Identification of seven <i>O-</i>glycosylated proteins with nine unique glycosylation sites by ZIC HILIC enrichment of tryptically digested <i>A. baumannii</i> ATCC 17978 membrane extracts.

<p>Identification of seven <i>O-</i>glycosylated proteins with nine unique glycosylation sites by ZIC HILIC enrichment of tryptically digested <i>A. baumannii</i> ATCC 17978 membrane extracts.</p

MS/MS of A1S_3626 and A1S_3744 showing glycosylation in <i>A. baumannii</i> with a pentasaccharide.

<p>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 <i>m/z</i> 2895.165 from A1S_3626 revealed the peptide <b>SAGDQAASDIATATDNASAK</b> with a pentasaccharide of HexNAc-Hex-Hex-(HexNAc)-300 attached. B) MS/MS of the precursor ion peak at <i>m/z</i> 3852.76 from A1S_3744 revealed the peptide <b>ETPKEEEQDKVETAVSEPQPQKPAK</b> with the same pentasaccharide attached. C) MALDI-TOF MS of Pronase E digested membrane proteins showed a precursor ion peak of <i>m/z</i> 1358.4 which MS/MS analysis demonstrated to be the previously identified <i>O</i>-glycan (HexNAc-Hex-Hex-(HexNAc)-300 attached to the peptide fragment “ATD”.</p

Characterization of <i>A. baumannii</i> ATCC 17978 pathogenesis in a murine septicemia model.

<p>A) Determination of the LD₅₀ of <i>A. baumannii</i> ATCC 17978. Groups of 5 mice were injected with serial dilutions of <i>A. baumannii</i> WT to determine the LD₅₀ which was calculated to be 6.49×10⁴ CFU @ 18 hrs post infection. B) Murine competition septicemia between <i>A. baumannii</i> WT and <i>ΔpglL</i>. Groups of 3 mice were injected with ∼1∶1 WT to Δ<i>pglL</i> CFU's and were sacrificed after 18 hrs, spleens were harvested, and bacterial load determined.</p

List of strains, plasmids, and primers used in this work.

<p>List of strains, plasmids, and primers used in this work.</p

¹H∶¹³C HSQC 2D NMR data for the characterization of the <i>A. baumannii</i> 17978 <i>O-</i>glycan.

*<p>Indicates impurity.</p

<i>A. baumannii</i> requires PglL_Ab for biofilm formation.

<p>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 (*, <i>P</i><0.005 [<i>t</i> test; <i>n</i> = 8]; **, <i>P</i><0.001 [<i>t</i> test; <i>n</i> = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, <i>ΔpglL</i>, <i>ΔpglL</i> pWH1266 and <i>ΔpglL</i> 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 (*, <i>P</i><0.05 [Mann-Whitney U test; <i>n</i> = 6]). C)–E) Image stacks of the WT, <i>ΔpglL</i>, <i>ΔpglL</i> pWH1266 and <i>ΔpglL</i> 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 (*, <i>P</i><0.05 [Mann-Whitney U test; <i>n</i> = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and <i>ΔpglL</i> 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 <i>ΔpglL</i> mutant biofilms, respectively.</p

Identification of additional glycoproteins in <i>A. baumannii</i> ATCC 17978.

<p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002758#ppat-1002758-t002" target="_blank">Table 2</a>. A) ITMS-CID of the precursor ion at <i>m/z</i> 1999.943 reveals the pentasaccharide attached to the peptide <b>AKPASTPAVK</b>. B) FTMS-HCD of the precursor ion at <i>m/z</i> 1999.943 reveals the peptide sequence <b>AKPASTPAVK</b>.</p

Comparison of <i>A. baumannii</i> WT and Δ<i>pglL</i> membrane extracts by 2D-DIGE.

<p>Analysis of the membrane proteome of <i>A. baumannii</i> WT strain (A), Δ<i>pglL</i> 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 Δ<i>pglL</i> strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.</p