Possible translocation mechanisms of C. albicans through IECs. (A) Schematic representation of possible routes of C. albicans translocation. Possible routes of C. albicans translocation are shown as follows: I, apoptosis; II, paracellular; III, transcellular with damage; IV, transcellular without damage. TJ, tight junctions; AJ, adherens junctions; CaL, candidalysin. (B) C2BBe1 IECs were infected with WT C. albicans SC5314 for 5 h and 24 h and differentially stained. Living cells (Hoechst 33342) (blue), apoptotic cells (FITC-annexin V) (green), necrotic cells (ethidium homodimer III) (red), and late apoptotic/necrotic cells (red/green) are indicated by the color(s) indicated. Colored arrows point to examples of the stained cells. (C) A summary of statistical analysis is presented (mean ± SD; n = 3) that quantifies the proportion of live-apoptotic-necrotic staining observed in the images in panel B. (D) Quantification of caspase 3/7 activity in C2BBe1 IECs infected for 5 h and 24 h with C. albicans SC5314. Staurosporine was used as a positive control for the induction of apoptosis. Data are presented as means ± SD from three independent experiments. Caspase 3/7 activity is shown in relative light units (RLU). Values that are not significantly different (ns) are indicated. (E) Transcellular growth of WT C. albicans (BWP17+Clp30) and ece1Δ/Δ mutant hyphae through C2BBe1 IECs. C2BBe1 cells were infected with C. albicans and differentially stained at 6 h p.i. Extracellular C. albicans (pink), C. albicans (blue), and actin (green) are indicated. The white arrows show the point of invasion. (F) TEM images of C2BBe1 IECs infected with WT C. albicans (BWP17+Clp30) or ece1Δ/Δ mutant. The black arrows point to the host membrane. C.a., C. albicans; Cyt, cytoplasm; ES, extracellular space.