FIG 6.

Multiphoton laser scanning microscopy (MPLSM) of SipA-phiLOV induction of caspase-3 activation ex vivo. (A) The SL1344, ΔSipA, and ΔSipA/SipA-phiLOV strains were used to infect an ileal loop model, and caspase-3 activation was detected with a laser scanning multiphoton microscope. Active caspase-3 was detected in epithelial cells at the tips of numerous villi in both SL1344- and ΔSipA/SipA-phiLOV-infected loops but was absent in ΔSipA and control loops. In ΔSipA/SipA-phiLOV-infected loops this signal colocalized with a strong green fluorescence, visible over native tissue fluorescence, and this was not seen in SL1344 infection (see Fig. S2 in the supplemental material for additional images). To determine the localization of food boluses observed in the control intestine and distinguish their autofluorescence from that attributable to phiLOV or active caspase-3, 3D videos to show their intraluminal location were prepared. Corresponding videos for each infection were also made (see Movies S1 to S4 in the supplemental material). (B) Using the Section View mode in Imaris software, distinct points within villi were inspected by showing the cut-through point in the x, y, and z axes simultaneously. Representative images are shown, demonstrating the strong intracellular signal attributable to SipA-phiLOV. This signal is absent in SL1344 villi where caspase-3 is present, and no visible active caspase-3 is present in ΔSipA-infected or control villi.