Silencing of <i>ROT2</i>, the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the <i>Sporothrix schenckii</i>–Host Interaction

<i>Sporothrix schenckii</i> is a member of the <i>Sporothrix</i> 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 <i>Sporothrix</i> cell wall is composed of structural polysaccharides and glycoproteins that are covalently modified with both <i>N</i>-linked and <i>O</i>-linked glycans. Thus far, little is known about the <i>N</i>-linked glycosylation pathway in this organism or its contribution to cell wall composition and interaction with the host. Here, we silenced <i>ROT2</i>, which encodes the catalytic subunit of the endoplasmic reticulum α-glucosidase II, a processing enzyme key for the <i>N</i>-linked glycan core processing. Silencing of <i>ROT2</i> led to the accumulation of the Glc₂Man₉GlcNAC₂ glycan core at the cell wall and a reduction in the total content of <i>N</i>-linked glycans found in the wall. However, the highly silenced mutants showed a compensatory mechanism with increased content of cell wall <i>O</i>-linked glycans. The phenotype of mutants with intermediate levels of <i>ROT2</i> 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, <i>S. schenckii ROT2</i> is required for proper <i>N</i>-linked glycosylation, cell wall organization and composition, and interaction with the host.