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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 to glycosylate membrane proteins.10 µg of membrane extract from A. baumannii strains (lanes 1,2,3,4) were resolved by SDS-PAGE. A) Carbohydrates were detected by PAS stain. B) Proteins were detected by Coomassie staining. Samples were as follows: lane 1 WT; lane 1a Proteinase K treated WT; lane 2 ΔpglL, lane 3 ΔpglL, pWH1266-pglL, 4-ΔpglL, pWH1266 control.
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ppat-1002758-g001: A. baumannii requires PglLAb to glycosylate membrane proteins.10 µg of membrane extract from A. baumannii strains (lanes 1,2,3,4) were resolved by SDS-PAGE. A) Carbohydrates were detected by PAS stain. B) Proteins were detected by Coomassie staining. Samples were as follows: lane 1 WT; lane 1a Proteinase K treated WT; lane 2 ΔpglL, lane 3 ΔpglL, pWH1266-pglL, 4-ΔpglL, pWH1266 control.

Mentions: Most of the Neisseria O-glycoproteins identified to date are associated to membranes [16]. Membrane extracts from wild type and ΔA1S_3176 A. baumannii strains were analyzed by SDS-PAGE followed by PAS staining, a technique that is specific for detecting glycans, but presents low sensitivity (Fig. 1). A broad band migrating from 25 to 35 kDa was visualized in the extract of A. baumannii WT. Although the membrane protein profile between the WT and the ΔA1S_3176 strains appeared similar, the band detected via PAS stain was not visible in the mutant strain, suggesting that A1S_3176 is required for glycosylation of at least one protein (Fig. 1B). The PAS-reactive band disappeared upon treatment with proteinase K, associating the glycan signal with proteinaceous material. Complementation of A1S_3176 was achieved in trans, and analysis of A. baumannii ΔA1S_3176-pWH1266-pglL membrane extract showed the reappearance of the PAS stained band. Due to the aforementioned similarity between O-OTases and ligases, we carried out a conventional LPS extraction and analyzed the extract of the different strains via SDS-PAGE. Silver stain showed no obvious differences in the carbohydrate pattern were observed, suggesting that A1S_3176 is not involved in LPS synthesis (Fig S1). To further determine if A1S_3716 effected LPS biosynthesis, whole cells were digested with proteinase K and analyzed by Silver stain and no differences were observed (data not shown). However, it has been reported that the O-antigen chains of certain A. baumannii strains are not detectable by Silver stain and therefore we cannot conclusively exclude a role of A1S_3176 in LPS synthesis [25]. Together these results suggest that A1S_3176 is an O-OTase responsible for O-glycosylation in A. baumannii and will be referred from here on as PglLAb, as per its N. meningitidis ortholog.


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 to glycosylate membrane proteins.10 µg of membrane extract from A. baumannii strains (lanes 1,2,3,4) were resolved by SDS-PAGE. A) Carbohydrates were detected by PAS stain. B) Proteins were detected by Coomassie staining. Samples were as follows: lane 1 WT; lane 1a Proteinase K treated WT; lane 2 ΔpglL, lane 3 ΔpglL, pWH1266-pglL, 4-ΔpglL, pWH1266 control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002758-g001: A. baumannii requires PglLAb to glycosylate membrane proteins.10 µg of membrane extract from A. baumannii strains (lanes 1,2,3,4) were resolved by SDS-PAGE. A) Carbohydrates were detected by PAS stain. B) Proteins were detected by Coomassie staining. Samples were as follows: lane 1 WT; lane 1a Proteinase K treated WT; lane 2 ΔpglL, lane 3 ΔpglL, pWH1266-pglL, 4-ΔpglL, pWH1266 control.
Mentions: Most of the Neisseria O-glycoproteins identified to date are associated to membranes [16]. Membrane extracts from wild type and ΔA1S_3176 A. baumannii strains were analyzed by SDS-PAGE followed by PAS staining, a technique that is specific for detecting glycans, but presents low sensitivity (Fig. 1). A broad band migrating from 25 to 35 kDa was visualized in the extract of A. baumannii WT. Although the membrane protein profile between the WT and the ΔA1S_3176 strains appeared similar, the band detected via PAS stain was not visible in the mutant strain, suggesting that A1S_3176 is required for glycosylation of at least one protein (Fig. 1B). The PAS-reactive band disappeared upon treatment with proteinase K, associating the glycan signal with proteinaceous material. Complementation of A1S_3176 was achieved in trans, and analysis of A. baumannii ΔA1S_3176-pWH1266-pglL membrane extract showed the reappearance of the PAS stained band. Due to the aforementioned similarity between O-OTases and ligases, we carried out a conventional LPS extraction and analyzed the extract of the different strains via SDS-PAGE. Silver stain showed no obvious differences in the carbohydrate pattern were observed, suggesting that A1S_3176 is not involved in LPS synthesis (Fig S1). To further determine if A1S_3716 effected LPS biosynthesis, whole cells were digested with proteinase K and analyzed by Silver stain and no differences were observed (data not shown). However, it has been reported that the O-antigen chains of certain A. baumannii strains are not detectable by Silver stain and therefore we cannot conclusively exclude a role of A1S_3176 in LPS synthesis [25]. Together these results suggest that A1S_3176 is an O-OTase responsible for O-glycosylation in A. baumannii and will be referred from here on as PglLAb, as per its N. meningitidis ortholog.

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