established at the polymerization stage, but two fundamentally different approaches have been described. The polymerization and export processes resemble those used in the production of teichoic acids and the glycans for S-layer glycoproteins (
      • Liston S.D.
      • Mann E.
      • Whitfield C.
      Glycolipid substrates for ABC transporters required for the assembly of bacterial cell-envelope and cell-surface glycoconjugates.
      ,
      • Mann E.
      • Kimber M.S.
      • Whitfield C.
      Bioinformatics analysis of diversity in bacterial glycan chain-termination chemistry and organization of carbohydrate binding modules linked to ABC transporters.
      ). ABC transporters are also used for the export of some capsular polysaccharides, but these are built on lipid carriers other than undecaprenol (
      • Whitfield C.
      • Wear S.S.
      • Sande C.
      Assembly of bacterial capsular polysaccharides and exopolysaccharides.
      ).
      Figure thumbnail gr3
      Figure 3O-PS biosynthesis in the ABC transporter–dependent pathway. A, cartoon representations of the pathway identifying the major protein classes. K. pneumoniae O2a and E. coli O9a are used to illustrate the pathway variants with different strategies for establishing O-PS chain-length distributions. Both complete polymerization in the cytoplasm and then export the product via the Wzm-Wzt ABC transporter (purple). In the more elaborate E. coli O9a mechanism, a chain-terminating protein (gray) adds methyl and phosphate residues to the nonreducing terminus, and an additional carbohydrate-binding module on the ABC transporter NBD (Wzt) recognizes the terminated O-PS substrate. B, conceptual model of the trimeric O2a polymerase (WbbM) based on the crystal structure of the catalytic domain and predicted organization of the unstructured linker (yellow) and membrane-tethering amphipathic helices. The catalytic modules with donor specificity for UDP-Galp (GT8) and UDP-Galf (GT111) are shown in orange and blue, respectively (modified from Ref.
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Sweeney R.P.
      • Kamski-Hennekam E.R.
      • Gitalis R.
      • Mallette E.
      • Kelly S.D.
      • Lowary T.L.
      • Kimber M.S.
      • Whitfield C.
      A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family.
      ). C, cartoon representation of the polymerization and chain termination mechanisms from E. coli O9a (left) and K. pneumoniae O12 (right). The model is based on the crystal structures of the termination catalytic modules (
      • Hagelueken G.
      • Clarke B.R.
      • Huang H.
      • Tuukkanen A.
      • Danciu I.
      • Svergun D.I.
      • Hussain R.
      • Liu H.
      • Whitfield C.
      • Naismith J.H.
      A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.
      ,
      • Williams D.M.
      • Ovchinnikova O.G.
      • Koizumi A.
      • Mainprize I.L.
      • Kimber M.S.
      • Lowary T.L.
      • Whitfield C.
      Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.
      ), and positioning of the polymerase is predicted from biochemical data (modified from Ref.
      • Williams D.M.
      • Ovchinnikova O.G.
      • Koizumi A.
      • Mainprize I.L.
      • Kimber M.S.
      • Lowary T.L.
      • Whitfield C.
      Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.
      ).
      For O-PS, two variations of the pathway have been described, and these differ in the presence or absence of a sophisticated chain-termination mechanism (Fig. 3A). The simpler system is exemplified by K. pneumoniae O2a, where O2a antigen (Fig. 1C) synthesis requires three GTs. Two adapter GTs add sequential Galp and Galf residues to Und-PP–GlcNAc (generated by WecA), directing it into the pathway, and the WbbM polymerase is solely responsible for extending the repeat-unit region of the glycan on this acceptor structure (
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Sweeney R.P.
      • Kamski-Hennekam E.R.
      • Gitalis R.
      • Mallette E.
      • Kelly S.D.
      • Lowary T.L.
      • Kimber M.S.
      • Whitfield C.
      A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family.
      ). WbbM is a dual GT domain protein that forms homotrimers, and the catalytic domains in each monomer are linked via a flexible tether to C-terminal membrane–associating amphipathic helices (
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Sweeney R.P.
      • Kamski-Hennekam E.R.
      • Gitalis R.
      • Mallette E.
      • Kelly S.D.
      • Lowary T.L.
      • Kimber M.S.
      • Whitfield C.
      A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family.
      ) (Fig. 3B). The six catalytic sites in the WbbM trimer are positioned on a single surface oriented toward the membrane where the Und-PP–glycan is anchored. WbbM interacts with other GTs in the pathway in a heterocomplex (
      • Kos V.
      • Whitfield C.
      A membrane-located glycosyltransferase complex required for biosynthesis of the d-galactan I lipopolysaccharide O antigen in Klebsiella pneumoniae.
      ), and the other enzymes presumably occupy space between the membrane and the WbbM catalytic sites. WbbM behaves like a distributive polymerase in vitro, and the chain-length distribution of native O2 O-PS (Fig. 1B) may be influenced by concentrating the WbbM reaction products in a localized space, as well as the inherent flexibility of the linker. Polymerization is terminated by export via the ABC transporter. In the absence of export, the O2a chains become aberrantly long, whereas overexpression of the transport results in chains with a shorter average size (
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
      ), but the mechanistic principles underpinning this type of regulation have not yet been established. In serotype O1, WbbM interacts with a second bifunctional polymerase (WbbY), which extends a polymer composed solely of Galp residues (Fig. 1B) on a short segment of O2a glycan (
      • Kelly S.D.
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Sweeney R.P.
      • Williamson M.L.
      • Lowary T.L.
      • Whitfield C.
      Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification.
      ). In O2c, two monofunctional GTs add a different disaccharide to an O2a acceptor, but WbbM does not interact with the additional GTs in two-hybrid experiments (
      • Kelly S.D.
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Sweeney R.P.
      • Williamson M.L.
      • Lowary T.L.
      • Whitfield C.
      Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification.
      ). The relative positioning of the various active sites in productive heterocomplex is an important question for further research.
      E. coli O9a and K. pneumoniae O12 show much finer control over O-PS chain length (Fig. 1B) and are leading examples of a more elaborate chain-length regulatory process. The O9a antigen (Fig. 1C) is part of a group of related O-PS structures found in E. coli, K. pneumoniae, and other bacteria, resulting from horizontal transfer of the genetic locus. The polymerization strategy is similar to that described above for K. pneumoniae O2a, with two adapter GTs participating with WecA to generate a dedicated acceptor, which is then extended by a dual GT-domain polymerase (WbdA) (
      • Greenfield L.K.
      • Richards M.R.
      • Li J.
      • Wakarchuk W.W.
      • Lowary T.L.
      • Whitfield C.
      Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases.
      ,
      • Greenfield L.K.
      • Richards M.R.
      • Vinogradov E.
      • Wakarchuk W.W.
      • Lowary T.L.
      • Whitfield C.
      Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens.
      ). In E. coli O9a, the N-terminal GT domain of WbdA adds two α1,2-linked Manp residues, and the C-terminal module adds two α1,3-linked residues to create the tetrasaccharide repeat unit (
      • Greenfield L.K.
      • Richards M.R.
      • Vinogradov E.
      • Wakarchuk W.W.
      • Lowary T.L.
      • Whitfield C.
      Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens.
      ,
      • Liston S.D.
      • Clarke B.R.
      • Greenfield L.K.
      • Richards M.R.
      • Lowary T.L.
      • Whitfield C.
      Domain interactions control complex formation and polymerase specificity in the biosynthesis of the Escherichia coli O9a antigen.
      ), but some wbdA mutations alter the precise activity of the N-terminal domain, and those proteins add either one or three α1,2-linked residues, changing the serotype (
      • Kido N.
      • Kobayashi H.
