Pathogenic characterization of Phialophora submersa, a new black yeast isolated from freshwater sediments in Spain

<i>Phialophora submersa</i> is a recently described black yeast species (<i>Chaetothyriales</i>), isolated from freshwater sediments in Catalonia (Spain). It is closely related to <i>P. americana</i> and <i>P. verrucosa</i>, two opportunistic pathogens known to cause subcutaneous infections in humans and animals. This study investigates the pathogenic potential of <i>P. submersa</i>, its <i>in vitro</i> susceptibility to clinically relevant antifungal agents, and its response to various cellular stressors. Using a murine macrophage (J774A.1) infection model, we evaluated phagocytosis, intracellular survival, cell damage, and the expression of six immune-related genes (<i>TNF-α</i>, <i>CCL20</i>, <i>RELA</i>, <i>TP53</i>, <i>NLRP3</i>, <i>IL-1β</i>), in comparison with <i>P. americana</i> and <i>P. verrucosa</i>. The results showed that <i>P. submersa</i> induced higher phagocytosis rates in murine macrophages than the <i>P. verrucosa</i>, although lower than <i>P. americana</i>. Cell damage, intracellular survival, and expression of the immune-related genes were higher after macrophage infection with <i>P. verrucosa</i> than with <i>P. submersa</i> and <i>P. americana</i>, which exhibited comparable profiles. All three species displayed similar antifungal susceptibility profiles, being susceptible to most azoles (except fluconazole), terbinafine, and echinocandins (with reduced efficacy against <i>P. verrucosa</i>), but showed moderate resistance to flucytosine, amphotericin B, and olorofim. The resistance of <i>P. submersa</i> to stress was strain-dependent, with only one strain exhibiting notable resistance to multiple stressors. This research provides new insights into the biology of <i>P. submersa</i>, including its potential as a human pathogen, and the molecular factors that could drive an infection process.