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Published on: September 30, 2011
Purification of a Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans
1Department of Microbiology, School of Medicine, State University of New York, Buffalo 14214.
Abstract:
Proteins of Streptococcus mutans MT703 were isolated by differential filtration from chemically defined culture medium following growth of the bacteria. Incubation of this preparation with cryostat-cut sections of fresh rabbit cardiac muscle resulted in deposition of streptococcal components on basement membranes of sarcolemmal sheaths and capillary walls, as indicated by indirect immunofluorescence assay. Binding of radioiodinated streptococcal proteins to heart in vitro was time dependent and saturable. Unlabeled S. mutans proteins competitively inhibited 72% of heart binding by the radiolabeled proteins, indicating a high level of binding specificity. A selection of components common to tissue basement membranes was tested for their abilities to inhibit the binding of streptococcal proteins to heart tissue. Of the glycosaminoglycans, heparin was the most effective inhibitor, followed by heparan sulfate and chondroitin sulfate. Hyaluronic acid was not inhibitory. Of the glycoproteins tested, laminin and collagen type IV were weakly inhibitory, whereas fibronectin was ineffective. A single polypeptide was purified to homogeneity by affinity chromatography on a column of heparin-agarose. Gel filtration chromatography of the purified protein under nondissociating conditions showed a single component at 31 kilodaltons (kDa), whereas in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one band appeared at 8 kDa. This indicates that the tissue-binding protein may either be a linear polypeptide or be released into the environment by the bacterium as a tetramer of the 8-kDa polypeptide. The purified protein had an isoelectric point of 9.5 and showed binding activity for basement membranes in thin sections of heart. Chemical analyses of the purified binding protein showed it to have high contents of lysine and alanine and to be devoid of half-cystine, methionine, tyrosine, histidine, and both neutral and amino sugars.
Insights
Streptococcus mutans proteins bind specifically to heart basement membranes. A purified 8-kDa polypeptide, possibly a tetramer, mediates this binding, with heparin showing inhibitory effects.
Area of Science:
- Microbiology
- Biochemistry
- Cardiovascular Research
Background:
- Streptococcus mutans is a bacterium associated with various infections.
- Bacterial adherence to host tissues is a critical step in pathogenesis.
- Understanding bacterial-host interactions is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the interaction of Streptococcus mutans proteins with cardiac tissue.
- To identify and characterize the specific bacterial components responsible for binding to heart basement membranes.
- To elucidate the molecular mechanisms underlying this interaction.
Main Methods:
- Isolation of Streptococcus mutans proteins using differential filtration.
- Incubation of bacterial proteins with rabbit cardiac muscle sections.
- Indirect immunofluorescence assay to detect bacterial component deposition.
- In vitro binding assays with radioiodinated proteins.
- Affinity chromatography and gel filtration for protein purification.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
- Isoelectric focusing and chemical analysis of the purified protein.
Main Results:
- Streptococcal components deposited on basement membranes of cardiac sarcolemma and capillaries.
- Binding of S. mutans proteins to heart tissue was time-dependent and saturable.
- Unlabeled S. mutans proteins inhibited radiolabeled protein binding, indicating specificity.
- Heparin was the most effective inhibitor of binding among tested basement membrane components.
- A single polypeptide of 31 kDa (nondissociating) or 8 kDa (SDS-PAGE) was purified.
- The purified protein bound to basement membranes and contained high lysine and alanine content.
Conclusions:
- Streptococcus mutans possesses proteins that specifically bind to cardiac basement membranes.
- A purified 8-kDa polypeptide, potentially existing as a tetramer, is involved in this binding.
- Heparin and related glycosaminoglycans can inhibit the binding of these bacterial proteins to heart tissue.
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