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

Abstract

Severe and often fatal opportunistic fungal infections arise frequently following mucosal damage caused by trauma or cytotoxic chemotherapy. Interaction of fungal pathogens with epithelial cells that comprise mucosae is a key early event associated with invasion, and, therefore, enhancing epithelial defense mechanisms may mitigate infection. Here, we establish a model of mold and yeast infection mediated by inducible epithelial cell loss in larval zebrafish. Epithelial cell loss by extrusion promotes exposure of laminin associated with increased fungal attachment, invasion, and larval lethality, whereas fungi defective in adherence or filamentation have reduced virulence. Transcriptional profiling identifies significant upregulation of the epidermal growth factor receptor ligand epigen (EPGN) upon mucosal damage. Treatment with recombinant human EPGN suppresses epithelial cell extrusion, leading to reduced fungal invasion and significantly enhanced survival. These data support the concept of augmenting epithelial restorative capacity to attenuate pathogenic invasion of fungi associated with human disease.

Keywords: EGF signaling; cell extrusion; epitheli; fungi; zebrafish.

Figure 1.. A model of invasive fungal infection mediated by inducible epithelial cell loss

(A) Schematic of a 4 days post fertilization (dpf) zebrafish larva (scale bar, 500 μm); scanning electron microscopy (SEM) image of Rhizopus arrhizus (clinical isolate 749) spores prior to immersion with larvae (scale bar, 5 μm); and timeline of addition and removal of MTZ to induce epithelial damage, addition of 5 × 10⁶/mL R. arrhizus spores for 16 h, and survival monitoring. W, triple wash step. (B–I) Maximum intensity projections of confocal images for (B–I) NTR-mCherry and (F–I) Rhizopus arrhizus-GFP in the orofacial region and tail fin of 5 dpf larvae under homeostatic conditions (B, C, F, and G) and after epithelial damage (D, E, H, and I). Arrowheads in (D) and (E) denote cell extrusion events, and arrowheads in (F) and (H) denote fungal spore attachment and hyphal growth, respectively. Scale bars, 40 μm. (J) Larva survival curves after Rhizopus infection under homeostatic conditions and with tissue damage (n = 27–28 per condition). (k) Survival rates of R. arrhizus-infected larvae with differential intensity of tissue damage (n = 43 per MTZ concentration). (L) Larva survival curves after tissue damage and exposure to differential amounts of R. arrhizus spores (n = 31–33 per inoculum). (M) Survival rates of larvae with mucosal damage infected with 5 × 10⁶ heat-inactivated (30 min, 100 C) or vital R. arrhizus 749 spores (n = 42 per condition). (J–M) Aggregated survival rates across three independent experiments are plotted. Error bars represent SD; Mantel-Cox log rank test.

Figure 2.. Continual epithelial cell extrusion compromises barrier integrity and leads to lethal fungal invasion

(A–C) Fluorescence images for NTR-mCherry and Rhizopus arrhizus-GFP in the tail fin of 5 dpf larvae after epithelial damage. Arrowheads denote epithelial tissue damage, and asterisks mark the fungus. Scale bar, 200 μm. (D) Mean number of hyphal invasion sites per larva. Hyphal invasion sites were quantified in 37 MTZ-treated larvae and 25 larvae during epithelial homeostasis from 2 independent experiments. Error bars represent SEM; unpaired two-sided t test. (E–G) Maximum intensity projections of confocal images of induced epithelial extrusion and subsequent tissue damage over time (NTR-mCherry and cldnB:lyn-GFP). Boxes denote regions shown below. Double arrowheads denote amounts of tissue contraction. Scale bars, 100 μm. (H and I) Maximum intensity projections of confocal images of homeostatic epithelium and epithelial tissue damage after MTZ treatment (NTR-mCherry and Krt4:GFP). Arrows denote areas of tissue contraction. Arrowheads denote regions with epithelial gaps. Scale bar, 20 μm. (J and K) SEM image of R. arrhizus 749 spores during invasion after mucosal damage. Scale bars, 10 μm. (L) Survival rates of larvae infected with R. arrhizus 749 spores immediately after epithelial damage or after a 24-h recovery period, a time point when the epithelial tissue tears and gaps have largely resolved. Aggregated survival rates based on 42–45 larvae per condition across three independent experiments are plotted. Error bars represent SD; Mantel-Cox log rank test.

