Characterization of the fibronectin-binding MSCRAMM BBK32 from Borrelia burgdorferi : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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College Station, Tex. :
Texas A&M University System Health Science Center,
2004.
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| Subjects: |
| Abstract: | ABSTRACT: BBK32 is a fibronectin (Fn)-binding MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecule) on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBk32 is composed to two domains: an N-terminal segment lacking well-defined secondary structures and a C-terminal segment composed largely of alpha-helices. Analysis of purified recombinant forms of the two domains by circular dichroism (CD) spectrometry, gel-permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal extended, unstructured segment and a C-terminal globular domain in BBK32. Solid phase binding experiments suggested that the Fn-binding activity is located to the unstructured N-terminal domain. Binding of the unstructured N-terminal domain is accompanied by a conformational change in the MSCRAMM as indicated by BBK32 antibodies recognizing Ligand Induced Binding Sites (LIBS) present in the Fn-MSCRAMM complex but not in the apoform of the proteins. Analysis of changes in CD of BBK32 upon Fn-binding revealed the formation of additional beta-strands in the complex. Examination of the sequence of BBK32 in more detail revealed that it contains segments homologous to Fn-binding motifs in MSCRAMMs from Gram-positive bacteria and the a Fn-binding sequence in collagen. Using a series of recombinant proteins containing single or multiple homology regions in BBK32, we found that the Borrelia MSCRAMM contained multiple Fn-binding motifs that contributed to the overall affinity of intact BBK32 for Fn. Furthermore, BBK32 recognized two distinct themolysing-generated Fn fragments; the N-terminal Heparin-binding domain and the Gelatin-binding domain. Analysis of the binding specificities of the recombinant truncates revealed that discrete regions in BBK32 bound to different Fn-domains. Isothermal Titration Calorimetry analysis of the interaction of the N-terminal extended domain of BBK32 and Fn in a 1:1 stochiometry. More extensive amino acid sequence analysis suggests that the segment of amino acid 116-210 in BBK32 contains first 5 Fn type I module specific residues in order. Taken together, these data suggest that BBK32 interacts with fibronectin in an anti-parallel manner through a mechanism resembling the tandem [beta]-zipper that was previously demonstrated for fibronectin-binding protein interactions of Gram-positive bacteria (Schwarz-Linek, U. et al. (2003) Nature 432, 177-181). |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Graduate School of Biomedical Sciences of The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy August 2004." Approved as to style and content by: Magnus Höök, eric L. Brown, Steven J. Norris, Richard R. Sinden, Jon T. Skare, Richard Finnell. |
| Physical Description: | xi, 114 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references. |