Common Polysaccharide Antigen Research Articles

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energise their transport into cells and catalyse their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Structural Characterization and Evaluation of an Epitope at the Tip of the A-Band Rhamnan Polysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Biosynthesis of the Pseudomonas aeruginosa common polysaccharide antigen by D-Rhamnosyltransferases WbpX and WbpY.

The Gram-negative bacterium Pseudomonas aeruginosa simultaneously expresses two O-antigenic glycoforms. While the O-specific antigen (OSA) is variable in composition, the common polysaccharide antigen (CPA) is highly conserved and is composed of a homopolymer of D-rhamnose (D-Rha) in trisaccharide repeating units [D-Rhaα1-2-D-Rhaα1-3-D-Rhaɑ1-3]n. We have previously reported that α3-D-Rha-transferase WbpZ transfers a D-Rha residue from GDP-D-Rha to D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl. Genes encoding two more D-Rha-transferases are found in the O antigen gene cluster (wbpX and wbpY). In this study we showed that WbpX and WbpY recombinantly expressed in E. coli differ in their donor and acceptor specificities and have properties of GT-B folded enzymes of the GT4 glycosyltransferase family. NMR spectroscopic analysis of the WbpY reaction product showed that WbpY transferred one D-Rha residue in α1-3 linkage to synthetic D-Rhaα1-3-D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl acceptor. WbpX synthesized several products that contained D-Rha in both α1-2 and α1-3 linkages. Mass spectrometry indicated that the mixture of WbpX and WbpY efficiently catalyzed the synthesis of D-Rha oligomers in a non-processive mechanism. Since O antigens are virulence factors, these findings open the door to advancing technology for antibacterial drug discovery and vaccine development.Supplementary informationThe online version contains supplementary material available at 10.1007/s10719-022-10040-4.

Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with KDs in the nM range. These HMW carbohydrates contain 74.9–80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewisx, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex.

SummaryThe common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l‐ and d‐rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o‐methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

Loss of wbpL disrupts O-polysaccharide synthesis and impairs virulence of plant-associated Pseudomonas strains.

SummaryDespite its importance for membrane stability and pathogenicity of mammalian pathogens, functions of the O‐polysaccharide (OPS) of lipopolysaccharide (LPS) remain unclear in plant‐associated bacteria. Genetic information about OPS biosynthesis in these bacteria is largely missing. Genome analysis of various plant‐associated Pseudomonas strains revealed that one of the two known OPS biosynthesis clusters from Pseudomonas aeruginosa PAO1, the common polysaccharide antigen (CPA) gene cluster, is only conserved in some strains of the Pseudomonas fluorescens group. For the O‐specific antigen (OSA) biosynthesis cluster, the putative genomic position could be identified, but orthologues of most functional important OSA biosynthesis enzymes could not be detected. Nevertheless, orthologues of the glycosyltransferase WbpL, required for initiation of CPA and OSA synthesis in P. aeruginosa PAO1, could be identified in the analysed Pseudomonas genomes. Knockout mutations of wbpL orthologues in Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas cichorii ATCC10857/DSM50259 (Pci) resulted in strains lacking the OPS. Infection experiments of Arabidopsis thaliana plants revealed a reduced entry into the leaf apoplast after spray inoculation and a reduced apoplastic amplification of Pst ∆wbpL. Stab and spray inoculation of lettuce (Lactuca sativa) leaves with Pci ∆wbpL causes reduced infection symptoms compared to the wild‐type strain. Furthermore, swarming motility was reduced in ∆wbpL mutants of Pst and Pci. This might be a possible reason for reduced bacterial titres after surface inoculation and reduced bacterial amplification in the plant. Our results imply that the presence of lipopolysaccharide OPS is required for efficient host colonization and full virulence of plant‐pathogenic Pseudomonas bacteria.

LlpB represents a second subclass of lectin-like bacteriocins.