      A single amino acid substitution in a mannosyltransferase, WbdA, converts the Escherichia coli O9 polysaccharide into O9a: generation of a new O-serotype group.
      ,
      • Guachalla L.M.
      • Stojkovic K.
      • Hartl K.
      • Kaszowska M.
      • Kumar Y.
      • Wahl B.
      • Paprotka T.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      • Szijártó V.
      Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3.
      ). The modular WbdA enzymes also operate in a distributive mechanism in vitro (
      • Greenfield L.K.
      • Richards M.R.
      • Vinogradov E.
      • Wakarchuk W.W.
      • Lowary T.L.
      • Whitfield C.
      Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens.
      ), and the relatively limited range of product chain lengths (Fig. 1B) is dictated by a dedicated chain-termination mechanism prior to export (
      • Clarke B.R.
      • Cuthbertson L.
      • Whitfield C.
      Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter.
      ). WbdD is the bifunctional chain terminator that operates by adding a nonreducing terminal methylphosphate cap to prevent further polymerization (
      • Clarke B.R.
      • Cuthbertson L.
      • Whitfield C.
      Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter.
      ,
      • Clarke B.R.
      • Richards M.R.
      • Greenfield L.K.
      • Hou D.
      • Lowary T.L.
      • Whitfield C.
      In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan.
      ,
      • Hagelueken G.
      • Huang H.
      • Clarke B.R.
      • Lebl T.
      • Whitfield C.
      • Naismith J.H.
      Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a.
      ). What determines when the terminator intervenes in the process to create the observed modality? Mathematical modeling of O-PS chain-length distributions observed under conditions where the ratios of polymerase and terminator were experimentally manipulated implicated a structural element in the regulation (
      • King J.D.
      • Berry S.
      • Clarke B.R.
      • Morris R.J.
      • Whitfield C.
      Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry in Escherichia coli O9a.
      ), and this was explained by the subsequent discovery of a molecular ruler built into the machinery (
      • Hagelueken G.
      • Clarke B.R.
      • Huang H.
      • Tuukkanen A.
      • Danciu I.
      • Svergun D.I.
      • Hussain R.
      • Liu H.
      • Whitfield C.
      • Naismith J.H.
      A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.
      ). The WbdD terminator exists as a trimer, where the catalytic domains are separated from a membrane-associated amphipathic helix by an extended coiled-coil structure (
      • Hagelueken G.
      • Clarke B.R.
      • Huang H.
      • Tuukkanen A.
      • Danciu I.
      • Svergun D.I.
      • Hussain R.
      • Liu H.
      • Whitfield C.
      • Naismith J.H.
      A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.
      ). The polymerase (WbdA) and the terminator (WbdD) form a membrane-bound complex where WbdA interacts with the noncatalytic membrane-associated C-terminal region of WbdD (
      • Clarke B.R.
      • Greenfield L.K.
      • Bouwman C.
      • Whitfield C.
      Coordination of polymerization, chain termination, and export in assembly of the Escherichia coli O9a antigen in an ABC transporter-dependent pathway.
      ), creating a physical separation of the polymerization and termination catalytic sites (Fig. 3C). With this organization, polymerization can progress until the O-PS length is minimally sufficient for interaction with the terminator. Experimentally varying the length of the coiled-coil region by insertion or deletion of the defining heptad motifs caused corresponding changes in the size distribution of O9a O-PS, validating this component as the molecular ruler (
      • Hagelueken G.
      • Clarke B.R.
      • Huang H.
      • Tuukkanen A.
      • Danciu I.
      • Svergun D.I.
      • Hussain R.
      • Liu H.
      • Whitfield C.
      • Naismith J.H.
      A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.
      ).
      What about other O-PS structures? The K. pneumoniae O12 O-PS repeat unit (Fig. 1C) is capped by a single β-Kdo residue (
      • Vinogradov E.
      • Frirdich E.
      • MacLean L.L.
      • Perry M.B.
      • Petersen B.O.
      • Duus J.Ø.
      • Whitfield C.
      Structures of lipopolysaccharides from Klebsiella pneumoniae. Elucidation of the structure of the linkage region between core and polysaccharide O chain and identification of residues at the non-reducing termini of the O chains.
      ), and its chain-length distribution is regulated by a conceptually similar strategy. In this case, a single protein (WbbB) possesses both polymerization and termination activity. Two GT modules near the C terminus perform polymerization, and they are separated from a chain-terminating β-Kdo GT by a coiled-coil molecular ruler (
      • Williams D.M.
      • Ovchinnikova O.G.
      • Koizumi A.
      • Mainprize I.L.
      • Kimber M.S.
      • Lowary T.L.
      • Whitfield C.
      Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.
      ,
      • Ovchinnikova O.G.
      • Mallette E.
      • Koizumi A.
      • Lowary T.L.
      • Kimber M.S.
      • Whitfield C.
      Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99).
      ) (Fig. 3C). In principle, this process does not have to be confined to O-PSs, and strong candidates are found among systems from diverse bacterial genera that build glycans that decorate paracrystalline protein layers (S-layers) on the surfaces of some Gram-positive bacteria (
      • Steiner K.
      • Novotny R.
      • Werz D.B.
      • Zarschler K.
      • Seeberger P.H.
      • Hofinger A.
      • Kosma P.
      • Schäffer C.
      • Messner P.
      Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus.
      ). A recent bioinformatics survey has identified candidates for similar biosynthetic strategies and glycan terminator chemistries in a wide range of bacteria from different environments, producing glycans whose structures and functions have yet to be studied (
      • Mann E.
      • Kimber M.S.
      • Whitfield C.
      Bioinformatics analysis of diversity in bacterial glycan chain-termination chemistry and organization of carbohydrate binding modules linked to ABC transporters.
      ). These must now be validated experimentally to discover whether additional mechanistic complexity exists.
      Core ABC transporter structures possess two transmembrane domains (TMDs) forming the membrane channel and two nucleotide-binding domains (NBDs) that turnover ATP to drive transport (reviewed in Refs.
      • Locher K.P.
      Mechanistic diversity in ATP-binding cassette (ABC) transporters.
      and
      • Ford R.C.
      • Beis K.
      Learning the ABCs one at a time: structure and mechanism of ABC transporters.
      ). Most O-PS transporters are formed from a complex of two TMD proteins (called Wzm) and two NBDs (Wzt) (reviewed in Ref.
      • Liston S.D.
      • Mann E.
      • Whitfield C.
      Glycolipid substrates for ABC transporters required for the assembly of bacterial cell-envelope and cell-surface glycoconjugates.
      ), and this is the format found in K. pneumoniae O2a (
      • Bronner D.
      • Clarke B.R.
      • Whitfield C.
      Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1.
      ). An exception has been reported in export of Lewis antigen-mimicking Helicobacter pylori O-PS (
      • Hug I.
      • Couturier M.R.
      • Rooker M.M.
      • Taylor D.E.
      • Stein M.
      • Feldman M.F.
      Helicobacter pylori lipopolysaccharide is synthesized via a novel pathway with an evolutionary connection to protein N-glycosylation.
      ). The exporter involved (Wzk) is composed of two identical polypepeptides, which each contain one TMD and one NBD, resembling the PglK exporter from the Campylobacter N-glycosylation pathway (
      • Perez C.
      • Gerber S.
      • Boilevin J.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.-L.
      • Locher K.P.
      Structure and mechanism of an active lipid-linked oligosaccharide flippase.
      ). A solved structure is available for an ABC transporter from Aquifex aeolicus that closely resembles the E. coli O9a O-PS transporter (
      • Bi Y.
      • Mann E.
      • Whitfield C.
      • Zimmer J.
      Architecture of a channel-forming O-antigen polysaccharide ABC transporter.
      ,
      • Caffalette C.A.
      • Corey R.A.
