Proceedings of the 10th International Colloquium on Paratuberculosis

#228 Mycobacterium avium ssp. paratuberculosis infection causes change in <strong>the</strong> epi<strong>the</strong>lial
cells permeability: correlation with <strong>the</strong> cell adhesion and tight junction molecule expression
Sangeeta Khare, Quynhtien Tran, L. Garry Adams
Texas A&M University, USA
Epi<strong>the</strong>lial cells constitute <strong>the</strong> first mechanical and immunological barrier against infection <strong>of</strong> <strong>the</strong> Mycobacterium
avium ssp. paratuberculosis (MAP). Earlier, we found that cell-adhesion molecules were one <strong>of</strong> <strong>the</strong> top-scoring
pathways affected by MAP infection during bovine ligated-ileal-loop experiment.
Objective: The objective for this study was to distinguish <strong>the</strong> role <strong>of</strong> epi<strong>the</strong>lial cells during MAP
interaction, and correlate it with cell-adhesion molecule gene expression and alteration in <strong>the</strong> tight junction and
permeability <strong>of</strong> infected cells.
Materials and Methods: The T84, a human cell line <strong>of</strong> colon carcinoma cells were polarized after seeding
<strong>the</strong> cells on <strong>the</strong> apical compartment <strong>of</strong> Transwell plates and adding medium to <strong>the</strong> basolateral compartment.
During 7-10 days, <strong>the</strong> cells develop a transepi<strong>the</strong>lial resistance (TER) that allows <strong>the</strong>m to mimic <strong>the</strong> conditions
<strong>of</strong> <strong>the</strong> intestinal epi<strong>the</strong>lium. A method for attaining <strong>the</strong> epi<strong>the</strong>lial permeability in vitro was achieved by <strong>the</strong> measurement
<strong>of</strong> electrical physical resistance to determine <strong>the</strong> TER. These cells developed a transepi<strong>the</strong>lial resistance
<strong>of</strong> 500-1500W/cm2. MAP were added at a multiplicity <strong>of</strong> infection ratio <strong>of</strong> 1:10 (cells to bacteria ratio).
The TER <strong>of</strong> Polarized T84 cells was measured at various times post-infection. The cells were also processed
to obtain RNA for <strong>the</strong> gene expression.
Results: The results show that MAP infection caused a decrease in <strong>the</strong> TER, thus increased <strong>the</strong> permeability
<strong>of</strong> <strong>the</strong> epi<strong>the</strong>lial cells for bacterial invasion. The polarity <strong>of</strong> <strong>the</strong> epi<strong>the</strong>lial cells was disturbed due to alteration
in <strong>the</strong> tight junction. Various cell adhesion molecules (Vascular, Intracellular, Neuronal and Junctional), that
play important role in <strong>the</strong> maintenance <strong>of</strong> <strong>the</strong> cellular architecture, showed a differential gene expression.
Conclusion: In conclusion, this study show that MAP infection to epi<strong>the</strong>lial cells causes increased permeability
by weakening <strong>of</strong> <strong>the</strong> tight junctions. Permeability <strong>of</strong> epi<strong>the</strong>lial cells is also correlated with <strong>the</strong> changes in
<strong>the</strong> gene expression <strong>of</strong> cell adhesion molecules.
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