FIGURE 1.

LoxB is up-regulated upon arachidonic acid exposure and promotes germination. (A) loxB but not loxA is expressed in glucose minimal media (GMM) +0.5% tergitol and RPMI. Different spore concentrations of 1 × 10⁶ and 1 × 10⁵ spores were tested to look at density-dependent effects on lipoxygenase expression after 48 h of growth at 37°C. loxB was up-regulated at 1 × 10⁶ spores/mL in RPMI media compared to GMM. Actin served as a loading control for semi-qPCR analysis. (B) Verification of LoxB enzymatic activity in novel fungal strains. The previously documented product of A. fumigatus LoxB (13-HODE) was assessed in WT, ΔloxB, and OE::loxB strains. While significant levels of 13-HODE were identified in OE::loxB supernatant, background levels equivalent to those identified in the blank were recorded in WT and ΔloxB supernatants. (C) Addition of 0.5 μg/μL arachidonic acid (AA) reduces the number of wild type swollen spores (SS) and germlings (GLs) compared to mock treatment, however, the same concentration of linoleic acid (LA) does not (D). (E) loxB is up-regulated in the presence of AA after 29 h at 37°C. Values are average (n = 3) fold expression ± SEM after standardization to wild type tissue from mock treatment (vehicle). (F) Germination assay for loxB strains grown in GMM + 0.5% tergitol in the presence and absence of AA. Addition of AA significantly delays SS and GL formation of all strains compared to the mock treatment. (G) Upon exposure to 0.5 μg/μL LA, no difference in growth is observed after 15 hpi between wild type, ΔloxB, and loxB(c) complement strains. However, the increase in SS and GLs when loxB is overexpressed is maintained as in (F), suggesting delayed germination of the ΔloxB strain is a function of AA treatment, whereas accelerated germination of the OE::loxB strain is not. Values represent average of n = 3 trials ± SEM and Student’s t-test was used to identify statistical differences, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.