      • Sansom M.S.P.
      • Stansfeld P.J.
      • Zimmer J.
      A lipid gating mechanism for the channel-forming O antigen ABC transporter.
      ). The transporter possesses a contiguous transmembrane channel to accommodate its glycan substrate, but it is notable that the O-PS substrates for these transporters are often linear glycans with side chains added post-transport (see below). The absence of elaborate side chains may reflect constraints imposed by the channel dimensions. An amphipathic gate helix at the NBD:TMD interface is thought to provide access to the channel for the substrate. The working model proposes iterative transport steps comparable with a translocase (like cellulose synthase, which catalyzes polymerization and export), rather than a conventional alternating access mechanism common in ABC transporters. The undecaprenyl lipid carrier is predicted to stay in the membrane phase, whereas the glycan traverses the membrane within the transporter lumen (
      • Bi Y.
      • Mann E.
      • Whitfield C.
      • Zimmer J.
      Architecture of a channel-forming O-antigen polysaccharide ABC transporter.
      ).
      The K. pneumoniae O2a ABC transporter transports Und-PP–linked products with no specificity for the glycan structure (
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
      ). In contrast, ABC transporters from systems that use a chain-termination mechanism possess an additional carbohydrate-binding module (CBM) that recognizes the terminal part of the glycan as a prerequisite for export (
      • Cuthbertson L.
      • Kimber M.S.
      • Whitfield C.
      Substrate binding by a bacterial ABC transporter involved in polysaccharide export.
      ,
      • Mann E.
      • Mallette E.
      • Clarke B.R.
      • Kimber M.S.
      • Whitfield C.
      The Klebsiella pneumoniae O12 ATP-binding cassette (ABC) transporter recognizes the terminal residue of its O-antigen polysaccharide substrate.
      ,
      • Mann E.
      • Kelly S.D.
      • Al-Abdul-Wahid M.S.
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Liu B.
      • Whitfield C.
      Substrate recognition by a carbohydrate-binding module in the prototypical ABC transporter for lipopolysaccharide O antigen from Escherichia coli O9a.
      ). In doing so, it ensures that the chain-length determination imparted during assembly is faithfully carried through to the final product on the cell surface. Exchange of the CBM confers new specificity on the transporter (
      • Cuthbertson L.
      • Powers J.
      • Whitfield C.
      The C-terminal domain of the nucleotide-binding domain protein Wzt determines substrate specificity in the ATP-binding cassette transporter for the lipopolysaccharide O antigens in Escherichia coli serotypes O8 and O9a.
      ). One mechanistic consequence of a separate substrate recognition event is that synthesis and transport can be temporally uncoupled in E. coli O9a; substrate engagement apparently only requires the terminating moiety. In contrast, the direct participation of the K. pneumoniae O2a ABC transporter in chain-length determination requires obligatory coupling of synthesis and transport (
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
      ). The A. aeolicus CBM was removed to facilitate crystallization of the ABC transporter, but it shares the same strand exchange–stabilized dimer organization and immunoglobulin-like structure seen in other homologues from E. coli O9a and K. pneumoniae O12 (
      • Cuthbertson L.
      • Kimber M.S.
      • Whitfield C.
      Substrate binding by a bacterial ABC transporter involved in polysaccharide export.
      ,
      • Mann E.
      • Mallette E.
      • Clarke B.R.
      • Kimber M.S.
      • Whitfield C.
      The Klebsiella pneumoniae O12 ATP-binding cassette (ABC) transporter recognizes the terminal residue of its O-antigen polysaccharide substrate.
      ,
      • Bi Y.
      • Zimmer J.
      Structure and ligand-binding properties of the O antigen ABC transporter carbohydrate-binding domain.
      ). The CBM is thought to increase the local concentration of substrate near the entrance into the transporter, but the presence of the CBM also significantly increases the ability of the transporter to turn over ATP in vitro, so its functional role is more complex than simple substrate recognition (
      • Bi Y.
      • Mann E.
      • Whitfield C.
      • Zimmer J.
      Architecture of a channel-forming O-antigen polysaccharide ABC transporter.
      ). After the ligase reaction transfers nascent O-PS to lipid A core, the spent undecaprenyl diphosphate is dephosphorylated and the monophosphate form is returned for another reaction cycle. Whereas the Wzm-Wzt structure represents a major advance, important questions still remain, including the mechanisms initiating transport and the reorientation of the undecaprenyl phosphate after ligation.

      An “outlier” assembly process in salmonella O:54

      The third O-PS assembly system is currently confined to a single example, S. enterica serovar Borreze O:54 (Fig. 4). The dedicated enzymes for this particular O-PS are encoded on a mobilizable plasmid, so the O:54 antigen can be coexpressed with a Wzx/Wzy pathway O-PS determined by the harboring isolate's chromosomal locus (
      • Keenleyside W.J.
      • Perry M.
      • Maclean L.L.
      • Poppe C.
      • Whitfield C.
      A plasmid-encoded rfbO:54 gene cluster is required for biosynthesis of the O:54 antigen in Salmonella enterica serovar Borreze.
      ). At the heart of the system is a single protein responsible for both chain extension and translocation of the O-PS to the periplasm, analogous to processes performed by bacterial synthases. Bacterial cellulose synthase offers the most detailed structural and biochemical understanding of these enzymes (
      • McNamara J.T.
      • Morgan J.L.W.
      • Zimmer J.
      A molecular description of cellulose biosynthesis.
      ). Like cellulose synthase, the O:54 synthase possesses a single-catalytic site GT module but has fewer TMHs, suggesting a topology closer to bacterial chitin and hyaluronan synthases, whose structures are not yet solved (
      • Wear S.S.
      • Hunt B.A.
      • Clarke B.R.
      • Whitfield C.
      Analysis of the topology and active-site residues of WbbF, a putative O-polysaccharide synthase from Salmonella enterica serovar Borreze.
      ). The polymerizing O:54 synthase activity requires a disaccharide acceptor synthesized by the WecA PGT as well as a dedicated adapter GT encoded on the plasmid (
      • Keenleyside W.J.
      • Whitfield C.
      A novel pathway for O-polysaccharide biosynthesis in Salmonella enterica serovar Borreze.
      ). The involvement of a PGT and WaaL to ligate the O-PS product to lipid A core dictates the involvement of Und-PP–linked intermediates, distinguishing it from other known synthases, which operate without lipid intermediates. It is unclear how this critical difference affects the translocation/flipping process. Cellulose synthase is a processive enzyme, and residues correlated with processivity are conserved in the O:54 enzyme, but how this class of enzymes determines glycan chain-length distribution remains unknown.
      Figure thumbnail gr4
      Figure 4O-PS biosynthesis in the synthase-dependent pathway. Shown is a cartoon representation of the pathway from the sole example, S. Borreze O:54, identifying the major protein classes. The characteristic synthase protein combines polymerase and export functions.

      Bacteriophages and the generation of O-PS diversity

      Bacteriophages are considered to be one of the main selective pressures driving bacterial surface glycan diversity (
      • Mostowy R.J.
      • Holt K.E.
      Diversity-generating machines: genetics of bacterial sugar-coating.
      ). Bacteriophage infection of Gram-negative bacteria involves complex interactions with cell-surface receptors, frequently outer membrane proteins or membrane-proximal parts of the LPS molecule (
      • Nobrega F.L.
      • Vlot M.
      • de Jonge P.A.
      • Dreesens L.L.
      • Beaumont H.J.E.
      • Lavigne R.
      • Dutilh B.E.
      • Brouns S.J.J.
      Targeting mechanisms of tailed bacteriophages.
