Identification of the Pseudomonas aeruginosa O17 and O15 O-Specific Antigen Biosynthesis Loci Reveals an ABC Transporter-Dependent Synthesis Pathway and Mechanisms of Genetic Diversity.

Many bacterial cell surface glycans, such as the O antigen component of lipopolysaccharide (LPS), are produced via the so-called Wzx/Wzy- or ABC transporter-dependent pathways. O antigens are highly diverse polysaccharides that protect bacteria from their environment and engage in important host-pathogen interactions. The specific structure and composition of O antigens are the basis of classifying bacteria into O serotypes. In the opportunistic pathogen Pseudomonas aeruginosa, there are currently 20 known O-specific antigen (OSA) structures. The clusters of genes responsible for 18 of these O antigens have been identified, all of which follow the Wzx/Wzy-dependent pathway and are located at a common locus. In this study, we located the two unidentified O antigen biosynthesis clusters responsible for the synthesis of the O15 and the O17 OSA structures by analyzing published whole-genome sequence data. Intriguingly, these clusters were found outside the conserved OSA biosynthesis locus and were likely acquired through multiple horizontal gene transfer events. Based on data from knockout and overexpression studies, we determined that the synthesis of these O antigens follows an ABC transporter-dependent rather than a Wzx/Wzy-dependent pathway. In addition, we collected evidence to show that the O15 and O17 polysaccharide chain lengths are regulated by molecular rulers with distinct and variable domain architectures. The findings in this report are critical for a comprehensive understanding of O antigen biosynthesis in P. aeruginosa and provide a framework for future studies.IMPORTANCEP. aeruginosa is a problematic opportunistic pathogen that causes diseases in those with compromised host defenses, such as those suffering from cystic fibrosis. This bacterium produces a number of virulence factors, including a serotype-specific O antigen. Here, we identified and characterized the gene clusters that produce the O15 and O17 O antigens and show that they utilize a pathway for synthesis that is distinct from that of the 18 other known serotypes. We also provide evidence that these clusters have acquired mutations in specific biosynthesis genes and have undergone extensive horizontal gene transfer within the P. aeruginosa population. These findings expand on our understanding of O antigen biosynthesis in Gram-negative bacteria and the mechanisms that drive O antigen diversity.

The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology.

The major constituent of the outer membrane of Gram-negative bacteria is lipopolysaccharide (LPS), which is comprised of lipid A, core oligosaccharide, and O antigen, which is a long polysaccharide chain extending into the extracellular environment. Due to the localization of LPS, it is a key molecule on the bacterial cell wall that is recognized by the host to deploy an immune defence in order to neutralize invading pathogens. However, LPS also promotes bacterial survival in a host environment by protecting the bacteria from these threats. This review explores the relationship between the different LPS glycoforms of the opportunistic pathogen Pseudomonas aeruginosa and the ability of this organism to cause persistent infections, especially in the genetic disease cystic fibrosis. We also discuss the role of LPS in facilitating biofilm formation, antibiotic resistance, and how LPS may be targeted by new antimicrobial therapies.

Unique Regions of the Polysaccharide Copolymerase Wzz2 from Pseudomonas aeruginosa Are Essential for O-Specific Antigen Chain Length Control.

The outer leaflet of the outer membrane of nearly all Gram-negative bacteria contains lipopolysaccharide (LPS). The distal end of LPS may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length. The chain length of the polysaccharide has many implications for bacterial survival and consequently is tightly controlled. In the Wzx/Wzy-dependent route of O antigen synthesis, one or more Wzz proteins determine the chain length via an unknown mechanism. To gain insight into this mechanism, we identified and characterized important regions of two Wzz proteins in Pseudomonas aeruginosa serotype O13, which confer the production of "long" (Wzz1) and "very long" (Wzz2) chain lengths, respectively. We found that compared to Wzz1, Wzz2 has distinct amino acid insertions in the central α-helices (insα6 and insα7) and in membrane-distal (insL4) and -proximal (insIL) loops. When these regions were deleted in Wzz2, the mutant proteins conferred drastically shortened chain lengths. Within these regions we identified several conserved amino acid residues that were then targeted for site-directed mutagenesis. Our results implicate an RTE motif in loop 4 and a "hot spot" of charged and polar residues in insα7 in the function of Wzz2 We present evidence that the functionally important residues of insα7 are likely involved in stabilizing Wzz through coiled-coil interactions.IMPORTANCE O antigen is an important virulence factor presented on the cell surface of Gram-negative bacteria that is critical for bacterial physiology and pathogenesis. However, some aspects of O antigen biosynthesis, such as the mechanisms for determining polysaccharide chain length, are poorly understood. In this study, we identified unique regions in the O antigen chain length regulators (termed Wzz) of the problematic opportunistic pathogen Pseudomonas aeruginosa We show that these regions are critical for determining O antigen chain length, which provides new insight into the model of the Wzz mechanism. Ultimately, our work adds knowledge toward understanding an important step in the biosynthesis of this virulence factor, which is applicable to a wide range of Gram-negative pathogens.

Membrane Translocation and Assembly of Sugar Polymer Precursors.

Bacterial polysaccharides play an essential role in cell viability, virulence, and evasion of host defenses. Although the polysaccharides themselves are highly diverse, the pathways by which bacteria synthesize these essential polymers are conserved in both Gram-negative and Gram-positive organisms. By utilizing a lipid linker, a series of glycosyltransferases and integral membrane proteins act in concert to synthesize capsular polysaccharide, teichoic acid, and teichuronic acid. The pathways used to produce these molecules are the Wzx/Wzy-dependent, the ABC-transporter-dependent, and the synthase-dependent pathways. This chapter will cover the initiation, synthesis of the various polysaccharides on the cytoplasmic face of the membrane using nucleotide sugar precursors, and export of the nascent chain from the cytoplasm to the extracellular milieu. As microbial glycobiology is an emerging field in Gram-positive bacteria research, parallels will be drawn to the more widely studied polysaccharide biosynthesis systems in Gram-negative species in order to provide greater understanding of these biologically significant molecules.

A Bacteriophage-Acquired O-Antigen Polymerase (Wzyß) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα.

Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and ß, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a ß-polymerase (Wzyß). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyß was determined using a dual phoA-lacZα reporter system wherein random 3' gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to ß-galactosidase activity. The topology of Wzyß developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg(254), Arg(270), Arg(272), and His(300) were found to be essential for Wzyß function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzy ß knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce ß-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzy ß under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and ß-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyß utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1.

O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein. Amino acid substitutions in CL6 were found to alter Wzz-mediated O-antigen modality, with evidence suggesting that these changes may perturb the C-terminal WzyPa tertiary structure. Together, these data suggest that the catch-and-release mechanism of O-Ag polymerization is widespread among bacteria and that regulation of polymer length is affected by interaction of Wzz with Wzy.