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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 pathogenesis is dependent on PglLAb in the Galleria mellonella virulence model.Representative data of survival rate of 3 biological replicates of 10 individual G. mellonella injected with 2.31±1.13×105 CFU of each strain in 5 µL of sterilized PBS and incubated @ 37°C. Survival was assayed by response to touch or discoloration. While killing by WT and ΔpglL-pglL was observed, no killing was observed in the ΔpglL and 20% killing was observed in the ΔpglL-pWH1266 strains up to 96 hours. No killing was observed in the PBS injection control for the length of the experiment.
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ppat-1002758-g006: A. baumannii pathogenesis is dependent on PglLAb in the Galleria mellonella virulence model.Representative data of survival rate of 3 biological replicates of 10 individual G. mellonella injected with 2.31±1.13×105 CFU of each strain in 5 µL of sterilized PBS and incubated @ 37°C. Survival was assayed by response to touch or discoloration. While killing by WT and ΔpglL-pglL was observed, no killing was observed in the ΔpglL and 20% killing was observed in the ΔpglL-pWH1266 strains up to 96 hours. No killing was observed in the PBS injection control for the length of the experiment.

Mentions: Two well-established virulence models for A. baumannii are the D. discoideum predation and the G. mellonella infection models [5], [29]–[33]. D. discoideum is an unicellular amoeba that feeds on bacteria and previous work has demonstrated similarity between phagocytosis of the amoebae and mammalian phagocytes [34]. We examined if protein glycosylation was required for virulence towards D. discoideum by co-incubation of A. baumannii strains with the amoebae on SM/5 nutrient agar. A. baumannii was previously shown to inhibit amoebae growth in the presence of 1% ethanol [5]. The WT strain was virulent and inhibited all D. discoideum growth in the presence of 1% ethanol, which resulted in no plaque being formed. However the ΔpglL strain was avirulent towards the amoeba, which resulted in plaque formation in the bacterial lawn within 48 hours and clearing of the plate within 4–5 days (Fig S5). G. mellonella have been used to study many host-pathogen interactions, and have several advantages over other virulence models including the presence of both humoral (ie. antimicrobial peptides) and cellular immune response systems (phagocytic cells) [32]. Most importantly, a correlation has been established between the virulence of several bacteria in G. mellonella and mammalian models [35], [36]. For the G. mellonella, while a similar bacterial load (2.31±1.13×105 CFU) was injected for each of the strains, only the WT and complemented strains were able to kill the wax moth larvae after 36 hours, (20% and 0% survival), whereas larvae injected with ΔpglL and the ΔpglL vector control strains had significantly higher survival rates (100% and 80%; Fig. 6). The LD50 of the WT and complemented strains were determined to be approximately 2.6×104 and 1.4×104 respectively after 36 hours. No additional killing was observed in the ΔpglL or vector control strains up to 96 hours. A PBS injected control maintained 100% survival throughout the length of the virulence assay. These results demonstrate a critical role for O-glycosylation in the virulence of A. baumannii in these two model systems.


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 pathogenesis is dependent on PglLAb in the Galleria mellonella virulence model.Representative data of survival rate of 3 biological replicates of 10 individual G. mellonella injected with 2.31±1.13×105 CFU of each strain in 5 µL of sterilized PBS and incubated @ 37°C. Survival was assayed by response to touch or discoloration. While killing by WT and ΔpglL-pglL was observed, no killing was observed in the ΔpglL and 20% killing was observed in the ΔpglL-pWH1266 strains up to 96 hours. No killing was observed in the PBS injection control for the length of the experiment.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002758-g006: A. baumannii pathogenesis is dependent on PglLAb in the Galleria mellonella virulence model.Representative data of survival rate of 3 biological replicates of 10 individual G. mellonella injected with 2.31±1.13×105 CFU of each strain in 5 µL of sterilized PBS and incubated @ 37°C. Survival was assayed by response to touch or discoloration. While killing by WT and ΔpglL-pglL was observed, no killing was observed in the ΔpglL and 20% killing was observed in the ΔpglL-pWH1266 strains up to 96 hours. No killing was observed in the PBS injection control for the length of the experiment.
Mentions: Two well-established virulence models for A. baumannii are the D. discoideum predation and the G. mellonella infection models [5], [29]–[33]. D. discoideum is an unicellular amoeba that feeds on bacteria and previous work has demonstrated similarity between phagocytosis of the amoebae and mammalian phagocytes [34]. We examined if protein glycosylation was required for virulence towards D. discoideum by co-incubation of A. baumannii strains with the amoebae on SM/5 nutrient agar. A. baumannii was previously shown to inhibit amoebae growth in the presence of 1% ethanol [5]. The WT strain was virulent and inhibited all D. discoideum growth in the presence of 1% ethanol, which resulted in no plaque being formed. However the ΔpglL strain was avirulent towards the amoeba, which resulted in plaque formation in the bacterial lawn within 48 hours and clearing of the plate within 4–5 days (Fig S5). G. mellonella have been used to study many host-pathogen interactions, and have several advantages over other virulence models including the presence of both humoral (ie. antimicrobial peptides) and cellular immune response systems (phagocytic cells) [32]. Most importantly, a correlation has been established between the virulence of several bacteria in G. mellonella and mammalian models [35], [36]. For the G. mellonella, while a similar bacterial load (2.31±1.13×105 CFU) was injected for each of the strains, only the WT and complemented strains were able to kill the wax moth larvae after 36 hours, (20% and 0% survival), whereas larvae injected with ΔpglL and the ΔpglL vector control strains had significantly higher survival rates (100% and 80%; Fig. 6). The LD50 of the WT and complemented strains were determined to be approximately 2.6×104 and 1.4×104 respectively after 36 hours. No additional killing was observed in the ΔpglL or vector control strains up to 96 hours. A PBS injected control maintained 100% survival throughout the length of the virulence assay. These results demonstrate a critical role for O-glycosylation in the virulence of A. baumannii in these two model systems.

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