Silencing of <i>MNT1</i> and <i>PMT2</i> Shows the Importance of <i>O</i>-Linked Glycosylation During the <i>Sporothrix schenckii</i>–Host Interaction

<i>Sporothrix schenckii</i> is a pathogenic fungus of worldwide distribution and one of the etiological agents of sporotrichosis. The cell wall is the first point of contact with host cells; therefore, its composition has been widely studied. It has a cell wall composed of chitin, β-glucans, and glycoproteins modified with <i>N-</i>linked and <i>O-</i>linked glycans. Protein <i>O-</i>linked glycosylation is mediated by two gene families, <i>PMT</i> and <i>MNT.</i> Therefore, we evaluated the relevance of protein <i>O-</i>linked glycosylation during the interaction of <i>S. schenckii</i> with the host. Independent silencing of the <i>MNT1</i> and <i>PMT2</i> was accomplished by interference RNA. Morphological analyses revealed defects in cell morphology in both yeast and mycelial cells; however, these defects differed between <i>MNT1</i> and <i>PMT2</i> silencing. Subsequently, the cell wall was characterized, and the silencing of these genes markedly changed cell wall organization. When the silenced strains interacted with human peripheral blood mononuclear cells, a reduced ability to stimulate the proinflammatory cytokines IL-6 and TNFα was found. However, the <i>PMT2-</i>silenced mutants also stimulated higher levels of IL-10 and IL-1β. Interaction with macrophages and neutrophils was also altered, with increased phagocytosis and decreased extracellular trap formation in both sets of silenced strains. Survival assays in <i>Galleria mellonella</i> larvae showed that silencing of any of these genes reduced the ability of <i>S. schenckii</i> to kill the host. In addition, the mutant strains showed defects in the adhesion to extracellular matrix proteins. These data indicate that <i>MNT1</i> and <i>PMT2</i> are relevant for cell wall synthesis and interaction with the host.