Silencing of <em>OCH1 </em>unveils the role of <em>Sporothrix schenckii</em> <em>N</em>-linked glycans during the host&ndash;fungus interaction

Abstract

BackgroundSporothrix schenckii is a neglected fungal pathogen for the human being and other mammals. In several fungal systems, Och1 is a Golgi α1,6-mannosyltransferase with a key function in the synthesis of N-linked glycans; which are important elements during the host-fungus interplay. The role of OCH1 in fungal virulence seems to be species-specific, being an essential component for Candida albicans virulence and dispensable during the interaction of Aspergillus fumigatus with the host.MethodsHere, we silenced S. schenckii OCH1 and characterized the phenotype of the mutant strains.ResultsThe mutant strains did not show defects in the cell or colony morphology, the growth rate or the ability to undergo dimorphism; but the cell wall changed in both composition and exposure of inner components at the surface. When interacting with human monocytes, the silenced strains had a reduced ability to stimulate TNFα and IL-6 but stimulated higher levels of IL-10. The interaction with human macrophages was also altered, with reduced numbers of silenced cells phagocytosed. These strains showed virulence attenuation in both Galleria mellonella and in the mouse model of sporotrichosis. Nonetheless, the cytokine levels in infected organs did not vary significantly when compared with the wild-type strain.ConclusionOur data demonstrate that OCH1 silencing affects different aspects of the S. schenckii-host interaction.

Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells

“…To remove N-linked mannans from the cell wall, yeast cells were incubated for 20 h at 37 • C with 25 U of endoglycosidase H (New England Biolabs, Ipswich, MA, USA) in 100 mM sodium acetate, pH 5.2 [33], while O-linked mannans were trimmed by resuspending cells in 20 mL 1 N NaOH and gently shaken for 20 h at room temperature [34]. In both cases, cells were pelleted by centrifuging, the supernatants collected, the pH neutralized, lyophilized, and used to quantify the sugar content with the phenol-sulfuric acid method [35,36].…”

“…The PBMC-yeast interactions were carried out in round-bottom 96-well plates containing aliquots of 100 µL of 5 × 10 6 PBMCs/mL in RPMI 1640 Dutch modification (added with 2 mM glutamine, 0.1 mM pyruvate, and 0.05 mg/mL gentamycin; all reagents from Sigma), and 100 µL of 1 × 10 6 yeast cells/mL. The interactions were incubated for 24 h at 37 • C with 5% (v/v) CO 2 , plates were centrifuged for 10 min at 874× g and 4 • C; the supernatants were recollected and kept at −20 • C until used [36]. The concentration of TNF-α, IL-6, and IL-10 was quantified by ELISA using the kit ABTS ELISA Development from Peprotech (Rocky Hill, NJ, USA).…”

“…The excess of dye was removed by washing cells twice with PBS and resuspended at a cell concentration of 3 × 10 7 yeast cells/mL. Interactions were performed in aliquots of 800 µL of DMEM (Sigma), in 6-well plates with a macrophage-yeast ratio of 1:6 [36]. Plates were incubated for 2 h at 37 • C and 5% (v/v) CO 2 .…”

“…Fluorescent signals were obtained using the FL1 (green) and FL3 (red) channels previously compensated with macrophage and yeast cells without labeling. Phagocytosis of yeast-like cells was analyzed from counted events in green (early stage of the phagocytosis process), in both green and red (intermediate stage of the phagocytosis process), and red (cells within acidified phagolysosomes, regarded as a late stage of the phagocytosis process) [36,48].…”

“…The last left pro-leg of larvae was disinfected with 70% (v/v) ethanol, and 2 × 10 7 yeast cells contained in 10 µL of PBS were injected with a Hamilton syringe and a 26-gauge needle. Animals were kept at 28 • C and survival was monitored daily for 2 weeks [36]. Loss of irritability and body melanization were taken as signs of animal death.…”