      ). O-PS layers can mask such receptors, and some phages require tail fibers with enzymatic (e.g. glycosidase or deacetylase) activities to infect the host, but their role may go beyond simply clearing access to the surface; in many cases, interaction with O-PS is a necessary step in the infection process (
      • Broeker N.K.
      • Barbirz S.
      Not a barrier but a key: how bacteriophages exploit host's O‐antigen as an essential receptor to initiate infection.
      ). Understanding these processes and the potential steps to resistance is central to efforts to deploy bacteriophage therapies. Resistance to bacteriophages can follow several routes, the simplest being replacement of all or part of the O-PS locus by horizontal gene transfer (
      • Mostowy R.J.
      • Holt K.E.
      Diversity-generating machines: genetics of bacterial sugar-coating.
      ) or mutation to alter the specificity of key enzymes (like the E. coli O9 example described above). Another strategy that is available for laboratory-grown bacteria is loss of the O-PS receptor, and O-PS–specific phages played a vital role in the selection and characterization of mutants during the development of our understanding of LPS genetics and biochemistry. However, this clearly comes at a fitness cost outside the laboratory, given the important roles of O-PS in microbial biology that will be discussed below. An elegant study in S. Typhimurium illustrates one strategy to balance these competing selective pressures. The horizontally acquired opvAB genes are phase-variable (transcriptionally on or off) (
      • Cota I.
      • Sánchez-Romero M.A.
      • Hernández S.B.
      • Pucciarelli M.G.
      • Portillo F.G.-D.
      • Casadesús J.
      Epigenetic control of Salmonella enterica O-antigen chain length: a tradeoff between virulence and bacteriophage resistance.
      ). They are regulated by Dam-mediated methylation of DNA adenine residues and by the OxyR transcriptional regulator, which responds to reactive oxygen species. In the “on” phase, the longer O-PS chains generated by WzzST are diminished at the expense of chains in the range of 3–8 repeat units. The proposed mechanism involves inhibition of WzzST (and presumably WzzFepE) function by the small 34-residue OpvA peptide and installation of OpvB (which shares sequence similarity with WzzST) as a replacement. The “on” phase thereby creates a subpopulation of bacteria resistant to O-PS–targeted phages; those cells are unable withstand complement or infect macrophages, but virulent “off” phase cells can reemerge as soon as phage selection is removed.
      Some bacteriophages exploit a conceptually similar “inhibit and replace” strategy to alter Wzy polymerase specificity and change O-PS structure, effectively precluding further infection by phages using the same O-PS receptor. This process falls under the category of “serotype conversion,” where structural modifications in the O-PS change its immunological epitopes and response to typing antisera. The impact of lysogenic bacteriophages on O-PS serology was recognized more than 60 years ago in Salmonella (
      • Uetake H.
      • Luria S.E.
      • Burrous J.W.
      Conversion of somatic antigens in Salmonella by phage infection leading to lysis or lysogeny.
      ), and the change in backbone linkage was subsequently identified (
      • Losick R.
      Isolation of a trypsin-sensitive inhibitor of O-antigen synthesis involved in lysogenic conversion by bacteriophage ε15.
      ). In both Salmonella and P. aeruginosa, the native polymerase is inhibited by a small polypeptide containing a single TMH. The P. aeruginosa protein shares sequence similarity with the N-terminal TMH of Wzz, suggesting that it exploits structural elements that might normally be used in Wzy:Wzz functional interactions and consistent with the observation that inhibition is more effective in the absence of both Wzz proteins (
      • Taylor V.L.
      • Udaskin M.L.
      • Islam S.T.
      • Lam J.S.
      The D3 bacteriophage α-polymerase-inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa.
      ). A precise mechanism will require solved structures of Wzy:inhibitor and Wzy:Wzz complexes. Inhibition of the native Wzy is accompanied by an alternate inhibitor-resistant Wzy protein encoded by the same bacteriophage to switch the anomeric configuration of the glycan linkage.
      Other prophage-encoded structural diversification strategies include the addition of O-acetyl, phosphoethanolamine, or glycose residues to an O-PS structure. For example, many S. flexneri O-serotypes are based on the same O-PS backbone (serotype Y), and serological diversity is created by modifications at different sites (
      • Knirel Y.A.
      • Sun Q.
      • Senchenkova S.N.
      • Perepelov A.V.
      • Shashkov A.S.
      • Xu J.
      O-antigen modifications providing antigenic diversity of Shigella flexneri and underlying genetic mechanisms.
      ) (Fig. 1C). Whereas phage-encoded O-acetylation appears to be a cytosolic activity, glycosylation involves a periplasmic modification with interesting parallels to compartmentally separated stages in protein N-glycosylation in eukaryotes (Fig. 5). Periplasmic glycosylation is performed by a system that acts as an accessory module to existing assembly pathways. These modules were initially found in Salmonella O-PS but now extend to other species and to glycosylation of different cell-surface glycans (reviewed in Ref.
      • Mann E.
      • Whitfield C.
      A widespread three-component mechanism for the periplasmic modification of bacterial glycoconjugates.
      ). In Salmonella and Shigella, the modification involves O-PS glucosylation (encoded by gtr genes), whereas K. pneumoniae perform galactosylation (gml genes). In some bacteria, the chromosomal glycosylation locus is not associated with a prophage, offering potential insight into the original source of these systems. Periplasmic glycosylation precludes the use of nucleotide sugars as donors, and the three-component systems that direct the process are characterized by Und-P–glycose direct donors. The first two components catalyze synthesis and export of the donor and are conserved and functionally interchangeable between serotypes that add the same glycose modification, whereas the final transfer reaction is structure-specific. Und-P–glycose donors are synthesized by enzymes related to eukaryotic dolichol phosphate mannose synthase 1 (DPM1) and possess a conventional N-terminal glycosyltransferase GT-A fold (family GT2) anchored in the membrane by two C-terminal transmembrane helices. In the prototype from Synechocystis, the protein forms a tetramer with cytosolic catalytic sites located 15 Å from the membrane, and the reaction is thought to involve partial extraction of Und-P substrate from the membrane (
      • Ardiccioni C.
      • Clarke O.B.
      • Tomasek D.
      • Issa H.A.
      • Alpen, von D.C.
      • Pond H.L.
      • Banerjee S.
      • Rajashankar K.R.
      • Liu Q.
      • Guan Z.
      • Li C.
      • Kloss B.
      • Bruni R.
      • Kloppmann E.
      • Rost B.
      • et al.
      Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.
      ). The product is then flipped to the periplasmic face of the membrane by a protein sharing topology with flippases from related glycosylation systems and with EmrE, a MATE (multi-antimicrobial extrusion) family transporter (
      • Mann E.
      • Whitfield C.
      A widespread three-component mechanism for the periplasmic modification of bacterial glycoconjugates.
      ). Glycosylation of the O-PS backbone is performed by GT-C-fold enzymes resembling eukaryotic protein mannosyltransferases, like ALG6, which operate in the ER lumen. ALG6 possesses one structurally conserved and one variable integral membrane domain, and catalytic residues are placed in extramembrane loops (
      • Bloch J.S.
      • Pesciullesi G.
      • Boilevin J.
      • Nosol K.
      • Irobalieva R.N.
      • Darbre T.
      • Aebi M.
      • Kossiakoff A.A.
      • Reymond J.-L.
      • Locher K.P.
      Structure and mechanism of the ER-based glucosyltransferase ALG6.
      ).
      Figure thumbnail gr5
      Figure 5Periplasmic glycosylation of O-PS. The cartoon identifies the major protein classes, and the example is glucosylation of S. Typhimurium O:4.
      Periplasmic glycosylation in Salmonella occurs on an Und-PP–linked O-PS substrate, prior to ligation to lipid A core (
      • Hämmerling G.
      • Lüderitz O.
      • Westphal O.
