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O antigen of Pseudomonas aeruginosa ..

Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage ..

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Lam JS Genetics of O-antigen biosynthesis in ..

. Basker MJ, Edmondson RA, Sutherland R. Comparative antibacterial activity of azlocillin, mezlocillin, carbenicillin, and ticarcillin and relative stability to beta-lactamases ofPseudomonas aeruginosa and Klebsiella aerogenes. Infection 1979;7:67-73. []

Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: ..
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Other organisms for which synergy seems to be important with regard to the penicillins includes Pseudomonas aeruginosa. Again, a combination of an antipseudomonal penicillin plus an aminoglycoside may result in increased bactericidal activity. This has been demonstrated in vitro and animal studies (, , ), but there is limited data in humans to support these findings. In vitro synergy between the extended spectrum penicillins (azlocillin, mezlocillin) and ciprofloxacin has also been demonstrated (, , ). Immunocompromised patients are a population who may benefit the most from antipseudomonal synergy. There is data to suggest that synergistic combination therapy results in increased survival versus non-synergistic combinations of drugs (, , ).

The genetics of O antigen biosynthesis in ..

Cloning and surface expression of Pseudomonas aeruginosa O antigen in ..
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Erwinia amylovora, the causal agent of fire blight, is an enterobacterial pathogen of Rosaceous plants including apple and pear. We have been studying the response of E. amylovora to oxidative stress because, during infection, the bacterium elicits an oxidative burst response in host plants. During the screening of a transposon mutant library for hydrogen peroxide sensitivity, we identified a mutant carrying an insertion in waaL, a gene involved in lipopolysaccharide biosynthesis, that was more sensitive to hydrogen peroxide than the parental wild-type strain. We also confirmed that a waaL mutant of Pseudomonas aeruginosa exhibited an increased sensitivity to hydrogen peroxide compared with the wild-type strain. The E. amylovora waaL mutant was also reduced in virulence, showed a decrease in twitching motility, and was more sensitive to polymyxin B than the wild type. Each of these phenotypes was complemented by the cloned waaL gene. Our results highlight the importance of the lipopolysaccharide layer to virulence in E. amylovora and the unexpected finding of an additional function of lipopolysaccharide in protection from oxidative stress in E. amylovora and P. aeruginosa.

N2 - Erwinia amylovora, the causal agent of fire blight, is an enterobacterial pathogen of Rosaceous plants including apple and pear. We have been studying the response of E. amylovora to oxidative stress because, during infection, the bacterium elicits an oxidative burst response in host plants. During the screening of a transposon mutant library for hydrogen peroxide sensitivity, we identified a mutant carrying an insertion in waaL, a gene involved in lipopolysaccharide biosynthesis, that was more sensitive to hydrogen peroxide than the parental wild-type strain. We also confirmed that a waaL mutant of Pseudomonas aeruginosa exhibited an increased sensitivity to hydrogen peroxide compared with the wild-type strain. The E. amylovora waaL mutant was also reduced in virulence, showed a decrease in twitching motility, and was more sensitive to polymyxin B than the wild type. Each of these phenotypes was complemented by the cloned waaL gene. Our results highlight the importance of the lipopolysaccharide layer to virulence in E. amylovora and the unexpected finding of an additional function of lipopolysaccharide in protection from oxidative stress in E. amylovora and P. aeruginosa.

The genetics of O antigen biosynthesis in P

The Pseudomonas aeruginosa serogroup O11 strain PA103 O antigen gene locus consists of ..
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Because of the sequence similarities between the two putative methyltransferase-encoding genes pa5457 and pa5459, with wbdD from E. coli O8, we had hypothesized that one or both of these genes might catalyze an analogous polysaccharide methylation reaction and that this methylation would be the signal recognized by the ABC transporter. Our data do not rule out this possibility, but it is clear that these P. aeruginosa methyltransferases do not have the same kind of chain length-regulating function as WbdD from E. coli O8 (WbdDO8). Overexpression of WbdD results in dramatic shortening of OPS chains, whereas overexpression of the CPA locus methyltransferase genes does not. The E. coli WbdD proteins are larger because they contain domains for interacting with the membrane and glycosyltransferase enzymes (), whereas these domains are not present in PA5457 and PA5459. The precise mechanism for coordination of elongation and termination of OPS synthesis in the E. coli model systems is still unknown, but it is clear that these interacting domains are important. Of the two putative methyltransferase-encoding genes in the CPA cluster, pa5459 seems to be more critical for biosynthesis of the CPA structure. Knocking out this gene, or overexpressing it, resulted in observable changes in CPA phenotypes, indicating an alteration in the chemical structure of the molecule. Although the phenotype of the pa5457 pa5459 double mutant confirms that both genes participate in CPA biosynthesis, mutation of pa5457 alone did not confer detectable phenotype change, and overexpression of this gene had only a very subtle effect on the chain length distribution. Possibly the SDS-PAGE−silver staining and Western immunoblotting methods we used to characterize LPS were not sensitive enough to detect changes caused by genetic experiments with pa5457, and it may yet be the case that the function of this gene is important biologically. The requirement for at least one of these putative methyltransferase-encoding genes for CPA production substantiates previous findings that methyl sugars are present in the structure of the CPA polysaccharide, although there is not perfect agreement about which methyl sugars are present (–). Future structural study of the wild-type and mutant forms of the CPA LPS is warranted and has the potential to illuminate the specific functions encoded by these genes. Since methylation appears to be important for completion of CPA biosynthesis, it seems probable that at least one methyl sugar fulfills a critical signal function to permit export and processing of the polymer. It is therefore possible that methylation is one of the features recognized by the ABC transporter before export is triggered as we had hypothesized.

