Ultrastructural analysis of voriconazole-induced changes on <i>Lomentospora prolificans</i> cells.

<p>Transmission electron microscopy images (A) and measurements of cell wall thickness (B) of non-treated and 2 μg/ml voriconazole-treated fungal cells. Black lines highlight the thickness of the outer fibrillar layer. Results are shown as mean ± SEM, n ≥ 20 cells. ***p<0.0001 compared to non-treated cells.</p

Morphological changes on <i>Lomentospora prolificans</i> cells caused by Voriconazole (VRC) exposure.

<p>Germination assays (A) were performed to analyze the effect of VRC on fungal cells. After 9 h of incubation cells were stained with calcofluor white and microscopically analysed (B) to determine their length (C), width (D), occupied area (E), and emitted fluorescence (F). Scale bar = 5 μm. Results are shown as mean ± SEM, n = 4. **p<0.01, ***p<0.0001 compared to non-treated cells. a.u., arbitrary units.</p

Immunoproteomics-Based Analysis of the Immunocompetent Serological Response to <i>Lomentospora prolificans</i>

The filamentous fungus <i>Lomentospora prolificans</i> is an emerging pathogen causing severe infections mainly among the immunocompromised population. These diseases course with high mortality rates due to great virulence of the fungus, its inherent resistance to available antifungals, and absence of specific diagnostic tools. Despite being widespread in humanized environments, <i>L. prolificans</i> rarely causes infections in immunocompetent individuals likely due to their developed protective immune response. In this study, conidial and hyphal immunomes against healthy human serum IgG were analyzed, identifying immunodominant antigens and establishing their prevalence among the immunocompetent population. Thirteen protein spots from each morph were detected as reactive against at least 70% of serum samples, and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Hence, the most seroprevalent antigens were WD40 repeat 2 protein, malate dehydrogenase, and DHN1, in conidia, and heat shock protein (Hsp) 70, Hsp90, ATP synthase β subunit, and glyceraldehyde-3-phosphate dehydrogenase, in hyphae. More interestingly, the presence of some of these seroprevalent antigens was determined on the cell surface, as Hsp70, enolase, or Hsp90. Thus, we have identified a diverse set of antigenic proteins, both in the entire proteome and cell surface subproteome, which may be used as targets to develop innovative therapeutic or diagnostic tools

Effect of voriconazole on proteomic profiles of the <i>Lomentospora prolificans</i> cell surface subproteome.

<p>Fungal cells were grown in absence (A) or presence (B) of 2 μg/ml voriconazole, and their surfaceomes resolved by two-dimensional electrophoresis. Arrows point to the most differentially expressed protein spots that were identified by LC-MS/MS (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174885#pone.0174885.t001" target="_blank">Table 1</a>).</p

Biochemical characterization of the carbohydrate composition of <i>Lomentospora prolificans</i> cell wall.

<p>Carbohydrate compositional analysis of whole cell wall (A) and cell wall surface (B) upon exposure to 2 μg/ml voriconazole. Results are shown as mean ± SEM, n = 3. *p<0.05 compared to non-treated cells. Percentage of monosaccharide content in the whole cell wall (C) and surface (D).</p

Effect of cell membrane- and wall-disturbing agents on <i>Lomentospora prolificans</i> in the presence of voriconazole.

<p>Decimal dilutions of conidial suspensions were spotted onto potato dextrose agar plates containing SDS (100 μg/ml), calcofluor white (500 μg/ml; CFW) or congo red (750 μg/ml; CR), and combined with 0, 2 or 4 μg/ml of voriconazole (VRC).</p

Identification of the most differentially expressed proteins on <i>Lomentospora prolificans</i> cells exposed to voriconazole.

<p>Identification of the most differentially expressed proteins on <i>Lomentospora prolificans</i> cells exposed to voriconazole.</p