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. 2020 Apr 2;21(7):2465.
doi: 10.3390/ijms21072465.

Glycomics Microarrays Reveal Differential In Situ Presentation of the Biofilm Polysaccharide Poly- N-acetylglucosamine on Acinetobacter baumannii and Staphylococcus aureus Cell Surfaces

Andrea Flannery  1   2 , Marie Le Berre  3 , Gerald B Pier  4 , James P O'Gara  2 , Michelle Kilcoyne  1   3
Affiliations

Glycomics Microarrays Reveal Differential In Situ Presentation of the Biofilm Polysaccharide Poly- N-acetylglucosamine on Acinetobacter baumannii and Staphylococcus aureus Cell Surfaces

Andrea Flannery et al. Int J Mol Sci. .

Abstract

The biofilm component poly-N-acetylglucosamine (PNAG) is an important virulence determinant in medical-device-related infections caused by ESKAPE group pathogens including Gram-positive Staphylococcus aureus and Gram-negative Acinetobacter baumannii. PNAG presentation on bacterial cell surfaces and its accessibility for host interactions are not fully understood. We employed a lectin microarray to examine PNAG surface presentation and interactions on methicillin-sensitive (MSSA) and methicillin-resistant S. aureus (MRSA) and a clinical A. baumannii isolate. Purified PNAG bound to wheatgerm agglutinin (WGA) and succinylated WGA (sWGA) lectins only. PNAG was the main accessible surface component on MSSA but was relatively inaccessible on the A. baumannii surface, where it modulated the presentation of other surface molecules. Carbohydrate microarrays demonstrated similar specificities of S. aureus and A. baumannii for their most intensely binding carbohydrates, including 3' and 6'sialyllactose, but differences in moderately binding ligands, including blood groups A and B. An N-acetylglucosamine-binding lectin function which binds to PNAG identified on the A. baumannii cell surface may contribute to biofilm structure and PNAG surface presentation on A. baumannii. Overall, these data indicated differences in PNAG presentation and accessibility for interactions on Gram-positive and Gram-negative cell surfaces which may play an important role in biofilm-mediated pathogenesis.

Keywords: Acinetobacter baumannii; PNAG; Staphylococcus aureus; bacterial adhesins; biofilm; glycomics microarrays; lectin; poly-N-acetylglucosamine; polysaccharide.

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Conflict of interest statement

Gerald B. Pier is an inventor of intellectual properties (human monoclonal antibody to PNAG and PNAG vaccines) that are licensed by Brigham and Women’s Hospital to Alopexx Vaccine, LLC, and Alopexx Pharmaceuticals, LLC, entities in which Gerald B. Pier also holds equity. As an inventor of intellectual properties, Gerald B. Pier also has the right to receive a share of licensing-related income (royalties, fees) through Brigham and Women’s Hospital from Alopexx Pharmaceuticals, LLC, and Alopexx Vaccine, LLC. Gerald B. Pier’s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners Healthcare in accordance with their conflict of interest policies. The other authors declare no competing financial interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Structure of PNAG and verification of PNAG production. (a) Structure of partially deacetylated PNAG. Modifications of PNAG such as deacetylation and O-succinylation vary depending on bacterial genus and strain [32]. (b) Dot blot of heat-killed S. aureus Mn8m, S. aureus 8325-4 and A. baumannii S1 WT and mutant strains cultured under PNAG-promoting conditions detected by anti-PNAG mAb. The same cell numbers were loaded for comparison between strains (approximately 2 × 106 cells).
Figure 2
Figure 2
Lectin recognition of PNAG alone. (a) Lectin microarray profile of fluorescently labelled PNAG purified from S. aureus Mn8m culture. Bars represent the binding intensity of the mean of three experiments with error bars of +/−1 standard deviation (SD) of the mean. (b) Nonlinear fit transformation of GlcNAc inhibition PNAG binding to sWGA intensity data. Data points are the mean of three experiments with error bars of +/−1 SD of the mean. (c) Nonlinear fit transformation of GlcNAc inhibition of PNAG binding to WGA intensity data. Data points are the mean of three experiments with error bars of +/−1 SD of the mean.
Figure 3
Figure 3
Surface glycosylation profiles of WT bacterial strains grown in BHI media supplemented with glucose or NaCl. Bar charts represent binding intensities of bacteria to lectins on the lectin microarray. (a) S. aureus BH1CC WT and ∆ica mutant bacterial strains grown in BHI media with 1% glucose, (b) S. aureus 8325-4 WT and ∆ica mutant bacterial strains grown in BHI media with 4% NaCl, (c) S. aureus Mn8m and Mn8 ∆ica mutant bacterial strains grown in BHI media with 1% glucose, and (d) A. baumannii WT and ∆pga grown in BHI media with 1% glucose. Bars represent the mean of three experiments with error bars of +/−1 SD of the mean. * represents significant difference (p ≤ 0.05, calculated by Student’s t-test, two tailed) in binding between WT and ∆pga/ ∆ica.
Figure 4
Figure 4
Carbohydrate microarray binding intensity profiles of S. aureus BH1CC WT and ∆ica grown in BHI supplemented with (a) 1% glucose and (b) 4% NaCl. Bars represent the mean of three experiments with error bars of +/−1 SD of the mean.
Figure 5
Figure 5
Carbohydrate binding intensities of bacterial strains. (a) Unsupervised hierarchical clustering of carbohydrate microarray binding intensities for S. aureus Mn8m, 8325-4, BH1CC and A. baumannii all grown in BHI supplemented with 1% glucose, and supplemented with 4% NaCl for S. aureus 8325-4 and BH1CC. Binding intensity data was scale-normalised to 20,000 RFU maximum and clustered using Hierarchical Clustering Explorer v3.0 with complete linkage and Euclidean distance. (b) Bar chart representing carbohydrate binding intensities of A. baumannii WT and ∆pga grown in BHI glucose. Bars represent the mean of three experiments with error bars of +/−1 SD of the mean. * represents significant difference (p ≤ 0.05, calculated by Student’s t-test, two tailed) in binding between WT and ∆pga.
Figure 6
Figure 6
Model of proposed presentation of PNAG on the surface of (a) methicillin-sensistive S. aureus, and (b) A. baumannii.
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