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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

Identification of additional glycoproteins in A. baumannii ATCC 17978.Tryptically digested membranes were enriched via ZIC-HILIC and analyzed by LC-MS and HCD MS-MS. All spectra were analyzed for the diagnostic oxonium ion of 301.10 m/z, and positive spectra were analyzed manually to identify the glycopeptide. This spectra is representative of each glycopeptide identified in Table 2. A) ITMS-CID of the precursor ion at m/z 1999.943 reveals the pentasaccharide attached to the peptide AKPASTPAVK. B) FTMS-HCD of the precursor ion at m/z 1999.943 reveals the peptide sequence AKPASTPAVK.
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ppat-1002758-g004: Identification of additional glycoproteins in A. baumannii ATCC 17978.Tryptically digested membranes were enriched via ZIC-HILIC and analyzed by LC-MS and HCD MS-MS. All spectra were analyzed for the diagnostic oxonium ion of 301.10 m/z, and positive spectra were analyzed manually to identify the glycopeptide. This spectra is representative of each glycopeptide identified in Table 2. A) ITMS-CID of the precursor ion at m/z 1999.943 reveals the pentasaccharide attached to the peptide AKPASTPAVK. B) FTMS-HCD of the precursor ion at m/z 1999.943 reveals the peptide sequence AKPASTPAVK.

Mentions: To determine if additional glycoproteins were present in A. baumannii ATCC 17978, we employed ZIC-HILIC glycopeptide enrichment. Utilizing membrane extracts previously shown to contain A1S_3626 and A1S_3744 putative glycopeptides were enriched and analyzed using an LTQ-Orbitrap Velos. HCD scans containing oxonium ion were manually inspected and searched using MASCOT resulting in the identification of at least 9 different glycosylation sites on 7 different glycoproteins in A. baumannii ATCC 17978 (Table 1; Fig. 4). This peptide-centric approach enabled multiple novel glycoproteins to be identified of which six of the seven proteins are annotated as uncharacterized hypothetical proteins, with the remaining being annotated as MotB (A1S_1193). (Table 1). This demonstrates that PglLAb is able to glycosylate multiple proteins in A. baumannii ATCC 17978.


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)

Identification of additional glycoproteins in A. baumannii ATCC 17978.Tryptically digested membranes were enriched via ZIC-HILIC and analyzed by LC-MS and HCD MS-MS. All spectra were analyzed for the diagnostic oxonium ion of 301.10 m/z, and positive spectra were analyzed manually to identify the glycopeptide. This spectra is representative of each glycopeptide identified in Table 2. A) ITMS-CID of the precursor ion at m/z 1999.943 reveals the pentasaccharide attached to the peptide AKPASTPAVK. B) FTMS-HCD of the precursor ion at m/z 1999.943 reveals the peptide sequence AKPASTPAVK.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002758-g004: Identification of additional glycoproteins in A. baumannii ATCC 17978.Tryptically digested membranes were enriched via ZIC-HILIC and analyzed by LC-MS and HCD MS-MS. All spectra were analyzed for the diagnostic oxonium ion of 301.10 m/z, and positive spectra were analyzed manually to identify the glycopeptide. This spectra is representative of each glycopeptide identified in Table 2. A) ITMS-CID of the precursor ion at m/z 1999.943 reveals the pentasaccharide attached to the peptide AKPASTPAVK. B) FTMS-HCD of the precursor ion at m/z 1999.943 reveals the peptide sequence AKPASTPAVK.
Mentions: To determine if additional glycoproteins were present in A. baumannii ATCC 17978, we employed ZIC-HILIC glycopeptide enrichment. Utilizing membrane extracts previously shown to contain A1S_3626 and A1S_3744 putative glycopeptides were enriched and analyzed using an LTQ-Orbitrap Velos. HCD scans containing oxonium ion were manually inspected and searched using MASCOT resulting in the identification of at least 9 different glycosylation sites on 7 different glycoproteins in A. baumannii ATCC 17978 (Table 1; Fig. 4). This peptide-centric approach enabled multiple novel glycoproteins to be identified of which six of the seven proteins are annotated as uncharacterized hypothetical proteins, with the remaining being annotated as MotB (A1S_1193). (Table 1). This demonstrates that PglLAb is able to glycosylate multiple proteins in A. baumannii ATCC 17978.

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