SummaryBacteriocins are secreted bacterial proteins that selectively kill related strains. Lectin‐like bacteriocins are atypical bacteriocins not requiring a cognate immunity factor and have been primarily studied in Pseudomonas. These so‐called LlpAs are composed of a tandem of B‐lectin domains. One domain interacts with d‐rhamnose residues in the common polysaccharide antigen of Pseudomonas lipopolysaccharide (LPS). The other lectin domain is crucial for interference with the outer membrane protein assembly machinery by interacting with surface‐exposed loops of its central component BamA. Via genome mining, we identified a second subclass of Pseudomonas lectin‐like proteins, termed LlpB, consisting of a single B‐lectin domain. We show that these proteins also display bactericidal activity. Among LlpB‐resistant transposon mutants of an LlpB‐susceptible Pseudomonas strain, a major subset was hit in an acyltransferase gene, predicted to be involved in LPS core modification, hereby suggesting that LlpBs equally attach to LPS for surface anchoring. This indicates that LPS binding and target strain specificity are condensed in a single B‐lectin domain. The identification of this second subclass of lectin‐like bacteriocins further expands the toolbox of antibacterial warfare deployed by bacteria and holds potential for their integration in biotechnological applications.

Hitting with a BAM: Selective Killing by Lectin-Like Bacteriocins.

ABSTRACTLectin-like bacteriocins (LlpAs) are secreted by proteobacteria and selectively kill strains of their own or related species, and they are composed of two B-lectin domains with divergent sequences. In Pseudomonas spp., initial binding of these antibacterial proteins to cells is mediated by the carboxy-terminal domain through d-rhamnose residues present in the common polysaccharide antigen of their lipopolysaccharide, whereas the amino-terminal domain accounts for strain selectivity of killing. Here, we show that spontaneous LlpA-resistant mutants carry mutations in one of three surface-exposed moieties of the essential β-barrel outer membrane protein insertase BamA, the core component of the BAM complex. Polymorphism of this loop in different Pseudomonas groups is linked to LlpA susceptibility, and targeted cells all share the same signature motif in this loop. Since heterologous expression of such a bamA gene confers LlpA susceptibility upon a resistant strain, BamA represents the primary bacteriocin selectivity determinant in pseudomonads. Contrary to modular bacteriocins that require uptake via the Tol or Ton system, parasitism of BamA as an LlpA receptor advocates a novel bacteriocin killing mechanism initiated by impairment of the BAM machinery.

Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa pathogenicity island 1 (PAPI-1) is one of the largest genomic islands of this important opportunistic human pathogen. Previous studies have shown that PAPI-1 encodes several putative virulence factors, including a major regulator of biofilm formation and antibiotic-resistance traits. PAPI-1 is horizontally transferable into recipient strains lacking this island via conjugation mediated by the specialized type IV pilus. The PAPI-1 encodes a cluster of ten genes associated with the synthesis and assembly of the type IV pilus. The PAPI-1 acquisition mechanism is currently not well understood.ResultsIn this study, we performed a series of conjugation experiments and identified determinants of PAPI-1 acquisition by analyzing transfer efficiency between the donor and a series of mutant recipient strains. Our data show that common polysaccharide antigen (CPA) lipopolysaccharide (LPS), a homopolymer of D-rhamnose, is required for initiating PAPI-1 transfer, suggesting that this structure acts as a receptor for conjugative type IV pilus in recipient strains. These results were substantiated by experimental evidence from PAPI-1 transfer assay experiments, in which outer membrane or LPS preparations from well-defined LPS mutants were added to the transfer mix to assess the role of P. aeruginosa LPS in PAPI-1 transfer and in vitro binding experiments between pilin fusion protein GST-pilV2’ and immobilized LPS molecules were performed. Our data also showed that P. aeruginosa strains that had already acquired a copy of PAPI-1 were unable to import additional copies of the island, and that such strains produced proportionally lower amounts of CPA LPS compared to the strains lacking PAPI-1.ConclusionsThese results suggest that a PAPI-1 exclusion mechanism exists in P. aeruginosa that might serve to regulate the avoidance of uncontrolled expansions of the bacterial genome.

Discovery, characterization and invivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa.

Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonasaeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity invivo in a murine model of P.aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P.aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right.

Polymyxin Susceptibility in Pseudomonas aeruginosa Linked to the MexXY-OprM Multidrug Efflux System.

The ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced the mexXY genes of the MexXY-OprM multidrug efflux system in Pseudomonas aeruginosa and increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci, arnBCADTEF and PA4773-74. Consistent with the SPC-promoted reduction in arn and PA4773-74 expression being linked to mexXY, expression of these LPS modification loci was moderated in a mutant constitutively expressing mexXY and enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lacking arnB and/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lacking mexXY was unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance 'mechanism' is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-type P. aeruginosa but not in the ΔmexXY mutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-induced mexXY expression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility.