      Structural investigations on the core polysaccharide of Salmonella typhimurium and the mode of attachment of the O-specific chains.
      ). The observation that the reducing terminal repeat unit is not glycosylated, and that LPS in wzy mutants is not modified, led to a conclusion that glycosylation required a minimum glycan chain length (
      • Hämmerling G.
      • Lüderitz O.
      • Westphal O.
      Structural investigations on the core polysaccharide of Salmonella typhimurium and the mode of attachment of the O-specific chains.
      ,
      • Wright A.
      Mechanism of conversion of the Salmonella O antigen by bacteriophage ε34.
      ,
      • Rudén U.
      • Mäkelä P.H.
      O-Acetylation and glucosylation of lipopolysaccharide in hydrids between Salmonella groups B and C2.
      ,
      • Helander I.M.
      • Moran A.P.
      • Mäkelä P.H.
      Separation of two lipopolysaccharide populations with different contents of O-antigen factor 122 in Salmonella enterica serovar Typhimurium.
      ). The structure of ALG6 suggests a reaction in close proximity to the membrane, implying the enzyme either lacks access to, or has low affinity for, shorter O-PS chains. Resolution of this question will require further biochemical investigation. The subsequent discovery of side-chain Gal addition in serotypes of K. pneumoniae (e.g. seroconversion of serotype O2a to O2afg, Fig. 1C) provides experimentally validated examples of three-component glycosylation of O-PSs from ABC transporter–dependent pathways (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ,
      • Mann E.
      • Ovchinnikova O.G.
      • King J.D.
      • Whitfield C.
      Bacteriophage-mediated glucosylation can modify lipopolysaccharide O-antigens synthesized by an ATP-binding cassette (ABC) transporter-dependent assembly mechanism.
      ,
      • Clarke B.R.
      • Ovchinnikova O.G.
      • Kelly S.D.
      • Williamson M.L.
      • Butler J.E.
      • Liu B.
      • Wang L.
      • Gou X.
      • Follador R.
      • Lowary T.L.
      • Whitfield C.
      Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.
      ). NMR data reveal that modification of these O-PSs can be stoichiometric when the modification genes are overexpressed, but they do not offer a reliable picture of the status of the terminal repeat units. The observed structure suggests a model where repeat units are modified as they emerge from the ABC transporter, and the associations of the GT-C enzyme with either polymerase or ABC transporter offer the next challenge in understanding this process.

      Impact of chain-length distribution on O-PS function

      Various functions have been assigned to O-PS, depending on the lifestyle of the bacterium. Most detailed (mechanistic) investigations have inevitably focused on human pathogens, but it is important to recognize that O-PS is equally important in plant pathogenesis (reviewed in Ref.
      • Lerouge I.
      • Vanderleyden J.
      O-antigen structural variation: mechanisms and possible roles in animal/plant–microbe interactions.
      ), interactions between plant pathogens such as Xylella fastidiosa and its insect vector (
      • Rapicavoli J.N.
      • Kinsinger N.
      • Perring T.M.
      • Backus E.A.
      • Shugart H.J.
      • Walker S.
      • Roper M.C.
      • Goodrich-Blair H.
      O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.
      ), and cell-cell interactions involving plant symbionts (
      • Russo D.M.
      • Abdian P.L.
      • Posadas D.M.
      • Williams A.
      • Vozza N.
      • Giordano W.
      • Kannenberg E.
      • Downie J.A.
      • Zorreguieta A.
      • Lovell C.R.
      Lipopolysaccharide O-chain core region required for cellular cohesion and compaction of in vitro and root biofilms developed by Rhizobium leguminosarum.
      ).
      Molecular simulations with E. coli LPS (
      • Blasco P.
      • Patel D.S.
      • Engström O.
      • Im W.
      • Widmalm G.
      Conformational dynamics of the lipopolysaccharide from Escherichia coli O91 revealed by nuclear magnetic resonance spectroscopy and molecular simulations.
      ) illustrate a model where the O-PS chains extend from the cell surface and their flexibility is constrained by adjacent LPS molecules. This offers a potential barrier impeding access to the cell surface by some larger molecules, underpinning many of the documented functions. The barrier has been visualized by EM and extends 31-36 nm from the outer membrane surface in P. aeruginosa (
      • Lam J.S.
      • Graham L.L.
      • Lightfoot J.
      • Dasgupta T.
      • Beveridge T.J.
      Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.
      ) and 35-70 nm in S. flexneri (
      • West N.P.
      • Sansonetti P.
      • Mounier J.
      • Exley R.M.
      • Parsot C.
      • Guadagnini S.
      • Prévost M.-C.
      • Prochnicka-Chalufour A.
      • Delepierre M.
      • Tanguy M.
      • Tang C.M.
      Optimization of virulence functions through glucosylation of Shigella LPS.
      ), depending on O-PS structure. Whereas O-PS functions vary with species and (in some cases) specific O-PS chemistry, they often contribute to protection against environmental threats including lysozyme (
      • Bao Y.
      • Zhang H.
      • Huang X.
      • Ma J.
      • Logue C.M.
      • Nolan L.K.
      • Li G.
      O-specific polysaccharide confers lysozyme resistance to extraintestinal pathogenic Escherichia coli.
      ), colicins (
      • Tran E.N.H.
      • Papadopoulos M.
      • Morona R.
      Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri.
      ), oxidative stress (
      • Zheng Y.
      • Wang H.
      • Huang L.
      • Zhang T.
      • Zong B.
      • Ren X.
      • Zhu Y.
      • Song F.
      • Wang X.
      • Chen H.
      • Tan C.
      Effect of O antigen ligase gene mutation on oxidative stress resistance and pathogenicity of NMEC strain RS218.
      ), and bile (
      • Crawford R.W.
      • Keestra A.M.
      • Winter S.E.
      • Xavier M.N.
      • Tsolis R.M.
      • Tolstikov V.
      • Bäumler A.J.
      Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance.
      ). They can also promote resistance to antimicrobial peptides (
      • Mercedes Pescaretti L.
      • de M.
      • López F.E.
      • Morero R.D.
      • Delgado M.A.
      The PmrA/PmrB regulatory system controls the expression of the wzzfepE gene involved in the O-antigen synthesis of Salmonella enterica serovar Typhimurium.
      ,
      • Thomassin J.-L.
      • Lee M.J.
      • Brannon J.R.
      • Sheppard D.C.
      • Gruenheid S.
      • Le Moual H.
      Both group 4 capsule and lipopolysaccharide O-antigen contribute to enteropathogenic Escherichia coli resistance to human α-defensin 5.
      ,
      • Günther S.D.
      • Fritsch M.
      • Seeger J.M.
      • Schiffmann L.M.
      • Snipas S.J.
      • Coutelle M.
      • Kufer T.A.
      • Higgins P.G.
      • Hornung V.
      • Bernardini M.L.
      • Höning S.
      • Krönke M.
      • Salvesen G.S.
      • Kashkar H.
      Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide.
      ), polymyxin (
      • Crawford R.W.
      • Keestra A.M.
      • Winter S.E.
      • Xavier M.N.
      • Tsolis R.M.
      • Tolstikov V.
      • Bäumler A.J.
      Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance.
      ), and colistin (
      • Ricci V.
      • Zhang D.
      • Teale C.
      • Piddock L.J.V.
      • Bonomo R.A.
      The O-antigen epitope governs susceptibility to colistin in Salmonella enterica.
      ), which is predominantly driven by lipid A modifications (
      • Simpson B.W.
      • Trent M.S.
      Pushing the envelope: LPS modifications and their consequences.
      ). Some O-PS structures can also contribute to the recognition of LPS by the innate immune system and offer additional elements to canonical immune responses under certain circumstances. Examples include recognition of O-PS by soluble CD14 (
      • Védrine M.
