Figure 5. Transcription of the C. perfringens tpeL toxin gene is not upregulated early during infection of Caco-2 cell cultures.

(A) PCR was performed using primers to specifically amplify either the C. perfringens tpeL gene (lanes 2 and 4) or a 3´ fragment of the C. difficile tcdA gene (lanes 3 and 5) that is not present in the tpeL gene (Amimoto et al., 2007). As template, DNA extracted from C. difficile strain 00030 (C. diff) or C. perfringens strain JGS1495 (C. per) was used. Lane 1 contains 100 bp ladder, selected marker size is shown at left of each gel. (B) RT-PCR for tpeL or polC transcripts. Type C strain JGS1495 was inoculated into a TGY, MEM or a Caco-2 cell culture and then incubated for 2 h at 37°C, or inoculated in TY and incubated for 24 h. Bacteria were collected from growing conditions and pelleted by centrifugation. Total RNA was extracted from those pellets and treated with DNase I. RT-PCR reactions were then performed with 20–100 ng of RNA (results shown are for 100 ng) and using a specific pair of primers to amplify either tpeL or the house keeping polC gene. Where indicated, reverse transcriptase (RT) was (+) or was not (−) added into the reaction tubes. As a control, DNA from JGS1495 was added into a reaction tube. Molecular markers were increments of a 100 bp ladder, size of selected markers, in bp, is shown at the left of the gel.