EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion

Abstract

EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasionGraphical abstract Highlights d Inducible epithelial cell loss allows modeling of fungal infection in zebrafish larvae d Extrusion of numerous cells exposes laminin and increases fungal adhesion and invasion d Cell extrusion stimulates epigen, mmp13a, and il1b expression and neutrophil recruitment d rhEPGN suppresses extrusion to provide protective effects against fungal invasion

Protocol for fungal infection following the induction of epithelial cell loss in larval zebrafish

“… Note: Extrusion and the extent of associated tissue damage can be assessed by using the Et(Gal4-VP16) zc1044a , Tg(UAS-1b:nsfB-mCherry) c264 line in conjunction with other transgenic lines such as, Tg(UAS-1b:Lifeact-EGFP) utm1 ; Tg( k rt4:GFP); and Tg(-8.0cldnb:lynEGFP) zf106 that allow for independent visualization of both the surface epithelial cells and cell-cell contacts. For additional details about quantification of the resulting epithelial tissue damage, please see ( Wurster et al., 2021 ). …”

“… Note: Considering a remainder of 100 μL E3 medium in the wells after the last wash step, addition of 1 mL of fresh E3 medium and the 100-μL volume of the spore inoculum itself, this inoculum results in our recommended standard spore concentration of 5×10 6 /mL. However, we have previously shown that the infection severity can be easily adjusted by using up to 10-fold lower or higher spore inoculums (as seen in Figure 1L in ( Wurster et al., 2021 ). CRITICAL: All experiments should include control groups of uninfected larvae with epithelial homeostasis and induced epithelial cell loss, respectively, as a sentinel for non-infection-attributable baseline mortality.…”

“… Incubate plates for 16 h (± 1 h) at 28°C on a nutator ( Figure 1 ). Optional: For defining interactions of fungal spores with the damaged epithelium, anesthetized (tricaine-S) infected and non-infected larvae can be mounted in a 4-well MatTek dish and high-resolution imaging can be performed using laser scanning or spinning disc confocal microscopy as in ( Atieh et al., 2021 ; Eisenhoffer and Rosenblatt, 2011 ) or anesthetized larvae can be mounted in 24-well plates on soft agar and imaged using the IncuCyte ZOOM microscope system ( Wurster et al., 2021 ). …”

“…Limitations of the protocol include potential antifungal toxicity and high fungal inoculums to induce lethal infection with some pathogenic fungal species. For complete details on the use and execution of this protocol, please refer to Wurster et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Wurster et al. (2021) .…”