      • Berthault C.
      • Leroux C.
      • Répérant-Ferter M.
      • Gitton C.
      • Barbey S.
      • Rainard P.
      • Gilbert F.B.
      • Germon P.
      Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.
      ), activating cytokine production via a non-TLR4 pathway in NK cells (
      • Kanevskiy L.M.
      • Erokhina S.A.
      • Streltsova M.A.
      • Ziganshin R.H.
      • Telford W.G.
      • Sapozhnikov A.M.
      • Kovalenko E.I.
      The role of O-antigen in LPS-induced activation of human NK cells.
      ), and recognition of O-PS by C-type lectin 2 (Dectin-2) (
      • Wittmann A.
      • Lamprinaki D.
      • Bowles K.M.
      • Katzenellenbogen E.
      • Knirel Y.A.
      • Whitfield C.
      • Nishimura T.
      • Matsumoto N.
      • Yamamoto K.
      • Iwakura Y.
      • Saijo S.
      • Kawasaki N.
      Dectin-2 recognizes mannosylated O-antigens of human opportunistic pathogens and augments lipopolysaccharide activation of myeloid cells.
      ).
      The most detailed description of the importance of O-PS chain length is of its interaction with the complement system. Classic studies from Leive and co-workers (
      • Grossman N.
      • Schmetz M.A.
      • Foulds J.
      • Klima E.N.
      • Jimenez-Lucho V.E.
      • Leive L.L.
      • Joiner K.A.
      • Jiminez V.
      Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo.
      ) with Salmonella and E. coli established that resistance to complement-mediated killing in the absence of antibodies correlates strongly with O-PS size and, to some extent, its distribution (i.e. the percentage of “capped” lipid A cores). In these bacteria, O-PS efficiently activates the complement system, but a population of long-chain molecules preferentially bind complement component C3 (
      • Grossman N.
      • Schmetz M.A.
      • Foulds J.
      • Klima E.N.
      • Jimenez-Lucho V.E.
      • Leive L.L.
      • Joiner K.A.
      • Jiminez V.
      Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo.
      ,
      • Joiner K.A.
      • Grossman N.
      • Schmetz M.
      • Leive L.
      C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo.
      ). The net effect is that steric hindrance prevents the downstream complement cascade product, C5b-9, from interacting with the outer membrane surface and forming a stable, lasting, hydrophobic interaction. The amount of bound membrane attack complex, C5b-9, is a correlate of serum-mediated bacterial killing (
      • Joiner K.A.
      • Schmetz M.A.
      • Goldman R.C.
      • Leive L.
      • Frank M.M.
      Mechanism of bacterial resistance to complement-mediated killing: inserted C5b-9 correlates with killing for Escherichia coli O111B4 varying in O-antigen capsule and O-polysaccharide coverage of lipid A core oligosaccharide.
      ) as it is a prerequisite for insertion of the membrane attack complex into the outer membrane (
      • Heesterbeek D.A.
      • Bardoel B.W.
      • Parsons E.S.
      • Bennett I.
      • Ruyken M.
      • Doorduijn D.J.
      • Gorham R.D.
      • Berends E.T.
      • Pyne A.L.
      • Hoogenboom B.W.
      • Rooijakkers S.H.
      Bacterial killing by complement requires membrane attack complex formation via surface‐bound C5 convertases.
      ). Some O-PSs influence the rate of complement activation via different efficiencies of C3 deposition or C3 convertase function (
      • Liang-Takasaki C.J.
      • Grossman N.
      • Leive L.
      Salmonellae activate complement differentially via the alternative pathway depending on the structure of their lipopolysaccharide O-antigen.
      ,
      • Grossman N.
      • Joiner K.A.
      • Frank M.M.
      • Leive L.
      C3b binding, but not its breakdown, is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from Salmonellae.
      ,
      • Grossman N.
      • Svenson S.B.
      • Leive L.
      • Lindberg A.A.
      Salmonella O antigen-specific oligosaccharide-octyl conjugates activate complement via the alternative pathway at different rates depending on the structure of the O antigen.
      ,
      • Jimenez-Lucho V.E.
      • Joiner K.A.
      • Foulds J.
      • Frank M.M.
      • Leive L.
      C3b generation is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from salmonellae.
      ), but it is uncertain whether these properties are direct effects of chemistry or indirect results of O-PS conformation. These examples all involve O-PS produced by Wzx/Wzy-dependent pathways, but the same principles likely apply to the products of ABC transporter–dependent mechanisms. Isolates of K. pneumoniae O1 are resistant to serum killing, but mutants defective in production of the O1 antigen become sensitive despite still producing O-PS composed solely of the O2a antigen (
      • McCallum K.L.
      • Schoenhals G.
      • Laakso D.
      • Clarke B.
      • Whitfield C.
      A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.
      ). The killing effect was abrogated by heat-treating the serum, suggesting a role for complement. The survival of O1 isolates could reflect differences in either chain length or conformation. Notably, the addition of a side-chain galactose to the O2a antigen also confers some protection in K. pneumoniae O2afg isolates, which predominate in the carbapenemase-resistant ST258 clone (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ). In this case, the PAGE profiles of the corresponding LPS molecules do not suggest a large change in apparent size with or without the side-chain galactose.
      Antibodies recognizing cell-surface epitopes promote bacterial killing by the alternative complement pathway without increasing the amount of bound C5b-9 (
      • Joiner K.A.
      • Goldman R.C.
      • Hammer C.H.
      • Leive L.
      • Frank M.M.
      Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F (ab′)2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition.
      ), but O-PS has a complex and species-dependent impact on the efficacy of antibodies targeting surface epitopes. Early investigations suggested that O-PS presents a barrier to antibodies recognizing underlying proteins (e.g. Refs.
      • Bentley A.T.
      • Klebba P.E.
      Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.
      and
      • van der Ley P.
      • Kuipers O.
      • Tommassen J.
      • Lugtenberg B.
      O-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae.
      ), but a recent study illustrates that the role of O-PS in this context can involve more than a simple steric barrier (
      • Domínguez-Medina C.C.
      • Pérez-Toledo M.
      • Schager A.E.
      • Marshall J.L.
      • Cook C.N.
      • Bobat S.
      • Hwang H.
      • Chun B.J.
      • Logan E.
      • Bryant J.A.
      • Channell W.M.
      • Morris F.C.
      • Jossi S.E.
      • Alshayea A.
      • Rossiter A.E.
      • et al.
      Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface.
      ). In this case, interactions between IgG and an outer membrane protein antigen were influenced by the physical space occupied by the antigen and its dynamic interaction with O-PS. The end result is that effective antibody binding required the cognate O-PS and outer membrane protein epitope. In Bordetella parapertussis, O-PS protects the bacterium against opsonization with antibodies induced against surface antigens by the acellular B. pertussis vaccine. Although these bacteria are taken up by polymorphonuclear leukocytes, they are targeted to lipid rafts and trafficked to nonbactericidal LAMP-negative phagosomes, where they survive (
      • Gorgojo J.
      • Lamberti Y.
      • Valdez H.
      • Harvill E.T.
      • Rodríguez M.E.
      Bordetella parapertussis survives the innate interaction with human neutrophils by impairing bactericidal trafficking inside the cell through a lipid raft-dependent mechanism mediated by the lipopolysaccharide O antigen.
      ). Perhaps surprisingly, high-titer IgG antibodies directed against O-PS can enhance complement resistance in Salmonella (
      • MacLennan C.A.
      • Gilchrist J.J.
      • Gordon M.A.
      • Cunningham A.F.
      • Cobbold M.
      • Goodall M.
      • Kingsley R.A.
      • van Oosterhout J.J.G.
      • Msefula C.L.
      • Mandala W.L.
