RESUMEN
Globo H (GH) is a tumor-associated carbohydrate antigen (TACA), and GH conjugations have been evaluated as potential cancer vaccines. However, like all carbohydrate-based vaccines, low immunogenicity is a major issue. Modifications of the TACA increase its immunogenicity, but the systemic modification on GH is challenging and the synthesis is cumbersome. In this study, we synthesized several azido-GH analogs for evaluation, using galactose oxidase to selectively oxidize C6-OH of the terminal galactose or N-acetylgalactosamine on lactose, Gb3, Gb4, and SSEA3 into C6 aldehyde, which was then transformed chemically to the azido group. The azido-derivatives were further glycosylated to azido-GH analogs by glycosyltransferases coupled with sugar nucleotide regeneration. These azido-GH analogs and native GH were conjugated to diphtheria toxoid cross-reactive material CRM197 for vaccination with C34 adjuvant in mice. Glycan array analysis of antisera indicated that the azido-GH glycoconjugate with azide at Gal-C6 of Lac (1-CRM197) elicited the highest antibody response not only to GH, SSEA3, and SSEA4, which share the common SSEA3 epitope, but also to MCF-7 cancer cells, which express these Globo-series glycans.
RESUMEN
Chili peppers are an important food additive used in spicy cuisines worldwide. However, the yield and quality of chilis are threatened by anthracnose disease caused by Colletotrichum acutatum. Despite the impact of C. acutatum on chili production, the genes involved in fungal development and pathogenicity in this species have not been well characterized. In this study, through T-DNA insertional mutagenesis, we identified a mutant strain termed B7, which is defective for the growth of C. acutatum on a minimal nutrient medium. Our bioinformatics analysis revealed that a large fragment DNA (19.8 kb) is deleted from the B7 genome, thus resulting in the deletion of three genes, including CaGpiP1 encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein, CaNRT2.1 encoding a membrane-bound nitrate/nitrite transporter, and CaRQH1 encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the CaHP1 gene encoding a hypothetical protein. Functional characterization of CaGpiP1, CaNRT2.1, and CaHP1 by targeted gene disruption and bioassays indicated that CaNRT2.1 is responsible for the growth-defective phenotype of B7. Both B7 and CaNRT2.1 mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to CaNRT2.1, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of CaGpiP1 and CaHP1 in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in C. acutatum. Collectively, in this study, our results identify the growth-defective strain B7 via T-DNA insertion and reveal the critical role of CaNRT2.1 in nitrate transportation for the fungal growth of C. acutatum.