Characterization of the interaction between Mycobacterium avium subsp. paratuberculosis and bovine epithelial cells in culture and identification of invasion-associated genes by transposon mutagenesis Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/st74cv012

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  • Mycobacterium avium subsp. paratuberculosis (MAP) is a cause of Johne's disease (JD) in cattle and other ruminants. MAP infection in the bovine host is not well characterized. It is assumed that crossing the bovine intestinal mucosa is important for MAP to establish infection. MAP's ability to infect bovine epithelial cells in culture was determined. Cultured monolayer ofMadin- Darby Bovine Kidney (MDBK) cells were infected by 0.90,1.25 and 3.37 % of initial inoculum after 1, 2 and 4 h of incubation, respectively. The results suggest that MAP can establish infection in bovine epithelial cells. Previous clinical studies on MAP infection have suggested that aftet; establishment of the infection in the intestinal mucosa, systemic spread of the infection can occur involving mammary gland. To test the ability ofMAP to infect bovine mammary epithelial cells, MAP culture was incubated with mammary epithelial cells (MAC-T). The results showed an uptake of 1.80,2.51 and 3.64 % of the bacterial inoculum after 1, 2 and 4 h of contact time, respectively, suggesting that MAP can infect mammary epithelial cells. MAP was also shown to translocate across MAC-T cells, from both the apical and the basolateral surfaces at approximately the same level of efficiency (0.11% after 24 h to 0.49% after 96 h). Influence of environmental conditions prior to infection were also investigated. Because MAP can be transmitted to the calf by milk, which is a hyperosmolar medium, it was investigated whether prior incubation in milk impacted the efficiency of invasion. The results indicated that MAP exposed to milk had greater invasion efficiency for MDBK cells compared to MAP exposed to broth or water (P <0.01). Prior intracellular incubation ofMAP in MAC-T cells resulted in an increased ability of MAP to enter bovine epithelial cells (P< 0.001). DNA microarray analysis of MAP genes expressed under intracellular incubation in MAC-T cells revealed that upregulation of genes may be associated with the invasive phenotype. To identify MAP factors associated with invasion of intestinal epithelial cells, a signaturetagged mutagenesis system was created. Individual screening of 600 mutant clones identified 4 mutants with impaired ability to enter cultured bovine epithelial cells (MDBK). Mutant clones 2C12, 265,286 and 2D1 showed significantly less invasive ability (P<0.01) compared to the wild type bacterium. Nucleotide sequencing of 4 mutant clones was carried out. The gene sequence analysis was carried out to identify the transposon insertion site and the interrupted gene in the mutant clones. The results indicated that mycobacterial cell entry (mcelD), oxidoreductase operon, NADH-ubiquinone oxidoreductase and potassium transporter (trkA) are invasion-associated genes for entry ofMAP into MDBK bovine epithelial cells in vitro. Complementation of oxidoreductase gene did not revert the invasive phenotype suggesting that other genes in the oxidoreductase operon may be associated with invasion.
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