FIG 2.

E. faecalis enhances EHEC T3SS activity. (A) A TEM-1 β-lactamase translocation assay was used to measure translocation of Tir. Reporter EHEC strains express either TEM-1 β-lactamase (bla) as a control or a tir-bla fusion that will be translocated into host cells via the T3SS. EHEC reporter strains were pregrown under T3SS-inducing conditions alone or in the presence of the indicated commensal strains and then used to infect HeLa cells. HeLa cells were then loaded with a fluorescent β-lactam compound whose emission spectrum is altered by β-lactamase cleavage. The 460/530 emission (em) ratio reflects the level of β-lactamase activity and therefore the level of Tir translocation into cells. The average and standard deviation of three replicates from a single experiment are plotted, and the experiment was repeated three times. (B) HeLa cells were infected with EHEC with or without E. faecalis strain V583 for 6 h; then cells were washed, fixed, and stained with FITC-phalloidin. Pedestals were visualized as green puncta of actin beneath attached red bacteria. The numbers of pedestals per field were counted for seven randomly selected fields, and the average and standard deviations are plotted. Scale bar, 10 μm. Two-tailed t tests were performed to determine significant differences compared to results for EHEC alone. **, P < 0.01; ***, P < 0.001.