Single-Nucleotide Polymorphisms Found in the migA and wbpX Glycosyltransferase Genes Account for the Intrinsic Lipopolysaccharide Defects Exhibited by Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa PA14 is widely used by researchers in many laboratories because of its enhanced virulence over strain PAO1 in a wide range of hosts. Although lipopolysaccharide (LPS) is an important virulence factor of all P. aeruginosa strains, the LPS of PA14 has not been characterized fully. A recent study showed that the structure of its O-specific antigen (OSA) belongs to serotype O19. We found that the OSA gene cluster of PA14 shares ∼99% identity with those of the O10/O19 group. These two serotypes share the same O-unit structure, except for an O-acetyl substitution in one of the sugars in O10. Here we showed that both PA14 and O19 LPS cross-reacted with the O10-specific monoclonal antibody MF76-2 in Western blots. Analysis by SDS-PAGE and silver staining showed that PA14 LPS exhibited modal chain lengths that were different from those of O19 LPS, in that only "very long" and "short" chain lengths were observed, while "medium" and "long" chain lengths were not detected. Two other novel observations included the lack of the uncapped core oligosaccharide epitope and of common polysaccharide antigen (CPA) LPS. The lack of the uncapped core oligosaccharide was caused by point mutations in the glycosyltransferase gene migA, while the CPA-negative phenotype was correlated with a single amino acid substitution, G20R, in the glycosyltransferase WbpX. Additionally, we showed that restoring CPA biosynthesis in PA14 significantly stimulated mature biofilm formation after 72 h, while outer membrane vesicle production was not affected. P. aeruginosa PA14 is a clinical isolate that has become an important reference strain used by many researchers worldwide. LPS of PA14 has not been characterized fully, and hence, confusion about its phenotype exists in the literature. In the present study, we set out to characterize the O-specific antigen (OSA), the common polysaccharide antigen (CPA), and the core oligosaccharide produced by PA14. We present evidence that PA14 produces an LPS consisting of "very-long-chain" and some "short-chain" OSA belonging to the O19 serotype but is devoid of CPA and the uncapped core oligosaccharide epitope. These intrinsic defects in PA14 LPS were due to single-nucleotide polymorphisms (SNPs) in the genes that encode glycosyltransferases in the corresponding biosynthesis pathways. Since sugars in CPA and the uncapped core are receptors for different bacteriocins and pyocins, the lack of CPA and an intact core may contribute to the increased virulence of PA14. Restoring CPA production in PA14 was found to stimulate mature biofilm formation.

Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-D-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-D-Rhamnosyltransferase WbpZ.

The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of D-rhamnose (D-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-D-Rha and enzymatically synthesized GDP-D-[(3)H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one D-Rha residue from GDP-D-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring D-mannose (D-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-D-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-rhamnosyltransferase that has significant activity of GDP-D-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a D-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. This is the first characterization of a D-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen.

O serotype-independent susceptibility of Pseudomonas aeruginosa to lectin-like pyocins.

Lectin-like bacteriocins of the LlpA family, originally identified in plant-associated bacteria, are narrow-spectrum antibacterial proteins composed of two tandemly organized monocot mannose-binding lectin (MMBL) domains. The LlpA-like bacteriocin of Pseudomonas aeruginosa C1433, pyocin L1, lacks any similarity to known P. aeruginosa bacteriocins. The initial interaction of pyocin L1 with target cells is mediated by binding to d-rhamnose, present in the common polysaccharide antigen of lipopolysaccharides (LPS), but the actual cytotoxic mechanism is unknown. In this study, we characterized the activity range of pyocin L1 and two additional L pyocins revealed by genome mining, representing two highly diverged LlpA groups in P. aeruginosa. The recombinant proteins exhibit species-specific antagonistic activities down to nanomolar concentrations against clinical and environmental P. aeruginosa strains, including several multidrug-resistant isolates. The overlap in target strain spectrum between two close homologues of the pyocin L1 group is only minimal, contrasting with the considerable spectral redundancy of LlpA proteins reported for other Pseudomonas species. No correlation was found between L pyocin susceptibility and phylogenetic relatedness of P. aeruginosa isolates. Sensitive strains were retrieved in 13 out of 15 O serotypes tested, excluding the possibility that the highly variable and immunogenic O serotype antigen of the LPS coating would represent a dominant susceptibility-discriminating factor.

