Article
Role of enterococcal cell wall polysaccharide structure in resistance to killing by neutrophils
Einfluss der Zellwandpolysaccharidstruktur von Enterokokken auf die Resistenz gegen Opsonophagozytose von neutrophilen Granulozyten
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Authors
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S.T. Geiß
- Uniklinik Freiburg, Innere Med. II, Freiburg, Germany
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P. Sanchez-Carballo
- Forschungszentrum Borstel, Strukturbiochemie, Borstel, Germany
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C. Theilacker
- Uniklinik Freiburg, Innere Med. II, Freiburg, Germany
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O. Holst
- Forschungszentrum Borstel, Strukturbiochemie, Borstel, Germany
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J. Hübner
- Uniklinik Freiburg, Innere Med. II, Freiburg, Germany
| Published: | June 2, 2010 |
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Outline
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Enterococci are a major cause of urinary tract infections, endocarditis and bacteriaemia. As many Streptococcus spp., Enterococcus faecalis expresses a complex carbohydrate known as the cell wall polysaccharide or type antigen. The cell wall polysaccharide is known to be involved in epithelial translocation, biofilm formation and resistance to killing by neutrophils. Little is known, however, how the cell wall carbohydrate impedes opsonophagocytic clearance.
We have generated an insertion mutant of the gene EF1172 in E. faecalis V583 (28% Id, 49% Si to tagB from B. subtilis 168). E. faecalis V583Δ1172 was sensitive to opsonophagocytic killing in the presence of 1.5% baby rabbit serum. SDS PAGE analysis of carbohydrates of cell walls after enzymatic digestion of peptidoglycan revealed a retarded migration of the cell wall polysaccharide of E. faecalis V583Δ1172 compared to the wild type strain. No difference in biofilm formation and adhesion to eukaryotic cell between wild type and mutant was observed.
To further investigate the structure of the cell wall polysaccharide from E. faecalis V583 wild type and V583Δ1172, we isolated the corresponding carbohydrates from both strains by size exclusion chromatography and anion-exchange chromatography. Both carbohydrates eluted at the same volume from a Sephacryl S-200 column, indicating comparable molecular mass of both polysaccharides. Compositional analysis revealed the presence of rhamnose, glucose and glucosamine in both samples. Galactosamine was only detected in the wild type polysaccharide but not in the polysaccharide obtained from V583D1172. Quantification of the neutral sugars revealed a different ratio of rhamnose to glucose in the polysaccharides; while the ratio of rhamnose:glucose was ≈2:1 in E. faecalis V583 wild type, it was ≈4:1 in V583D1172. Also, cell wall polysaccharide of the wild type strain contained twice as much phosphate as the corresponding carbohydrate from V583D1172.
In summary, we found that only minor changes in the composition of the cell wall polysaccharide of E. faecalis profoundly affect complement resistance. Further investigations of the chemical structure are needed to understand the molecular basis of complement resistance mediated by this cell wall polymer in E. faecalis.
