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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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MS/MS of A1S_3626 and A1S_3744 showing glycosylation in A. baumannii with a pentasaccharide.Spots excised from the 2D DIGE, digested with trypsin, and analyzed by MALDI-TOF-MS. Peaks not corresponding to peptide fragmentation were analyzed for glycosylation. A) MS/MS of the precursor ion peak at m/z 2895.165 from A1S_3626 revealed the peptide SAGDQAASDIATATDNASAK with a pentasaccharide of HexNAc-Hex-Hex-(HexNAc)-300 attached. B) MS/MS of the precursor ion peak at m/z 3852.76 from A1S_3744 revealed the peptide ETPKEEEQDKVETAVSEPQPQKPAK with the same pentasaccharide attached. C) MALDI-TOF MS of Pronase E digested membrane proteins showed a precursor ion peak of m/z 1358.4 which MS/MS analysis demonstrated to be the previously identified O-glycan (HexNAc-Hex-Hex-(HexNAc)-300 attached to the peptide fragment “ATD”.
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ppat-1002758-g003: MS/MS of A1S_3626 and A1S_3744 showing glycosylation in A. baumannii with a pentasaccharide.Spots excised from the 2D DIGE, digested with trypsin, and analyzed by MALDI-TOF-MS. Peaks not corresponding to peptide fragmentation were analyzed for glycosylation. A) MS/MS of the precursor ion peak at m/z 2895.165 from A1S_3626 revealed the peptide SAGDQAASDIATATDNASAK with a pentasaccharide of HexNAc-Hex-Hex-(HexNAc)-300 attached. B) MS/MS of the precursor ion peak at m/z 3852.76 from A1S_3744 revealed the peptide ETPKEEEQDKVETAVSEPQPQKPAK with the same pentasaccharide attached. C) MALDI-TOF MS of Pronase E digested membrane proteins showed a precursor ion peak of m/z 1358.4 which MS/MS analysis demonstrated to be the previously identified O-glycan (HexNAc-Hex-Hex-(HexNAc)-300 attached to the peptide fragment “ATD”.

Mentions: Analysis of the MALDI-TOF MS spectra of a tryptic digest of WT1 (A1S_3626) revealed a peptide fragment of 2895.24 Da that was absent in MT1 (Fig. 3A). MALDI-TOF-TOF MS/MS of this ion determined that in the wild-type strain the peptide SAGDQAASDIATATDNASAK was linked to the glycan HexNAc-Hex-Hex-(HexNAc)-300, where 300 corresponded to an unknown residue of m/z 300, whereas the same peptide was unmodified in ΔpglL sample (Fig S3A). Similarly, MALDI-TOF MS analysis of a tryptic digest of WT2 (A1S_3744) revealed a peptide fragment of 3852.69 Da that was absent in MT2 (Fig. 3B). MALDI-TOF-TOF MS/MS of the 3852.69 Da peak revealed the same pentasaccharide identified on A1S_3626 on the peptide ETPKEEEQDKVETAVSEPQPQKPAK (2822.33 Da), whereas the same peptide was unmodified in ΔpglL sample (Fig S3B).. We next purified membranes from A. baumannii, digested the sample with Pronase E, and enriched glycosylated peptides using activated charcoal microspin columns. We identified a peak in the MALDI-TOF MS of 1358.4 m/z that was subsequently analyzed by MALDI-TOF/TOF MS/MS (Fig. 3C). Manual peak annotation identified the previously characterized pentasaccharide attached to a sodiated tripeptide containing the amino acids A, T and D. Overall, these results demonstrate that PglLAb glycosylates at least two different proteins with a pentasaccharide with a preliminary structure of HexNAc-Hex-Hex-(HexNAc)-300.


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)

MS/MS of A1S_3626 and A1S_3744 showing glycosylation in A. baumannii with a pentasaccharide.Spots excised from the 2D DIGE, digested with trypsin, and analyzed by MALDI-TOF-MS. Peaks not corresponding to peptide fragmentation were analyzed for glycosylation. A) MS/MS of the precursor ion peak at m/z 2895.165 from A1S_3626 revealed the peptide SAGDQAASDIATATDNASAK with a pentasaccharide of HexNAc-Hex-Hex-(HexNAc)-300 attached. B) MS/MS of the precursor ion peak at m/z 3852.76 from A1S_3744 revealed the peptide ETPKEEEQDKVETAVSEPQPQKPAK with the same pentasaccharide attached. C) MALDI-TOF MS of Pronase E digested membrane proteins showed a precursor ion peak of m/z 1358.4 which MS/MS analysis demonstrated to be the previously identified O-glycan (HexNAc-Hex-Hex-(HexNAc)-300 attached to the peptide fragment “ATD”.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369928&req=5

ppat-1002758-g003: MS/MS of A1S_3626 and A1S_3744 showing glycosylation in A. baumannii with a pentasaccharide.Spots excised from the 2D DIGE, digested with trypsin, and analyzed by MALDI-TOF-MS. Peaks not corresponding to peptide fragmentation were analyzed for glycosylation. A) MS/MS of the precursor ion peak at m/z 2895.165 from A1S_3626 revealed the peptide SAGDQAASDIATATDNASAK with a pentasaccharide of HexNAc-Hex-Hex-(HexNAc)-300 attached. B) MS/MS of the precursor ion peak at m/z 3852.76 from A1S_3744 revealed the peptide ETPKEEEQDKVETAVSEPQPQKPAK with the same pentasaccharide attached. C) MALDI-TOF MS of Pronase E digested membrane proteins showed a precursor ion peak of m/z 1358.4 which MS/MS analysis demonstrated to be the previously identified O-glycan (HexNAc-Hex-Hex-(HexNAc)-300 attached to the peptide fragment “ATD”.
Mentions: Analysis of the MALDI-TOF MS spectra of a tryptic digest of WT1 (A1S_3626) revealed a peptide fragment of 2895.24 Da that was absent in MT1 (Fig. 3A). MALDI-TOF-TOF MS/MS of this ion determined that in the wild-type strain the peptide SAGDQAASDIATATDNASAK was linked to the glycan HexNAc-Hex-Hex-(HexNAc)-300, where 300 corresponded to an unknown residue of m/z 300, whereas the same peptide was unmodified in ΔpglL sample (Fig S3A). Similarly, MALDI-TOF MS analysis of a tryptic digest of WT2 (A1S_3744) revealed a peptide fragment of 3852.69 Da that was absent in MT2 (Fig. 3B). MALDI-TOF-TOF MS/MS of the 3852.69 Da peak revealed the same pentasaccharide identified on A1S_3626 on the peptide ETPKEEEQDKVETAVSEPQPQKPAK (2822.33 Da), whereas the same peptide was unmodified in ΔpglL sample (Fig S3B).. We next purified membranes from A. baumannii, digested the sample with Pronase E, and enriched glycosylated peptides using activated charcoal microspin columns. We identified a peak in the MALDI-TOF MS of 1358.4 m/z that was subsequently analyzed by MALDI-TOF/TOF MS/MS (Fig. 3C). Manual peak annotation identified the previously characterized pentasaccharide attached to a sodiated tripeptide containing the amino acids A, T and D. Overall, these results demonstrate that PglLAb glycosylates at least two different proteins with a pentasaccharide with a preliminary structure of HexNAc-Hex-Hex-(HexNAc)-300.

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