      • Leyton D.L.
      • Marshall J.L.
      • Gondwe E.N.
      • Bobat S.
      • López-Macías C.
      • et al.
      Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults.
      ), Pseudomonas aeruginosa (
      • Wells T.J.
      • Whitters D.
      • Sevastsyanovich Y.R.
      • Heath J.N.
      • Pravin J.
      • Goodall M.
      • Browning D.F.
      • O'Shea M.K.
      • Cranston A.
      • De Soyza A.
      • Cunningham A.F.
      • MacLennan C.A.
      • Henderson I.R.
      • Stockley R.A.
      Increased severity of respiratory infections associated with elevated anti-LPS IgG2 which inhibits serum bactericidal killing.
      ), and uropathogenic Escherichia coli (UPEC) (
      • Coggon C.F.
      • Jiang A.
      • Goh K.G.K.
      • Henderson I.R.
      • Schembri M.A.
      • Wells T.J.
      A novel method of serum resistance by Escherichia coli that causes urosepsis.
      ). The underlying mechanism appears to involve a barrier of bound antibodies on long chain O-PS that may further encourage complement deposition away from the outer membrane and physically blocks access of other protective antibodies.
      E. coli, Salmonella, and P. aeruginosa possess multimodal O-PS chain-length distributions due to the possession of two or more chain-length regulatory proteins, and this offers a mechanistic avenue to modulate chain-length distributions and contribute differentially to the biology of the organism. In Salmonella, the O-PS pattern is influenced by the level of expression of the O-PS biosynthesis locus, which is regulated by the transcriptional antiterminator RfaH (
      • Wang L.
      • Jensen S.
      • Hallman R.
      • Reeves P.R.
      Expression of the O antigen gene cluster is regulated by RfaH through the JUMPstart sequence.
      ) and by σ factors RpoN and RpoS via RfaH (
      • Bittner M.
      • Saldías S.
      • Altamirano F.
      • Valvano M.A.
      • Contreras I.
      RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi.
      ) as well as its balance with Wzz proteins, which affect wzy expression and Wzy activity (
      • Carter J.A.
      • Blondel C.J.
      • Zaldívar M.
      • Álvarez S.A.
      • Marolda C.L.
      • Valvano M.A.
      • Contreras I.
      O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene.
      ,
      • da Silva P.
      • Manieri F.Z.
      • Herrera C.M.
      • Trent M.S.
      • Moreira C.G.
      • Raffatellu M.
      Novel role of VisP and the Wzz system during O-antigen assembly in Salmonella enterica serovar Typhimurium pathogenesis.
      ). “Long” O-PS is generally considered to protect Salmonella against serum killing and is dictated by WzzST. wzzST expression is regulated by the RcsAB two-component system associated with stress response and swarming behavior (
      • Delgado M.A.
      • Mouslim C.
      • Groisman E.A.
      The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant.
      ) and depends on Dam methylation (
      • Sarnacki S.H.
      • Castañeda M.D.R.A.
      • Noto Llana M.
      • Giacomodonato M.N.
      • Valvano M.Á.
      • Cerquetti M.C.
      Dam methylation participates in the regulation of PmrA/PmrB and RcsC/RcsD/RcsB two component regulatory systems in Salmonella enterica serovar Enteritidis.
      ). “Very long” O-PS, directed by WzzFepE, can confer a fitness advantage on serovar Typhimurium in the inflamed intestine by offering resistance to the elevated bile levels in the lumen during colitis (
      • Crawford R.W.
      • Keestra A.M.
      • Winter S.E.
      • Xavier M.N.
      • Tsolis R.M.
      • Tolstikov V.
      • Bäumler A.J.
      Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance.
      ). Interestingly, S. Typhi does not cause acute colitis, and the absence of very-long O-PS (due to wzzFepE being a pseudogene in this species) enhances immune evasion enabled by the capsular polysaccharide known as Vi-antigen produced by serovar Typhi (
      • Crawford R.W.
      • Wangdi T.
      • Spees A.M.
      • Xavier M.N.
      • Tsolis R.M.
      • Bäumler A.J.
      Loss of very-long O-antigen chains optimizes capsule-mediated immune evasion by Salmonella enterica serovar Typhi.
      ).
      The role of O-PS in macrophage interactions is complex and may involve further fine tuning of O-PS size and structure. In bacteria like E. coli, Salmonella, and Shigella, interpretations are complicated by the production of so-called group 4 capsules. These redirect O-PS glycan from Wzx/Wzy-dependent pathways to the cell surface by a separate translocation machinery without ligation to lipid A core (
      • Whitfield C.
      Biosynthesis and assembly of capsular polysaccharides in Escherichia coli.
      ). They can contribute to the same biological properties and functions as LPS-linked O-PS but in ways that are sometimes different and often poorly understood. Here we confine the discussion to the LPS-linked forms. Production of “very long” O-PS is also important for uptake of Salmonella by macrophages (
      • Murray G.L.
      • Attridge S.R.
      • Morona R.
      Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement.
      ) as well as survival and replication within the Salmonella-containing vacuole (
      • da Silva P.
      • Manieri F.Z.
      • Herrera C.M.
      • Trent M.S.
      • Moreira C.G.
      • Raffatellu M.
      Novel role of VisP and the Wzz system during O-antigen assembly in Salmonella enterica serovar Typhimurium pathogenesis.
      ). Growth conditions that mimic those within the macrophage lead to enhanced production of “very long” O-PS (
      • Daines D.A.
      • Silver R.P.
      Evidence for multimerization of Neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors.
      ). In serovar Typhimurium, expression of pbgE2 and pbgE3 (associated with “short” O-PS), wzzST, and wzzFepE is regulated by the PmrAB two-component system (
      • Farizano J.V.
      • Pescaretti M.d. L.
      • López F.E.
      • Hsu F.-F.
      • Delgado M.A.
      The PmrAB system-inducing conditions control both lipid A remodeling and O-antigen length distribution, influencing the Salmonella Typhimurium-host interactions.
      ,
      • Mercedes Pescaretti L.
      • de M.
      • López F.E.
      • Morero R.D.
      • Delgado M.A.
      The PmrA/PmrB regulatory system controls the expression of the wzzfepE gene involved in the O-antigen synthesis of Salmonella enterica serovar Typhimurium.
      ,
      • Delgado M.A.
      • Mouslim C.
      • Groisman E.A.
      The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant.
      ). Subtle differences in regulation may occur between serovars, assisting niche adaptation. For example, PmrA apparently represses wzzST in serovar Enteritidis and induces wzzST in Typhimurium (
      • Sarnacki S.H.
      • Castañeda M.D.R.A.
      • Noto Llana M.
      • Giacomodonato M.N.
      • Valvano M.Á.
      • Cerquetti M.C.
      Dam methylation participates in the regulation of PmrA/PmrB and RcsC/RcsD/RcsB two component regulatory systems in Salmonella enterica serovar Enteritidis.
      ). The involvement of PmrAB integrates modulation of O-PS chain length into a coordinated response to polycations and iron that also includes regulated modifications of lipid A. The interdependence extends to an effect of WzzST on the balance of lipid A modifications (
      • Farizano J.V.
      • Pescaretti M.d. L.
      • López F.E.
      • Hsu F.-F.
      • Delgado M.A.
      The PmrAB system-inducing conditions control both lipid A remodeling and O-antigen length distribution, influencing the Salmonella Typhimurium-host interactions.
      ). The broader connection is reinforced by VisP, a periplasmic protein that affects outer membrane structure and integrity and works together with WzzST and WzzFepE to affect wzy expression (
      • da Silva P.
      • Manieri F.Z.
      • Herrera C.M.
      • Trent M.S.
      • Moreira C.G.
