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Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.

Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, Stratilo C, Reiz B, Cordwell SJ, Whittal R, Schild S, Feldman MF - PLoS Pathog. (2012)

Bottom Line: This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms.Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis.These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Alberta Glycomics Centre, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

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A. baumannii requires PglLAb for biofilm formation.A) Quantitative biofilm formation on polystyrene 96 well plates by strains incubated without perturbation in LB at 30°C. The bars indicate the means for 8 replicates. The error bars indicate the standard deviation of the means. Asterisks indicate significant differences (*, P<0.005 [t test; n = 8]; **, P<0.001 [t test; n = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL was determined by the COMSTAT software. For each strain at least six micrographs from three independent experiments were analyzed. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). C)–E) Image stacks of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL biofilms grown in flow cells for 24 h were analyzed for the biomass as well as the maximum and average thickness using the COMSTAT software. Shown are the medians of at least six image stacks from three independent experiments for each strain. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and ΔpglL mutant (lower row) biofilms grown in flow cells for 24 h. The first three images represent horizontal (xy, large panel) and vertical (xz and yz, side panels) projections at different z-levels (from left to right 0.2 µm, 3 µm and 6 µm). The fourth micrograph of each row represents a three-dimensional image analyzed by the AMIRA software package of the WT and ΔpglL mutant biofilms, respectively.
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ppat-1002758-g005: A. baumannii requires PglLAb for biofilm formation.A) Quantitative biofilm formation on polystyrene 96 well plates by strains incubated without perturbation in LB at 30°C. The bars indicate the means for 8 replicates. The error bars indicate the standard deviation of the means. Asterisks indicate significant differences (*, P<0.005 [t test; n = 8]; **, P<0.001 [t test; n = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL was determined by the COMSTAT software. For each strain at least six micrographs from three independent experiments were analyzed. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). C)–E) Image stacks of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL biofilms grown in flow cells for 24 h were analyzed for the biomass as well as the maximum and average thickness using the COMSTAT software. Shown are the medians of at least six image stacks from three independent experiments for each strain. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and ΔpglL mutant (lower row) biofilms grown in flow cells for 24 h. The first three images represent horizontal (xy, large panel) and vertical (xz and yz, side panels) projections at different z-levels (from left to right 0.2 µm, 3 µm and 6 µm). The fourth micrograph of each row represents a three-dimensional image analyzed by the AMIRA software package of the WT and ΔpglL mutant biofilms, respectively.

Mentions: It has been suggested that biofilm formation is important for A. baumannii virulence [28]. We tested if O-glycosylation has an impact on biofilm formation in this organism. Biofilm formation was detected using crystal violet staining and quantitatively analyzed by comparing the ratio between cell growth (OD600) and biofilm formation (OD580) at 30°C after 48 hours incubation (Fig. 5A). High absorbance values corresponding to a strong ability to create biofilms (1.23±0.48 and 1.12±0.40) were obtained for the WT strain and the ΔpglL strain complemented in trans respectively. On the contrary, the ΔpglL strain and the ΔpglL strain transformed with pWH1266 exhibited severely reduced levels of absorbance (0.18±0.07 and 0.20±0.04). Similar results were also observed at 37°C (data not shown). We further characterized the role of O-glycosylation in biofilm formation by employing a flow cell system. A. baumannii strains were stained with the green fluorescent stain SYTO 9, visualized by confocal laser scanning microscopy, and quantitative analysis of the biofilms was performed with COMSTAT. Assessment of the initial attachment after 2 hours shows that ΔpglL strain and vector control had significantly less surface coverage (4.12% and 2.32% respectively) than the WT and in trans complemented strain (6.41% and 6.45% respectively; Fig. 5B). Confocal microscopy and subsequent analysis of biofilms biomass, as well as average and maximal thickness after 24 hours showed significantly higher levels for the WT compared to the ΔpglL strain, and the phenotype was restored to WT levels when pglLAb was complemented in trans (Fig. 5 C, D, E, F; *P<0.05). These data indicate that the A. baumannii strain defective in O-glycosylation has a severely diminished capacity to form biofilms.


Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.

Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, Stratilo C, Reiz B, Cordwell SJ, Whittal R, Schild S, Feldman MF - PLoS Pathog. (2012)

A. baumannii requires PglLAb for biofilm formation.A) Quantitative biofilm formation on polystyrene 96 well plates by strains incubated without perturbation in LB at 30°C. The bars indicate the means for 8 replicates. The error bars indicate the standard deviation of the means. Asterisks indicate significant differences (*, P<0.005 [t test; n = 8]; **, P<0.001 [t test; n = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL was determined by the COMSTAT software. For each strain at least six micrographs from three independent experiments were analyzed. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). C)–E) Image stacks of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL biofilms grown in flow cells for 24 h were analyzed for the biomass as well as the maximum and average thickness using the COMSTAT software. Shown are the medians of at least six image stacks from three independent experiments for each strain. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and ΔpglL mutant (lower row) biofilms grown in flow cells for 24 h. The first three images represent horizontal (xy, large panel) and vertical (xz and yz, side panels) projections at different z-levels (from left to right 0.2 µm, 3 µm and 6 µm). The fourth micrograph of each row represents a three-dimensional image analyzed by the AMIRA software package of the WT and ΔpglL mutant biofilms, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369928&req=5

ppat-1002758-g005: A. baumannii requires PglLAb for biofilm formation.A) Quantitative biofilm formation on polystyrene 96 well plates by strains incubated without perturbation in LB at 30°C. The bars indicate the means for 8 replicates. The error bars indicate the standard deviation of the means. Asterisks indicate significant differences (*, P<0.005 [t test; n = 8]; **, P<0.001 [t test; n = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL was determined by the COMSTAT software. For each strain at least six micrographs from three independent experiments were analyzed. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). C)–E) Image stacks of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL biofilms grown in flow cells for 24 h were analyzed for the biomass as well as the maximum and average thickness using the COMSTAT software. Shown are the medians of at least six image stacks from three independent experiments for each strain. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and ΔpglL mutant (lower row) biofilms grown in flow cells for 24 h. The first three images represent horizontal (xy, large panel) and vertical (xz and yz, side panels) projections at different z-levels (from left to right 0.2 µm, 3 µm and 6 µm). The fourth micrograph of each row represents a three-dimensional image analyzed by the AMIRA software package of the WT and ΔpglL mutant biofilms, respectively.
Mentions: It has been suggested that biofilm formation is important for A. baumannii virulence [28]. We tested if O-glycosylation has an impact on biofilm formation in this organism. Biofilm formation was detected using crystal violet staining and quantitatively analyzed by comparing the ratio between cell growth (OD600) and biofilm formation (OD580) at 30°C after 48 hours incubation (Fig. 5A). High absorbance values corresponding to a strong ability to create biofilms (1.23±0.48 and 1.12±0.40) were obtained for the WT strain and the ΔpglL strain complemented in trans respectively. On the contrary, the ΔpglL strain and the ΔpglL strain transformed with pWH1266 exhibited severely reduced levels of absorbance (0.18±0.07 and 0.20±0.04). Similar results were also observed at 37°C (data not shown). We further characterized the role of O-glycosylation in biofilm formation by employing a flow cell system. A. baumannii strains were stained with the green fluorescent stain SYTO 9, visualized by confocal laser scanning microscopy, and quantitative analysis of the biofilms was performed with COMSTAT. Assessment of the initial attachment after 2 hours shows that ΔpglL strain and vector control had significantly less surface coverage (4.12% and 2.32% respectively) than the WT and in trans complemented strain (6.41% and 6.45% respectively; Fig. 5B). Confocal microscopy and subsequent analysis of biofilms biomass, as well as average and maximal thickness after 24 hours showed significantly higher levels for the WT compared to the ΔpglL strain, and the phenotype was restored to WT levels when pglLAb was complemented in trans (Fig. 5 C, D, E, F; *P<0.05). These data indicate that the A. baumannii strain defective in O-glycosylation has a severely diminished capacity to form biofilms.

Bottom Line: This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms.Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis.These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Alberta Glycomics Centre, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

Show MeSH
Related in: MedlinePlus