Alberta Glycomics Centre, Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
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.
13C NMR, 1H NMR, NMR-2D, SDS-PAGE, Western blotting, MALDI-TOF MS, composition analysis, genetic methods
NMR data for the A. baumannii O-glycan (D2O, 28°C, 600 MHz). NAc: 1.95/23.0; 2.00/23.3; 2.04/23.5; 2.04/23.5 ppm, all C-1 at 175.9 ppm. OAc: 2.06/21.3, 174.0 ppm. The amino acids attached to the pentasaccharide were determined to be Ser-Glu-Ala (order not determined).
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6
?,?,3,3,6,4,2 Ac
?,?,3,3,6,4,3 Ac
?,?,3,3,6,4,4 Ac
?,?,3,3,6,4 bDGlcpN3NA 103.4 54.5 53.9 71.9 76.0 174.4
?,?,3,3,6,6,2 Ac
?,?,3,3,6,6 bDGlcpN 103.1 56.7 75.2 71.0 77.0 61.8
?,?,3,3,6 aDGalp 99.3 69.8 70.6 79.1 70.8 72.3
?,?,3,3 bDGlcp 105.7 74.0 76.9 70.6 75.5 66.8
?,?,3,2 Ac
?,?,3 bDGalpN 101.9 52.0 81.4 69.0 76.1 62.2
?,? x?Ser 178.2 54.3 68.1
? x?Glu 178.4 54.2 27.7 31.4 ?
x?Ala? 175.9 51.0 17.7
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6
?,?,3,3,6,4,2 Ac
?,?,3,3,6,4,3 Ac
?,?,3,3,6,4,4 Ac
?,?,3,3,6,4 bDGlcpN3NA 4.84 3.92 4.20 4.96 3.99 -
?,?,3,3,6,6,2 Ac
?,?,3,3,6,6 bDGlcpN 4.50 3.70 3.52 3.45 3.45 3.77-3.94
?,?,3,3,6 aDGalp 4.95 3.68 3.94 4.12 4.02 3.78-4.12
?,?,3,3 bDGlcp 4.53 3.30 3.47 3.50 3.61 3.69-3.88
?,?,3,2 Ac
?,?,3 bDGalpN 4.58 4.07 3.87 4.14 3.70 3.77-3.80
?,? x?Ser - 4.30 4.14-4.14
? x?Glu - 4.45 2.02-2.14 2.51-2.51 -
x?Ala? - 4.42 1.42
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
?,?,3,3,6,4,2 Ac
?,?,3,3,6,4,3 Ac
?,?,3,3,6,4,4 Ac
?,?,3,3,6,4 bDGlcpN3NA 103.4/4.84 54.5/3.92 53.9/4.20 71.9/4.96 76.0/3.99
?,?,3,3,6,6,2 Ac
?,?,3,3,6,6 bDGlcpN 103.1/4.50 56.7/3.70 75.2/3.52 71.0/3.45 77.0/3.45 61.8/3.77-3.94
?,?,3,3,6 aDGalp 99.3/4.95 69.8/3.68 70.6/3.94 79.1/4.12 70.8/4.02 72.3/3.78-4.12
?,?,3,3 bDGlcp 105.7/4.53 74.0/3.30 76.9/3.47 70.6/3.50 75.5/3.61 66.8/3.69-3.88
?,?,3,2 Ac
?,?,3 bDGalpN 101.9/4.58 52.0/4.07 81.4/3.87 69.0/4.14 76.1/3.70 62.2/3.77-3.80
?,? x?Ser 54.3/4.30 68.1/4.14-4.14
? x?Glu 54.2/4.45 27.7/2.02-2.14 31.4/2.51-2.51
x?Ala? 51.0/4.42 17.7/1.42
report error
Found 1 record.
Displayed record 1
]; infection due to Acinetobacter baumannii [ICD11: XN8LS 
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