Abstract

Mycobacterium avium paratuberculosis (MAP) is an acid-fast bacillus that causes a fatal granulomatous ileocolitis called Johne’s Disease that affects primarily ruminants. The impact of MAP infection in the US dairy industry is particularly severe, with up to 70.4% of the herds being infected and annual losses as high as $1.5 billion. MAP is capable of persisting for months in the livestock environment and surviving the pasteurization process. While several vaccines are available, none can prevent infection and treatment with antimycobacterial chemotherapy does not result in a cure. Management and control of MAP infection requires characterization of host-pathogen interaction at the earliest stages in pathogenesis. It is known that establishment of infection requires MAP to bind to M cells found in the bovine ileal epithelial layer covering Peyer’s patches, a process that is mediated by a fibronectin (FN) protein bridge between host cell and MAP fibronectin attachment protein (FAP-P). Although, MAP and other mycobacteria express several FN binding proteins, interference or blocking FAP-P’s ability to bind FN virtually eliminates M cell targeting and intestinal tissue invasion. The binding motif on FN that is recognized by FAP-P has not been demonstrated definitively. Identifying this location may enable the use of peptides that can be added to milk replacer to prevent MAP infection in calves. In this study, we have used a peptide sequence corresponding to the FN binding domain of FAP-P in a luminescence assay to probe a series of FN fragments that together span the length of the FN monomer to identify which of the fragments contained the FAP-P binding site. FN fragments 2, 3, and 4 which contained 550-650 residues and did not overlap. Fragment 1 contained 1959 residues and completely overlapped fragments 2 and 3 and with a partial 78-residue overlap of fragment 4. Significant signal was observed when FN fragments 1 and 4 were probed. This indicated that the site on FN recognized by FAP-P was within this 78-residue region. Further investigation with heat-denatured fragment 4 resulted in reduced probe binding, suggesting that FAP-P binds to a linear sequence of amino acids. Future work will include competition experiments in cell culture.

Advisor

Timothy Secott

Committee Member

Robert Sorensen

Committee Member

Allison Land

Date of Degree

2023

Language

english

Document Type

Thesis

Degree

Master of Science (MS)

Program of Study

Biology

Department

Biological Sciences

College

Social and Behavioral Sciences

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In Copyright