Citation Hao Y, King JD, Huszczynski S, Kocíncová D, Lam JS. 2013. Five new genes are important for common polysaccharide antigen biosynthesis in Pseudomonas aeruginosa. mBio 4(1):e00631-12. doi:10.1128/mBio.00631-12

of O-antigen in Pseudomonas aeruginosa ..
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Penicillins - Infectious Disease and Antimicrobial Agents

Pseudomonas aeruginosa is an important opportunistic human pathogen that can infect immunocompromised individuals such as patients with cystic fibrosis (CF), cancer, AIDS, or burn wounds. Among the virulence factors P. aeruginosa produces, LPS has been shown to be a major factor (, ). LPS can be conceptually divided into three structural domains: lipid A, which anchors the LPS in the outer membrane; a long-chain O polysaccharide (OPS); and a core oligosaccharide that links the lipid A and the OPS. Most P. aeruginosa strains simultaneously produce two distinct forms of OPS. One of these is a heteropolymer composed of repeating units of two to five distinct sugars and is termed O-specific antigen (OSA; formerly called B band). The other form of OPS contains a homopolymer of -rhamnose (-Rha). Since this structure is produced by the majority of P. aeruginosa strains, it has been named the common polysaccharide antigen (CPA; historically called A band) (). Of the two O polysaccharides, CPA chains are shorter and less immunogenic than OSA, likely due to the neutral charge of the rhamnan. There is evidence that CPA is a virulence factor in the context of P. aeruginosa infections. During chronic lung infection in CF patients, there is a progressive decrease in the production of the OSA, while LPS-containing CPA is maintained on the cell surface of P. aeruginosa (). It has also been demonstrated that CPA plays an important role in the attachment of P. aeruginosa to human airway epithelial cells in vitro ().

Publications | Laboratory for Bioanalytical Spectroscopy

To examine whether these site-directed mutations of wbpZ affected the functions of this enzyme in vivo, the P. aeruginosa PAO1 wbpZ::Gm strain, which lacks CPA biosynthesis due to an insertion mutation in the wbpZ gene, was complemented with either the wild-type or the site-directed mutant wbpZ gene and analyzed for the ability to restore the biosynthesis of CPA LPS. It was found that recombinant forms of wbpZ with mutations C26A, D174A, D253A, and D256A were still functional in vivo and capable of rescuing CPA biosynthesis to the wild-type level. Thus, in spite of the much lower -Rha-transferase activity of the D256A mutant observed in vitro, this mutant construct was obviously still sufficiently active to act in the CPA synthesis pathway. However, the D172A and D254A mutants of wbpZ were not able to rescue the CPA biosynthesis (). This indicates that the D172A and D254A residues are crucial for the in vivo activity of wbpZ during O-antigen synthesis.

DNA is a long polymer made from repeating units called nucleotides

Lipopolysaccharide (LPS) is an important cell surface structure of Gram-negative bacteria. The human opportunistic pathogen Pseudomonas aeruginosa simultaneously produces an O-antigen-specific (OSA) form and a common polysaccharide antigen (CPA) form of LPS. CPA, the focus of this study, is composed of α-1-2, α1-3-linked -rhamnose sugars and has been shown to be important for attachment of the bacteria to human airway epithelial cells. Genome sequencing of this species revealed a new five-gene cluster that we predicted to be involved in CPA biosynthesis and modification. In this study, we have generated chromosomal knockouts by performing in-frame deletions and allelic replacements. Characterizing the function of each of the five genes is important for us to better understand CPA biosynthesis and the mechanisms of chain length termination and regulation of this unique D-rhamnan polysaccharide.

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