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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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Related in: MedlinePlus

Comparison of A. baumannii WT and ΔpglL membrane extracts by 2D-DIGE.Analysis of the membrane proteome of A. baumannii WT strain (A), ΔpglL strain (B), and merge (C). Spots WT1 and WT2 only present in the WT strain (green) whereas MT1 and MT2 were only present in the ΔpglL strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.
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ppat-1002758-g002: Comparison of A. baumannii WT and ΔpglL membrane extracts by 2D-DIGE.Analysis of the membrane proteome of A. baumannii WT strain (A), ΔpglL strain (B), and merge (C). Spots WT1 and WT2 only present in the WT strain (green) whereas MT1 and MT2 were only present in the ΔpglL strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.

Mentions: To identify the glycoprotein(s) in A. baumannii, we performed two dimensional in-gel electrophoresis (2D-DIGE) experiments [26]. Membrane samples of both WT and ΔpglL were isolated by ultracentrifugation and the lipidic components were removed as previously described [27]. Most of the signals corresponding to the wild type (Fig. 2A, green) and ΔpglL (Fig. 2B, red) proteins co-localized in the gel (Fig. 2C, yellow), indicating that these proteins were likely not glycosylated. However, a few proteins exhibited differential electrophoretic behavior (Fig. 2). These proteins spots were excised, in-gel digested, and analyzed by MALDI-TOF/TOF MS and MS/MS. We identified two separate pairs of proteins, which according to their electrophoretic migration, appeared to be larger and more acidic in the WT strain (WT1 and WT2) than in the ΔpglL strain (MT1 and MT2). Mass spectrometric analysis determined WT1 and MT1 samples to be A1S_3626 protein, whereas WT2 and MT2 were identified as A1S_3744 protein. Both, A1S_3626 and A1S_3744 are annotated as hypothetical proteins, and BLAST searches yielded homologues exclusively within the Acinetobacter genus.


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)

Comparison of A. baumannii WT and ΔpglL membrane extracts by 2D-DIGE.Analysis of the membrane proteome of A. baumannii WT strain (A), ΔpglL strain (B), and merge (C). Spots WT1 and WT2 only present in the WT strain (green) whereas MT1 and MT2 were only present in the ΔpglL strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369928&req=5

ppat-1002758-g002: Comparison of A. baumannii WT and ΔpglL membrane extracts by 2D-DIGE.Analysis of the membrane proteome of A. baumannii WT strain (A), ΔpglL strain (B), and merge (C). Spots WT1 and WT2 only present in the WT strain (green) whereas MT1 and MT2 were only present in the ΔpglL strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.
Mentions: To identify the glycoprotein(s) in A. baumannii, we performed two dimensional in-gel electrophoresis (2D-DIGE) experiments [26]. Membrane samples of both WT and ΔpglL were isolated by ultracentrifugation and the lipidic components were removed as previously described [27]. Most of the signals corresponding to the wild type (Fig. 2A, green) and ΔpglL (Fig. 2B, red) proteins co-localized in the gel (Fig. 2C, yellow), indicating that these proteins were likely not glycosylated. However, a few proteins exhibited differential electrophoretic behavior (Fig. 2). These proteins spots were excised, in-gel digested, and analyzed by MALDI-TOF/TOF MS and MS/MS. We identified two separate pairs of proteins, which according to their electrophoretic migration, appeared to be larger and more acidic in the WT strain (WT1 and WT2) than in the ΔpglL strain (MT1 and MT2). Mass spectrometric analysis determined WT1 and MT1 samples to be A1S_3626 protein, whereas WT2 and MT2 were identified as A1S_3744 protein. Both, A1S_3626 and A1S_3744 are annotated as hypothetical proteins, and BLAST searches yielded homologues exclusively within the Acinetobacter genus.

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