Emerging Evidence Suggests That N-Glycosylation Pathway Perturbation Has a Greater Impact than Microbial Depletion on α-Gal Antigen Biogenesis in Lone Star Tick (Amblyomma americanum) Salivary Glands

Alpha-gal syndrome (AGS) is an emerging tick-associated allergy characterized by IgE-mediated hypersensitivity to galactose-α-1,3-galactose (α-gal). Although α-gal antigens have been identified in Amblyomma americanum salivary glands, the mechanisms governing their production during feeding remain unresolved. Here, we investigated whether the tick core microbes or endogenous tick glycosylation pathways contribute to α-gal antigen biogenesis. Microbial depletion was induced by feeding larvae and nymphs on tetracycline-treated α-gal-deficient mice, followed by molting into adults; adult ticks were subsequently injected with tetracycline and maintained under microbiota-reduced conditions. In parallel, inhibition of N-linked glycosylation and galactosyltransferase activity using tunicamycin and 2-deoxy-2-fluoro-D-galactose (dGalF), respectively, was used to assess glycosylation-dependent mechanisms. α-gal-associated antigens were further evaluated by immunoblotting and confocal microscopy using an anti-α-gal antibody. Despite a marked reduction in bacterial burden in immature and adult ticks, tetracycline treatment α-gal-associated antigen remained detectable in partially fed nymph and adult salivary glands. Treatment with tunicamycin or dGalF reduced α-gal-associated antigen levels, as determined by Western blot analysis, whereas microbiota reduction did not measurably affect α-gal-associated antigen levels under the conditions tested. These preliminary findings suggest that endogenous glycosylation-dependent processes primarily contribute to α-gal antigen biogenesis in A. americanum salivary glands.