Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2-5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA-wbpN. A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequences, IS 1209, is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.
A-band, a D-rhamnose-containing common lipopolysaccharide antigen isolated from Pseudomonas aeruginosa AK1401, was found to be a receptor for bacteriophage A7. The phage-borne rhamnanase was capable of hydrolyzing the A-band to expose core-lipid A containing only two or three rhamnose repeats. Interaction of the hydrolyzed A-band with core-or lipid A-specific monoclonal antibodies revealed that common epitopes exist in the inner core and lipid A regions, while the outer core of A-band appears to be different from that of the serotype-specific (B-band) lipopolysaccharide.Lipopolysaccharide (LPS), a major component of the gram-negative outer membrane (17), is composed of heteropolysaccharide covalently linked to lipid A (9, 21). The heteropolysaccharide consists of three regions: a diglucosamine backbone, the oligosaccharide core, and the 0-antigenic polysaccharide chain. Specific structures within the LPS molecule serve as receptors for a variety of bacteriophages (15,29). Phage adsorption to its receptor is highly specific (15). Phage resistance that results from changes in LPS structure usually indicates that the LPS is the surface receptor (29), and the structural change identifies the region of the LPS comprising the receptor. Since the structure of the 0-serotype-specific antigen varies from strain to strain, the host range of a phage whose receptor is the 0 polysaccharide is rather narrow (9,15). One characteristic of 0-specific phage is that during infection they often hydrolyze the 0 antigen, destroying the initial receptor (29). In many strains of Pseudomonas aeruginosa, a second LPS species whose polysaccharide chain differs serologically and structurally from the 0-antigen chain is present (10, 14, 16, 22-24, 30, 31). This LPS has been termed A-band or common antigen LPS. The common antigen polysaccharide of P. aeruginosa is a regular homopolymer of rhamnose. On the basis of nuclear magnetic resonance and chemical analyses, the structure has been shown to consist of the repeating unit (2,10,30,31). Interestingly, the structure proposed for the repeating unit of the rhamnan chain in the common antigen LPS of P. aeruginosa is identical to that reported for the O-polysaccharide chain in the LPS of Pseudomonas syringae pv. morsprunorum C28 (26).The 0 polysaccharide of P. syringae pv. morsprunorum C28 LPS, which is composed entirely of rhamnose (26), is specifically cleaved and released as oligosaccharides by the action of a rhamnanase borne on the typing phage A7 (25). This phage uses the LPS as its initial binding site (20,25). Thus, it is expected that the common antigen (A-band) LPS from P. aeruginosa will also serve as a substrate for phage A7. This phage will serve as a tool to remove the polyrhamnose chain from the A-band LPS for detailed analysis of the core and lipid A of this molecule. In this report, we present evidence to show that phage A7 binds to and hydrolyzes the * Corresponding author. polyrhamnose chain of the A-band LPS from P. aeruginosa AK1401 and that the phage-digested A-band LPS ca...
Several strains of Pseudomonas aeruginosa exhibited the ability to hemagglutinate erythrocytes. Hydrophobic bond-breaking agents, but not sugars and saccharides, were effective inhibitors of hemagglutination. The results suggest the involvement of hydrophobic bonds in hemagglutination reactions of P. aeruginosa.
Outer membrane derived 'ghosts' can be readily generated from both smooth and deep rough (heptose-deficient LPS) strains of Escherichia coli 08. MORPHOlogical and biochemical studies confirmed that 'ghosts' of both strains are composed of protein (four major proteins), LPS, and phospholipid (cardiolipin and phosphatidylethanolamine) in the form of a single membrane of roughly the same shape as intact normal cells. The ghost membrane cleaves only slightly in freeze-etch preparations of ghosts derived from the smooth strain as compared to the extensive cleavage plane of ghosts derived from the rough strain. The asymmetrical distribution of ghost proteins was visualized, by critical point drying and shadowing with platinum, as a relatively smooth outer surface with some discernible particles (10-15 nm) and an extremely particulate inner surface (10-15-mm particles. Ghosts derived from the smooth strain retained their structure following chloroform-methanol extraction, while ghosts derived from the rough strain fragmented with chloroform-methanol extraction. Evidence is presented that LPS-protein interactions as well as protein-protein interactions are significant in maintaining the ghost structure.
The molecular nature of the murine Ab response to Pseudomonas aeruginosa LPS was examined using a panel of 10 well-defined anti-LPS mAbs. Abs to P. aeruginosa LPS are encoded by diverse V-genes, with at least five VH and four V kappa gene families represented in these Abs. The Abs that bind to hydrophilic O-polysaccharide side chains of B-band LPS and A-band LPS are encoded by VH J558, SM7, and J606 gene families, while Abs to hydrophobic core and lipid A regions are encoded by X24, SM7, and Q52 gene families. All active JH and only two J kappa (J kappa 2 and J kappa 5) germ-line genes are utilized in the anti-LPS Abs examined. Four of six anti-P. aeruginosa mAbs used diversity genes of the DSP2 gene family. Interestingly, JH1 and JH2 use was observed in three mAbs that reacted with hydrophilic LPS epitopes (O-polysaccharide, A-band LPS), whereas JH3 and JH4 use was observed in three mAbs that bound to the more hydrophobic regions of LPS (core, lipid A). Point mutations were observed in framework and complementarity-determining regions (CDRs) of VH and VL genes, suggesting an Ag-driven maturation process in response to P. aeruginosa LPS. Mutations occurred in all heavy chain CDRs, as well as in CDR1 and CDR3 of the light chain, indicating an important role of these regions in binding to LPS. These data suggest that diverse VH and V kappa genes encode Abs to LPS from P. aeruginosa.
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