FIG. 3.

Epithelial cell contact triggers upregulation of MAP3464 gene expression. (A) The expression of the MAP3464 and 16S genes in wt M. avium subsp. paratuberculosis upon contact with MDBK cells was examined by semiquantitative RT-PCR. After 0, 15, 30, and 60 min of infection, RNA was extracted from extracellular bacteria and cDNA was synthesized as described in Materials and Methods (lanes 1, 2, 3, and 4). For RT-PCR, the MAP3464 and 16S RNA genes were amplified from 1 μl of cDNA by use of specific primers. (B) Upregulation of MAP3464 gene expression (n-fold) upon 15, 30, and 60 min of infection with wt M. avium subsp. paratuberculosis as determined by real-time RT-PCR. The data represent the average of three independent experiments ± standard deviation. The levels of MAP3464 gene expression at 30 and 60 min after infection were comparable. (C) The expression of the MAP3464 and 16S genes in wt M. avium subsp. paratuberculosis and the ΔOx mutant upon 1 h of contact with MDBK cells was examined by semiquantitative RT-PCR. The expression of MAP3464 increased in wt M. avium subsp. paratuberculosis after 1 h of infection (lane 2) compared with what was seen for bacteria incubated in HBSS at 37°C for 1 h (lane 1). As shown in lanes 3 and 4, MAP3464 was not expressed in the ΔOx mutant in the tested conditions. The 16S RNA gene was used as a control because it is constitutively expressed. MW, molecular weight marker.