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Glycogenomics and glycobiotechnology of mannosylated glycoconjugates

Final Activity Report Summary - MANNOGLYC (Glycogenomics and glycobiotechnology of mannosylated glycoconjugates)

In the course of the MANNOGLYC Marie Curie Excellence Grant, research was performed along three major lines, the results of which can be summarised as follows:
1) We studied the importance of mannosylated lipoarabinomannan (ManLAM) as a factor in interaction of pathogenic Mycobacteria with their host cells. To do this, we generated transposon insertion mutants in key genes in the biosynthetic pathway of ManLAM and used the resulting mutants in studies of uptake and phagocytosis by macrophages and dendritic cells, as well as in vivo behaviour. Whereas previous studies had concluded that ManLAM is an important mediator of the phagosomal maturation block in macrophages, we found little or no evidence for such role of the molecule, when assessed in the context of an otherwise intact bacterium. Subsequently, we expanded our studies to other pathways which had been indirectly implicated in phagosomal maturation blockade by Mycobacteria. The PI3P phosphatase SapM, when mutated, also did not yield a strong defect in this aspect of pathogenesis. Furthermore, utilizing the M. bovis BCG transposon insertion library which was generated in these studies, we initiated genetic screens to identify mutants specifically deficient in intraphagosomal aspects of pathogenesis.

2) HIV-1 has a densely glycosylated surface glycoprotein and its glycans to a large extent consist of high-mannose glycans. Betting on the similarity between these glycans and the often immunodominant fungal cell surface glycans, we explored whether it would be possible to derive antigens from fungal organisms which would induce an immune response that was cross-reactive with the HIV-1 conserved N-glycan shield. To this end, we first profiled the N-glycome of several hundred non-pathogenic yeast species and selected a small number of yeast species that captured the largest diversity in these profiles. N-glycans were extracted from these yeast species' cell walls and conjugated to a carrier protein for immunization in rabbits. A strong antibody-mediated immune response was observed which cross-reacted in several cases with HIV-1 gp120, especially of the C-clade of viruses, but none of these antibodies was neutralizing HIV-1 infectivity in vitro. These findings are consistent with those of other colleagues who followed similar approaches. While promising, it is clear that this anti-glycan approach to HIV-1 vaccination will require much more work.

3) In a third work package, the unfolded protein response was inducibly engineered in Pichia pastoris and Yarrowia lipolytica as a tool to increase heterologous protein production, especially membrane proteins. The UPR-master regulator HAC1 was cloned from both organisms and expression studies completed for a variety of membrane proteins and secreted proteins. It was found that inducible HAC1 expression always led to a stronger phenotype than constitutive expression, but with mixed results as to improvement of expression levels (ranging from much improvement to deterioration).