FIG 4.
Sialidase activity, sialic acid release, and nanI gene expression of F4969, the F4969 nanI null mutant, and complementing strain when cocultured in PBS buffer with the HT29 or MTX-E12 cell lines. (A) Culture supernatant sialidase activity measured for 4, 8, or 24 h coculture of F4969 or its derivatives in PBS buffer and HT29 or MTX-E12 cells. Results shown are the average of three repetitions; error bars indicate the SD. Blue asterisks indicate a P value of <0.05 relative to nanI null mutant cultured with HT29 cell line. Orange asterisks indicate a P value of <0.05 relative to nanI null mutant cultured with MTX-E12 cell line. #, P < 0.05 relative to culture with HT29 cell line. (B) Sialic acid release by NanI when F4969 cocultures in PBS buffer with the HT29 or MTX-E12 cell lines at 6 h and 24 h. Sialic acid release due to NanI was calculated as the difference between the concentration of sialic acid released by coculture with F4969 versus the F4969 nanI null mutant strain. Shown is the mean of three repetitions. Error bars indicate SD. *, P < 0.05 relative to culture with HT29 cells. (C) Quantitative qRT-PCR analyses of nanI transcription was performed using 20 ng of F4969 RNA isolated from culture with HT29 or MTX-E12 cells for 4 h at 37°C. Average threshold cycle (CT) values were normalized to the value for the housekeeping 16S RNA gene, and the fold differences were calculated using the comparative CT (2−ΔΔCT) method. Shown is the mean of three repetitions. Error bars indicate SD. *, P < 0.05 relative to culture with HT29 cells.