Lectin-Like Bacteriocins from Pseudomonas spp. Utilise D-Rhamnose Containing Lipopolysaccharide as a Cellular Receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA(-) and OSA(-) cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA(-) cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.

A convenient synthesis of GDP-d-rhamnose: The donor substrate for d-rhamnosyltransferase WbpZ from Pseudomonas aeruginosa PAO1

Gram negative bacteria have lipopolysaccharides (LPS) that are critical for their survival. LPS molecules are composed of antigenic exopolysaccharide chains (O antigens). We are interested in discovering the enzymes involved in the biosynthesis of O antigens in Pseudomonas aeruginosa. The common polysaccharide antigen contains α-linked d-rhamnose residues. We have now synthesized GDP-d-rhamnose by a convenient synthesis in aqueous solution, and have shown that it can be used without extensive purification as the donor substrate for d-rhamnosyltransferase (WbpZ) from the P. aeruginosa strain PAO1. The availability of this nucleotide sugar preparation allows for characterization of d-rhamnosyltransferases.

Five New Genes Are Important for Common Polysaccharide Antigen Biosynthesis in Pseudomonas aeruginosa

ABSTRACTCommon polysaccharide antigen (CPA) is a conserved cell surface polysaccharide produced by Pseudomonas aeruginosa. It contains a rhamnan homopolymer and is one of the two forms of O polysaccharide attached to P. aeruginosa lipopolysaccharide (LPS). Our laboratory has previously characterized an eight-gene cluster (pa5447-pa5454 in P. aeruginosa PAO1) required for biosynthesis of CPA. Here we demonstrate that an adjacent five-gene cluster pa5455-pa5459 is also involved. Using reverse transcriptase PCR (RT-PCR), we showed that the original eight-gene cluster and the new five-gene cluster are both organized as operons. We have analyzed the LPS phenotypes of in-frame deletion mutants made in each of the five genes, and the results verified that these five genes are indeed required for CPA biosynthesis, extending the CPA biosynthesis locus to contain 13 contiguous genes. By performing overexpression experiments of different sets of these biosynthesis genes, we were able to obtain information about their possible functions in CPA biosynthesis.

Genetic and Functional Diversity of Pseudomonas aeruginosa Lipopolysaccharide.

Lipopolysccharide (LPS) is an integral component of the Pseudomonas aeruginosa cell envelope, occupying the outer leaflet of the outer membrane in this Gram-negative opportunistic pathogen. It is important for bacterium–host interactions and has been shown to be a major virulence factor for this organism. Structurally, P. aeruginosa LPS is composed of three domains, namely, lipid A, core oligosaccharide, and the distal O antigen (O-Ag). Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band). Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa. The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa. The underlying factors contributing to this diversity will be thoroughly discussed and presented in the context of its contributions to host–pathogen interactions and the control/prevention of infection.

Review: Lipopolysaccharide biosynthesis inPseudomonas aeruginosa

Pseudomonas aeruginosa causes serious nosocomial infections, and an important virulence factor produced by this organism is lipopolysaccharide (LPS). This review summarizes knowledge about biosynthesis of all three structural domains of LPS - lipid A, core oligosaccharide, and O polysaccharides. In addition, based on similarities with other bacterial species, this review proposes new hypothetical pathways for unstudied steps in the biosynthesis of P. aeruginosa LPS. Lipid A biosynthesis is discussed in relation to Escherichia coli and Salmonella, and the biosyntheses of core sugar precursors and core oligosaccharide are summarised. Pseudomonas aeruginosa attaches a Common Polysaccharide Antigen and O-Specific Antigen polysaccharides to lipid A-core. Both forms of O polysaccharide are discussed with respect to their independent synthesis mechanisms. Recent advances in understanding O-polysaccharide biosynthesis since the last major review on this subject, published nearly a decade ago, are highlighted. Since P. aeruginosa O polysaccharides contain unusual sugars, sugar-nucleotide biosynthesis pathways are reviewed in detail. Knowledge derived from detailed studies in the O5, O6 and O11 serotypes is applied to predict biosynthesis pathways of sugars in poorly-studied serotypes, especially O1, O4, and O13/O14. Although further work is required, a full understanding of LPS biosynthesis in P. aeruginosa is almost within reach.