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, 8 (6), e1002758

Identification of a General O-linked Protein Glycosylation System in Acinetobacter Baumannii and Its Role in Virulence and Biofilm Formation

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Identification of a General O-linked Protein Glycosylation System in Acinetobacter Baumannii and Its Role in Virulence and Biofilm Formation

Jeremy A Iwashkiw et al. PLoS Pathog.

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. Comparison of A. baumannii WT and ΔpglL membrane extracts by 2D-DIGE.
Analysis of the membrane proteome of A. baumannii WT strain (A), ΔpglL strain (B), and merge (C). Spots WT1 and WT2 only present in the WT strain (green) whereas MT1 and MT2 were only present in the ΔpglL strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.
Figure 3
Figure 3. 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”.
Figure 4
Figure 4. Identification of additional glycoproteins in A. baumannii ATCC 17978.
Tryptically digested membranes were enriched via ZIC-HILIC and analyzed by LC-MS and HCD MS-MS. All spectra were analyzed for the diagnostic oxonium ion of 301.10 m/z, and positive spectra were analyzed manually to identify the glycopeptide. This spectra is representative of each glycopeptide identified in Table 2. A) ITMS-CID of the precursor ion at m/z 1999.943 reveals the pentasaccharide attached to the peptide AKPASTPAVK. B) FTMS-HCD of the precursor ion at m/z 1999.943 reveals the peptide sequence AKPASTPAVK.
Figure 5
Figure 5. A. baumannii requires PglLAb for biofilm formation.
A) Quantitative biofilm formation on polystyrene 96 well plates by strains incubated without perturbation in LB at 30°C. The bars indicate the means for 8 replicates. The error bars indicate the standard deviation of the means. Asterisks indicate significant differences (*, P<0.005 [t test; n = 8]; **, P<0.001 [t test; n = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL was determined by the COMSTAT software. For each strain at least six micrographs from three independent experiments were analyzed. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). C)–E) Image stacks of the WT, ΔpglL, ΔpglL pWH1266 and ΔpglL ppglL biofilms grown in flow cells for 24 h were analyzed for the biomass as well as the maximum and average thickness using the COMSTAT software. Shown are the medians of at least six image stacks from three independent experiments for each strain. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, P<0.05 [Mann-Whitney U test; n = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and ΔpglL mutant (lower row) biofilms grown in flow cells for 24 h. The first three images represent horizontal (xy, large panel) and vertical (xz and yz, side panels) projections at different z-levels (from left to right 0.2 µm, 3 µm and 6 µm). The fourth micrograph of each row represents a three-dimensional image analyzed by the AMIRA software package of the WT and ΔpglL mutant biofilms, respectively.
Figure 6
Figure 6. 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.
Figure 7
Figure 7. 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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