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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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Characterization of A. baumannii ATCC 17978 pathogenesis in a murine septicemia model.A) Determination of the LD50 of A. baumannii ATCC 17978. Groups of 5 mice were injected with serial dilutions of A. baumannii WT to determine the LD50 which was calculated to be 6.49×104 CFU @ 18 hrs post infection. B) Murine competition septicemia between A. baumannii WT and ΔpglL. Groups of 3 mice were injected with ∼1∶1 WT to ΔpglL CFU's and were sacrificed after 18 hrs, spleens were harvested, and bacterial load determined.
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ppat-1002758-g007: Characterization of A. baumannii ATCC 17978 pathogenesis in a murine septicemia model.A) Determination of the LD50 of A. baumannii ATCC 17978. Groups of 5 mice were injected with serial dilutions of A. baumannii WT to determine the LD50 which was calculated to be 6.49×104 CFU @ 18 hrs post infection. B) Murine competition septicemia between A. baumannii WT and ΔpglL. Groups of 3 mice were injected with ∼1∶1 WT to ΔpglL CFU's and were sacrificed after 18 hrs, spleens were harvested, and bacterial load determined.

Mentions: We then tested A. baumannii ΔpglL virulence in vivo using a previously described murine septicemia competition model [37]–[39]. We first determined the LD50 of A. baumannii ATCC 17978 strain by injecting groups of 5 BALB/c mice with serially diluted bacteria cultures (Fig. 7A). A very small dose range between full survival and full killing was observed, and the LD50 was determined to be 6.49×104 CFU/mouse. The competition index (CI) was defined as the number of ΔpglL CFUs recovered/number of WT CFUs recovered, divided by the number of ΔpglL CFUs inoculated/number of WT CFUs inoculated. Cultures of each strain were mixed at a ratio of 1∶1, serial diluted, and plated to determine the initial CI. 1×105 CFU of the mixed strains were injected intraperitoneally into the BALB/c mice, which were subsequently sacrificed 18 hrs post injection. The spleens were aseptically harvested, serial diluted, and plated. All of the mice had a high spleen CFU load of 3.75±2.37×108 CFU/gram and were moribund at the time of sacrifice. While the initial prescreen showed a CI of 1.18±0.21 favoring the ΔpglL mutant, the spleen counts after 18 hrs showed a CI of 0.10±0.03 (Fig. 7B). This data suggests that Ab ΔpglL has a competitive disadvantage as compared to the WT strain. Together, these results indicate that A. baumannii strains lacking O-glycosylation are attenuated in mice.


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)

Characterization of A. baumannii ATCC 17978 pathogenesis in a murine septicemia model.A) Determination of the LD50 of A. baumannii ATCC 17978. Groups of 5 mice were injected with serial dilutions of A. baumannii WT to determine the LD50 which was calculated to be 6.49×104 CFU @ 18 hrs post infection. B) Murine competition septicemia between A. baumannii WT and ΔpglL. Groups of 3 mice were injected with ∼1∶1 WT to ΔpglL CFU's and were sacrificed after 18 hrs, spleens were harvested, and bacterial load determined.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002758-g007: Characterization of A. baumannii ATCC 17978 pathogenesis in a murine septicemia model.A) Determination of the LD50 of A. baumannii ATCC 17978. Groups of 5 mice were injected with serial dilutions of A. baumannii WT to determine the LD50 which was calculated to be 6.49×104 CFU @ 18 hrs post infection. B) Murine competition septicemia between A. baumannii WT and ΔpglL. Groups of 3 mice were injected with ∼1∶1 WT to ΔpglL CFU's and were sacrificed after 18 hrs, spleens were harvested, and bacterial load determined.
Mentions: We then tested A. baumannii ΔpglL virulence in vivo using a previously described murine septicemia competition model [37]–[39]. We first determined the LD50 of A. baumannii ATCC 17978 strain by injecting groups of 5 BALB/c mice with serially diluted bacteria cultures (Fig. 7A). A very small dose range between full survival and full killing was observed, and the LD50 was determined to be 6.49×104 CFU/mouse. The competition index (CI) was defined as the number of ΔpglL CFUs recovered/number of WT CFUs recovered, divided by the number of ΔpglL CFUs inoculated/number of WT CFUs inoculated. Cultures of each strain were mixed at a ratio of 1∶1, serial diluted, and plated to determine the initial CI. 1×105 CFU of the mixed strains were injected intraperitoneally into the BALB/c mice, which were subsequently sacrificed 18 hrs post injection. The spleens were aseptically harvested, serial diluted, and plated. All of the mice had a high spleen CFU load of 3.75±2.37×108 CFU/gram and were moribund at the time of sacrifice. While the initial prescreen showed a CI of 1.18±0.21 favoring the ΔpglL mutant, the spleen counts after 18 hrs showed a CI of 0.10±0.03 (Fig. 7B). This data suggests that Ab ΔpglL has a competitive disadvantage as compared to the WT strain. Together, these results indicate that A. baumannii strains lacking O-glycosylation are attenuated in mice.

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