Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii -Host Interaction.

Sporothrix schenckii is a member of the Sporothrix pathogenic clade and one of the most common etiological agents of sporotrichosis, a subcutaneous fungal infection that affects both animal and human beings. Like other fungal pathogens, the Sporothrix cell wall is composed of structural polysaccharides and glycoproteins that are covalently modified with both N -linked and O -linked glycans. Thus far, little is known about the N -linked glycosylation pathway in this organism or its contribution to cell wall composition and interaction with the host. Here, we silenced ROT2 , which encodes the catalytic subunit of the endoplasmic reticulum α-glucosidase II, a processing enzyme key for the N -linked glycan core processing. Silencing of ROT2 led to the accumulation of the Glc 2 Man 9 GlcNAC 2 glycan core at the cell wall and a reduction in the total content of N -linked glycans found in the wall. However, the highly silenced mutants showed a compensatory mechanism with increased content of cell wall O -linked glycans. The phenotype of mutants with intermediate levels of ROT2 silencing was more informative, as they showed changes in the cell wall composition and exposure of β-1.3-glucans and chitin at the cell surface. Furthermore, the ability to stimulate cytokine production by human mononuclear cells was affected, along with the phagocytosis by human monocyte-derived macrophages, in a mannose receptor-, complement receptor 3-, and TLR4-dependent stimulation. In an insect model of experimental sporotrichosis, these mutant cells showed virulence attenuation. In conclusion, S. schenckii ROT2 is required for proper N -linked glycosylation, cell wall organization and composition, and interaction with the host.