Figure 3.. Fungal spores adhere to damaged epithelia and initiate hyphal growth

(A and B) Maximum intensity projections of confocal images of R. arrhizus-GFP spores adhered to areas of damaged epithelial tissue with exposed laminin compared with homeostatic epithelium. Scale bar, 20 μm. (C) Quantification of spore adherence to wells coated with increasing concentrations of laminin. (D and E) Time-lapse imaging of R. arrhizus-GFP spores adhered at sites of epithelial damage undergoing germination. Arrowheads denote germlings/early hyphae. Asterisks denote epithelial cell extrusion. Scale bar, 20 μm. (F) Survival rates of larvae with induced cell loss infected with R. arrhizus 749 spores pre-coated or not with 0.2 or 2 μg/mL laminin. Aggregated survival rates based on 75 larvae per condition across five independent experiments, and SDs are plotted. (G) Survival rates of larvae with induced cell loss after addition of CotH-depleted R. arrhizus (CotHRNAi) and the corresponding wild-type control (control RNAi). (H) Survival rates of larvae with induced cell loss after addition efg1, cph1, or efg1/cph1 C. albicans mutants compared with an isogenic control (SC5314). Aggregated survival rates based on 44–45 larvae per condition across three independent experiments are plotted. Error bars represent SD. (I) Survival rates of larvae after induced cell loss after R. arrhizus infection and treatment with voriconazole (VRC), amphotericin B (AMB), and posaconazole (PCZ), normalized to the number of animals alive at the time of treatment (16 h post infection, n = 50–52 across five independent experiments). (F–I) Mantel-Cox log rank test.

Figure 4.. Treatment with rhEPGN suppresses epithelial cell extrusion and attenuates invasive fungal infection

(A) Heatmap of differentially expressed genesbetween homeostatic conditions (no MTZ) and induced epithelial cell loss by extrusion (MTZ-treated). q < 0.05, adjusted p value, Deseq2 and Benjamini-Hochberg method for FDR correction, three independent biological replicates, 15–30 larvae per replicate. (B) Gene Ontology (GO) analysis of gene categories enriched after epithelial cell loss. (C) Maximum intensity projections of fluorescent in situ hybridization for epigen in larvae with induced cell loss compared with homeostasis. Scale bar, 20 μm. (D–F) Larval survival after treatment with rhEPGN (D), rhEPGN and the EGFR inhibitor AG1478 (E), or rhEPGN and the MEK inhibitor U0126 (F) at different times during induction of damage and infection. Aggregated survival rates based on five (D, n = 72–74 per condition) or three independent experiments (E and F, n = 42–43 per condition), respectively, are plotted. Error bars represent SD; Mantel-Cox log rank test. (G) Quantification of the number of extruding cells with and without rhEPGN or rhEGF treatment during induced damage. Data are from three independent experiments, and error bars represent SD; ***p < 0.001; ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. (H–K) Maximum intensity projections of confocal still images of a time-lapse sequence after induction of epithelial cell loss (H and I) and during treatment with rhEPGN (J) or rhEGF (K). Scale bar, 50 μm.

Figure 5.. Neutrophils are recruited to sites of mucosal damage and invasive fungal infection

(A–C) Maximum intensity projections of confocal images of larvae stained for the neutrophil marker mpx (green) during homeostasis (A), after mucosal damage (B), and mucosal damage and Rhizopus infection. Scale bar, 20 μm. (D) Quantification of the total number of neutrophils present in the epithelium (n = 108 number of animals). (E–G) Still images from time-lapse videos of neutrophil dynamics after cell loss-induced mucosal damage and after treatment with hrEPGN to suppress extrusion. Scale bar, 50 μm. (H) Quantification of the number of circulating neutrophils with and without rhEPGN or rhEGF treatment during induced damage. Data are from 38 animals from three independent experiments. Error bars represent SD. (I) Still images from time-lapse videos of a neutrophil migrating to a site of extrusion (denoted by asterisks). (J and K) Maximum intensity projection images of fluorescent in situ hybridization for mmp13a and il1b in larvae with induced cell loss compared with those treated with hrEPGN. Scale bar, 20 μm.