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

Murphy, Kathleen; Park, Amber J.; Hao, Youai; Brewer, Dyanne; Lam, Joseph S.; Khursigara, Cezar M.

doi:10.1128/jb.01463-13

Cited by 107 publications

(112 citation statements)

References 58 publications

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“…Similarly, while Pseudomonas O-antigen mutants have no effect on the rate of OMV release, they do affect both their contents and size (43). The LPS of V. fischeri cells includes an unusual O antigen that plays a critical role in both flagellar motility and efficient colonization (18), but there is little understanding of this relationship or its relationship to broader changes in either the cell's surface or its OMVs.…”

Section: Discussionmentioning

confidence: 99%

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

et al. 2016

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Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCEDetermining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

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Section: Discussionmentioning

confidence: 99%

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

et al. 2016

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“…OMV were isolated mainly following the procedure described previously (30). Briefly, different P. aeruginosa strains were grown in 800 ml TSB (Becton, Dickinson, Mississauga, Ontario, Canada) for 16 h at 37°C in a rotary shaker at 200 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…Biofilm development was assayed according to previously described methods (30,44). Briefly, each PAO1 migA containing two point mutations, R92Q and V170M; the amino acid sequence is the same as that encoded by PA14 migA This study pHERDrmd pHERD20T vector containing the PA14 rmd gene This study strain was grown in tryptic soy broth (TSB) to an optical density at 600 nm (OD 600 ) of 0.5.…”

Section: Methodsmentioning

confidence: 99%

“…CPA was found to play important biological roles as a receptor for bacteriophage A7 (27) and lectin-like bacteriocins (28) and as a surface antigen for P. aeruginosa that is important for adherence to human bronchial epithelial cells (29). In a recent study, Murphy et al (30) showed that CPA is required for strain PAO1 to develop mature and robust biofilms. When we performed a BLASTN analysis of the genome sequence of PA14, we found that PA14 possesses the eightgene and five-gene CPA biosynthesis clusters.…”

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confidence: 99%

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

Hao

Murphy

et al. 2015

J Bacteriol

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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 ϳ99% 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. IMPORTANCEP. 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. P seudomonas aeruginosa is an important opportunistic pathogen that infects a wide range of organisms, including humans. It can result in fatal consequences in compromised individuals with cancer, AIDS, burn wounds, or cystic fibrosis (CF). It infects the lungs of ϳ80% of CF patients, and the infection usually becomes chronic, resulting in death in a majority of these patients (1). Two P. aeruginosa strains, PAO1 and PA14, have been us...

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“…It has been shown that alterations of a single band or both bands of the O-antigen of P. aeruginosa PAO1 can give rise to mutants with increased cytotoxicity mediated by the type III secretion system (Augustin et al, 2007). In addition, changes in O-polysaccharide expression in PAO1 affect the size and protein content of outer membrane vesicles, and the formation of a robust biofilm (Murphy et al, 2014).…”

Section: Cross-resistance and Reversibility Of Mutantsmentioning

confidence: 99%

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

Latino

2016

Microbiology

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Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10 25 for single phage infection and 10 26 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure.

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

Cited by 107 publications

References 58 publications

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

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

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

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