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. 2021 Sep 10;17(9):e1009884. doi: 10.1371/journal.ppat.1009884

Fig 6. The ECE1 P2-P3 sequence combination is critical for peak immunopathology and candidalysin secretion.

Fig 6

(A) Schematic showing ECE1 open reading frames containing SC5314 or 529L P2, P3, or P2-P3 sequences that were restored in an isogenic ece1Δ/Δ strain. Strains were intravaginally inoculated into estrogen-treated C57BL/6 mice. Mice (n = 12 per group) underwent vaginal lavage at d 3 post-infection and lavage fluids assessed for (B) CFU by microbiological plating (median ± IQR), (C) PMN recruitment by microscopy (median ± IQR), (D) IL-1β by ELISA (median ± IQR), and (E) tissue damage by LDH assay (median ± IQR). Statistical significance was assessed using Kruskal-Wallis and Dunn’s post-test. A431 vaginal epithelial cells were challenged with these same strains and (F) IL-1β measured by ELISA (mean ± SD) and (G) LDH release assessed (mean ± SD). (H) Similar strains harboring C-terminal candidalysin HiBiT tags were grown overnight in RPMI-1640 medium and cell-free supernatants assessed for luciferase activity (mean ± SD). Statistical significance was assessed by one-way ANOVA and Dunnet’s post-test. (I) P2-P3 sequences were parsed using ProP 1.0 Server to determine cleavage scores. All in vitro experiments were conducted in biological triplicate. *, p < 0.05, ** p < 0.01, *** p < 0.001.