Rhamnose-containing glycans and lipids differentially contribute to the Sporothrix-host interaction

Background Rhamnose is a distinctive component of the cell wall of pathogenic Sporothrix species and plays important roles in host–pathogen interactions, including immune recognition, phagocytosis, and virulence. However, the mechanisms responsible for its incorporation into different surface glycoconjugates remain poorly understood. This study investigated the role of the putative rhamnosyltransferases RHT1 and RHT2 in Sporothrix schenckii and Sporothrix brasiliensis . Methods RHT1 - and RHT2 -silenced mutants were generated in both species and analyzed for rhamnosyltransferase activity, cell wall composition, adhesion, biofilm formation, interactions with human immune cells, and virulence in the Galleria mellonella model. Cytokine production, TLR4-dependent recognition, and phagocytosis were evaluated to determine the contribution of distinct rhamnosylated glycoconjugates to host–pathogen interactions. Results Silencing of either gene reduced total rhamnosyltransferase activity but produced distinct effects on cell wall glycoconjugates. RHT2 silencing selectively decreased rhamnose incorporation into N -linked and O -linked glycans, whereas RHT1 silencing primarily altered the carbohydrate composition of a surface glycolipid, indicating functional specialization. RHT1 -silenced mutants displayed reduced adhesion to HeLa cells and impaired biofilm formation in both species. Immune interaction assays revealed species-specific effects: in S. schenckii, RHT2 silencing had a stronger impact on cytokine production, TLR4-dependent recognition, and phagocytosis, whereas in S. brasiliensis , both RHT1 and RHT2 significantly contributed to these processes. Virulence assays showed that silencing either gene reduced virulence in S. schenckii , while virulence attenuation in S. brasiliensis was primarily associated with RHT1 silencing. Conclusions RHT1 and RHT2 perform specialized, non-redundant functions in the biosynthesis of rhamnosylated glycoconjugates in Sporothrix . The distribution of rhamnose between glycoproteins and glycolipids differentially influences immune recognition, host colonization, and virulence, revealing species-specific contributions to the pathogenicity of S. schenckii and S. brasiliensis .