Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions.

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2'-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy

ESI-MS (positive ion mode, Li+ adducts) analysis of the GlcCer species of <i>S. apiospermum</i>.

<p>(A) MS1 spectrum. (B) ESI-MS2 of the ions species with <i>m/z</i> 734.9 observed in (A) and proposed structures for the major GlcCer species in <i>S. apiospermum.</i> (C) HPTLC plate of monosaccharides from <i>S. apiospermum</i> CMH. 1. Galactose, glucose, mannose and rhamnose standards; 2. Glucose from CMH. The sugars were detected using the orcinol-sulfuric acid spray reagent.</p

Cytotoxic activity of <i>S. apiospermum</i> CMH.

<p>Cytotoxic assay of <i>S. apiospermum</i> CMH on L929 and RAW cells during 48 h of incubation. Cell viability was measured by adding a neutral-red solution and measuring the absorbance at 490 nm using a spectrophotometer. The presence of <i>S. apiospermum</i> CMH in the culture supernatant was analyzed using TLC.</p

Reactivity of fungal CMH with the anti-CMH MAb.

<p>(A) ELISA analysis of the binding of Mabs to <i>S. apiospermum</i> CMH. The amount of antibody bound to CMHs was determined by incubation with a rabbit anti-mouse IgG. CMH from <i>S. apiospermum</i> (closed circle); CMH from <i>A. fumigatus</i> (closed square); CMH from <i>S. apiospermum</i> + unrelated antibody (open circle); negative control (open square). Inset: HPTLC of <i>S. apiospermum</i> CMH (spots a,b,c), which was developed in CHCl3: MeOH: 2 M NH4OH (40:10:1 v/v). Lane 1: stained with orcinol/ H2SO4; Lane 2: immunostaining with the anti-CMH MAb. Indirect immunofluorescence analysis of conidial (B and C) and mycelial (D and E) forms of <i>S. apiospermum</i> using the anti-CMH MAb. B and D: phase contrast; C and E: fluorescence signal.</p

Synergistic effect of combining the anti-CMH Mab with antifungal drugs.

<p>The viability of <i>S. apiospermum</i> conidia was analyzed in the presence of the anti-CMH Mab (50 µg/ml) and different concentrations (0.5 -16 µg/ml) of itraconazole (A) or amphotericin B (B). The values shown are means of three independent experiments.</p

HPTLC analysis of CMH released by <i>S. apiospermum</i> in the culture supernatant.

<p>Lane 1: Purified CMHs from <i>S. apiospermum</i>; Lane 2: CMH released in the supernatant; Lane 3: culture medium supernatant (control). The solvent used was chloroform: methanol: 2 M NH₄OH (40:10:1 v/v). Detection was performed using iodine and the orcinol-sulfuric acid reagent.</p

MAb to CMH affect phagocytosis.

<p>Influence of the Mab to CMH on the phagocytosis of <i>S. apiospermum</i> conidia cells by peritoneal macrophages (A) and on phagocyte antimicrobial activity. Fungal cells treated with the MAb against GlcCer were more efficiently internalized, as demonstrated by the phagocytic indices. (B) Microbial killing by macrophages was enhanced after treatment with antibodies to CMH.</p

Effect of the anti-CMH Mab on the germination and viability of <i>S. apiospermum</i> conidial cells.

<p>(A) Germinated conidia in the presence of different concentrations of anti-CMH Mab over 24 h were counted by optical microscopy. At least 100 conidia per field were counted, and the mean value of three independent counts was calculated. (B) Viability assays were performed using <i>S. apiospermum</i> conidia and the anti-CMH Mab over 24 h and evaluated using MTT. The absorbance at 570 nm was measured using a spectrophotometer.</p

Proposed nomenclature for Pseudallescheria, Scedosporium and related genera

As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.10 page(s