Generation of a bloodstream formTrypanosoma bruceidouble glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

Abstract The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake; i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis. Author Summary Blood-stage trypanosome parasites have a specialised invagination on the cell surface named the flagellar pocket (FP), where invariant essential nutrient receptors are located. The pocket houses diverse proteins, including a transferrin receptor (TfR), which facilitates uptake of host transferrin-bound iron for survival. Several FP proteins, including TfR, are linked to complex sugar molecules (carbohydrates), the functions of which are not well understood. Complex carbohydrates are made by enzymes called glycosyltransferases (GTs) and previously we partially inhibited complex carbohydrate synthesis by deletion of either TbGT8 or TbGT10. However, mutant parasites lacking either one of these enzymes survived, suggesting functional redundancy. Here, we created a parasite mutant that lacks both TbGT8 and TbG10 to understand the combined effect of losing both enzymes. The mutant parasites showed a decreased ability to uptake tomato lectin, a protein that specifically binds to these sugar conjugates in the FP, indicating a reduction in carbohydrate complexity. Despite reduced complexity in the sugar structures attached to TfR, its critical function in transferrin/iron uptake remained effective. Furthermore, the mutants remained viable in culture and in animal models, challenging previous assumptions about the necessity and function of these carbohydrate conjugates. Our findings imply a greater flexibility and redundancy in the carbohydrate complex roles than previously appreciated. Competing Interest Statement The authors have declared no competing interest. Abbreviations - VSG - variant surface glycoprotein - GPI - glycosylphosphatidylinositol - N-glycans - asparagine-linked glycans - BSF - bloodstream form - PCF - procyclic form - pNAL - poly-N-acetyl-lactosamine - N-acetyllactosamine, LacNAc - Galβ1-4GlcNAc - lacto-N-biose, LNB - Galβ1-3GlcNAc - GTs - glycosyltransferases - CAZY - Carbohydrate Active enZYmes - WGA - wheat germ agglutinin - GNTI - GlcNAc-transferase I - GNTII - GlcNAc-transferase II - RCA - Ricinius communis agglutinin - LAMP - lysosomal membrane protein - TfR - Transferrin receptor - BSD - blasticidine deaminase - HYG-TK - hygromycin phosphotransferase-thymidylate kinase - SSU - ribosomal small subunit.