      • Raffatellu M.
      Novel role of VisP and the Wzz system during O-antigen assembly in Salmonella enterica serovar Typhimurium pathogenesis.
      ). Exactly how these processes are functionally integrated is an important question for further investigation.
      Differences between the O-PS in Shigella sonnei and S. flexneri contribute to the reduced uptake and vacuolar escape observed for S. sonnei, in turn leading to less inflammatory cell death in infected macrophages (
      • Watson J.L.
      • Sanchez-Garrido J.
      • Goddard P.J.
      • Torraca V.
      • Mostowy S.
      • Shenoy A.R.
      • Clements A.
      Shigella sonnei O-antigen inhibits internalization, vacuole escape, and inflammasome activation.
      ). S. flexneri uses the needle structures of type-3 protein secretion systems (T3SS) to inject effector proteins as an essential step in the invasion of epithelial cells and evasion of innate immunity, but the efficiency of T3SS function is influenced by O-PS structure (
      • West N.P.
      • Sansonetti P.
      • Mounier J.
      • Exley R.M.
      • Parsot C.
      • Guadagnini S.
      • Prévost M.-C.
      • Prochnicka-Chalufour A.
      • Delepierre M.
      • Tanguy M.
      • Tang C.M.
      Optimization of virulence functions through glucosylation of Shigella LPS.
      ). Constitutive periplasmic O-PS glycosylation can affect the conformation of the closest linkage in the O-PS backbone in molecular modeling studies (
      • Theillet F.-X.
      • Simenel C.
      • Guerreiro C.
      • Phalipon A.
      • Mulard L.A.
      • Delepierre M.
      Effects of backbone substitutions on the conformational behavior of Shigella flexneri O-antigens: implications for vaccine strategy.
      ), leading to up to 50% reduction in the physical length of the O-PS chain in S. flexneri (
      • West N.P.
      • Sansonetti P.
      • Mounier J.
      • Exley R.M.
      • Parsot C.
      • Guadagnini S.
      • Prévost M.-C.
      • Prochnicka-Chalufour A.
      • Delepierre M.
      • Tanguy M.
      • Tang C.M.
      Optimization of virulence functions through glucosylation of Shigella LPS.
      ). Although this does not appear to affect resistance to serum killing, it does have a marked influence on the exposure and function of T3SS. Whereas glucosylation is constitutive in Shigella, O-PS glucosylation in laboratory-grown S. Typhimurium only occurs at low levels because it is subject to genetic (form) variation in this species (
      • Mäkelä P.H.
      Glucosylation of lipopolysaccharide in Salmonella: mutants negative for O antigen factor 122.
      ), and the ability to switch glucosylation on/off promotes long-term colonization of the intestine (
      • Bogomolnaya L.M.
      • Santiviago C.A.
      • Yang H.-J.
      • Baumler A.J.
      • Andrews-Polymenis H.L.
      Form variation” of the O12 antigen is critical for persistence of Salmonella Typhimurium in the murine intestine.
      ). After macrophage infection, S. flexneri uses O-PS to block apoptosis by inhibiting caspase activity (
      • Günther S.D.
      • Fritsch M.
      • Seeger J.M.
      • Schiffmann L.M.
      • Snipas S.J.
      • Coutelle M.
      • Kufer T.A.
      • Higgins P.G.
      • Hornung V.
      • Bernardini M.L.
      • Höning S.
      • Krönke M.
      • Salvesen G.S.
      • Kashkar H.
      Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide.
      ). O-PS–dependent anti-apoptotic function is not confined to Shigella. It has been proposed that Porphyromonas gingivalis O-PS interacts with bacterial (gingipain) proteases to promote their involvement in anti-apoptotic pathways (
      • Soto C.
      • Bugueño I.
      • Hoare A.
      • Gonzalez S.
      • Venegas D.
      • Salinas D.
      • Melgar-Rodríguez S.M.
      • Vernal R.
      • Gamonal J.
      • Quest A.F.G.
      • Pérez-Donoso J.M.P.
      • Bravo D.
      The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.
      ). The influence of O-PS may also extend beyond the intact bacterial cell, as O-PS in some E. coli isolates may enhance the efficiency and uptake of outer membrane vesicles by host cells by enabling raft-dependent endocytosis (
      • O'Donoghue E.J.
      • Sirisaengtaksin N.
      • Browning D.F.
      • Bielska E.
      • Hadis M.
      • Fernandez-Trillo F.
      • Alderwick L.
      • Jabbari S.
      • Krachler A.M.
      Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells.
      ).
      Most research concerning the biological impact O-PS chain length is directed at bacterial pathogenesis. However, there is also an interesting role played in cell-surface architecture in some bacteria that produce paracrystalline protein arrays (S-layers) to form a molecular sieve that covers the cell and protects it against various environmental stresses. Caulobacter crescentus O-PS plays a pivotal role in nucleating and binding S-layer to the surface. During assembly, the S-layer monomers bind to a specific structural element in the O-PS repeat units in a Ca2+-dependent process and then migrate to oligomerize at the tips of O-PS to form the intact layer (
      • Comerci C.J.
      • Herrmann J.
      • Yoon J.
      • Jabbarpour F.
      • Zhou X.
      • Nomellini J.F.
      • Smit J.
      • Shapiro L.
      • Wakatsuki S.
      • Moerner W.E.
      Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly.
      ,
      • von Kügelgen V,. A.
      • Tang H.
      • Hardy G.G.
      • Kureisaite-Ciziene D.
      • Brun Y.V.
      • Stansfeld P.J.
      • Robinson C.V.
      • Bharat T.A.M.
      In situ structure of an intact lipopolysaccharide-bound bacterial surface layer.
      ). Optimal assembly in this system is presumably aided by the tight modal chain-length distribution of O-PS in this species (
      • Walker S.G.
      • Karunaratne D.N.
      • Ravenscroft N.
      • Smit J.
      Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer.
      ), and the process appears to involve an ABC transporter–dependent pathway (
      • Awram P.
      • Smit J.
      Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus.
      ).

      Conclusions

      In the last few years, we have seen major advances in our understanding of O-PS biosynthesis, due to the combined application of biochemistry and structural biology. Access to large amounts of genome sequence data are offering an opportunity to understand the distribution of prototypical systems and components as well as reveal new systems for investigation that do not conform to currently recognized parameters. This information is already being used to dissect broader functions of O-PS to focus on the roles of specific structures, chain lengths, and regulatory systems. An important application of the mechanistic insight into O-PS assembly lies in the expanding field of glycoengineering, which was founded on the discovery of strategies to transfer heterologous O-PS to protein carriers by exploiting the Campylobacter N-glycosylation system (reviewed in Ref.
      • Harding C.M.
      • Feldman M.F.
      Glycoengineering bioconjugate vaccines, therapeutics, and diagnostics in E. coli.
      ). The approach has been expanded to include different bacterial glycans and human glycan mimics in search of novel vaccines, therapeutics, and diagnostic tools. Bacterial glycan biosynthesis machinery is important because of the vast diversity of enzyme activities and availability of homologs that may have more advantageous properties. Central to these exciting applications is a fundamental understanding of how glycan assembly systems work, how components from different assembly pathways can be productively combined for new functionalities, and whether the critical specificity of a particular enzyme is affected by its use outside the natural context. There are still important questions to resolve around the structure and mechanism of key membrane proteins, the architecture of multienzyme complexes, and the cellular factors that determine the cellular distribution of these complexes and the allocation of shared Und-P carrier. However, the framework is established, and the necessary tools are now available to answer these questions.

      Acknowledgments

      We thank Dr. Olga Ovchinnikova and Prof. Robert Ford for help with figures and members of the Whitfield laboratory for comments on drafts of the manuscript.

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