Abstract
Fibronectin attachment protein (FAP)- mediated binding of Mycobacterium avium subspecies paratuberculosis (MAP) to M cells is necessary for the early development of Johne's disease. Fibronectin (FN) plays a key role in this interaction by serving as a molecular bridge, connecting MAP-FAP to β1 integrins on M cells that cover Peyer's patches. The region on FN that is required for this attachment was previously described at the molecular level to be confined to a 78-amino acid region found within the fourth fragment of FN. The goal of this study was to further characterize this binding at the organism level by developing an assay to demonstrate FAP-mediated binding and to inhibit this interaction with a competitor peptide.In the integration assay, Mycobacterium smegmatis (MS), used as a proxy, was pretreated with biotinylated fibronectin (bFN) and exposed to streptavidin-coated magnetic beads. The MS+bFN+Beads complex was labeled with anti-FN, fixed with paraformaldehyde (para-f), and stained with propidium iodide (PI). In the competition experiment, MS was simultaneously pretreated with the competitor fibronectin (cFN) containing the 78-amino-acid sequence and bFN at a 1:1 and 1:2 mass ratios, respectively. The organism was exposed to magnetic beads, probed with anti-FN, fixed with para-f, and labeled with PI. The binding interaction and complex formation were analyzed by flow cytometry, and bead-associated aggregates and bacterial capture were detected using fluorescence profiles and scatter properties. Flow cytometry analysis of the integration assay shows beads with dual (red and green) fluorescence after exposure and attachment to bFN-bound MS. There was increased forward scatter (FSC) and side scatter (SSC), indicating formation of larger and more complex MS+bFN+Beads aggregates. Statistical analysis using the Kruskal-Wallis test showed a significant difference among groups (p = 0.0002), supporting the role of fibronectin in mediating bacterial capture. The competition assay resulted in decreased bead capture of bFN-bound MS and, consequently, reduced dual fluorescence relative to the control. There was no significant difference in bead capture of bFN-bound MS when the concentration of bFN was increased from 1:1 to 1:2 mass ratio. This indicates that the cFN fragment containing the 78-amino acid region competitively inhibited the binding of whole bFN to MS, thereby blocking its capture and attachment to the magnetic beads. These results suggest that at the organism level, the MS-FAP binding domain on FN lies within the 78-amino acid region on the fourth FN fragment, and is required for FAP-mediated binding. Future work can identify the peptide's core binding motif to develop milk replacers.
Advisor
Timothy Secott
Committee Member
Cecelia Noecker
Committee Member
Robert Sorensen
Date of Degree
2026
Language
english
Document Type
Thesis
Degree
Master of Science (MS)
Program of Study
Biology
Department
Biological Sciences
College
Science, Engineering and Technology
Recommended Citation
Boachie, E. A. (2026). Characterization of the interaction of Mycobacterium avium subspecies paratuberculosis Fibronectin attachment protein with Fibronectin at the organism level [Master’s thesis, Minnesota State University, Mankato]. Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato. https://cornerstone.lib.mnsu.edu/etds/1633/
Rights Statement
