Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

Purpose: Interactions with tumor-associated microglia and macrophages (TAM) are critical for glioblastoma progression. Polysialic acid (polySia) is a tumor-associated glycan, but its frequency of occurrence and its prognostic value in glioblastoma are disputed. Through interactions with the opposing immune receptors Siglec-11 and Siglec-16, polySia is implicated in the regulation of microglia and macrophage activity. However, due to a nonfunctional SIGLEC16P allele, SIGLEC16 penetrance is less than 40%. Here, we explored possible consequences of SIGLEC16 status and tumor cell–associated polySia on glioblastoma outcome. Experimental Design: Formalin-fixed paraffin-embedded specimens of two independent cohorts with 70 and 100 patients with newly diagnosed glioblastoma were retrospectively analyzed for SIGLEC16 and polySia status in relation to overall survival. Inflammatory TAM activation was assessed in tumors, in heterotypic tumor spheroids consisting of polySia-positive glioblastoma cells and Siglec-16–positive or Siglec-16–negative macrophages, and by exposing Siglec-16–positive or Siglec-16–negative macrophages to glioblastoma cell–derived membrane fractions. Results: Overall survival of SIGLEC16 carriers with polySia-positive tumors was increased. Consistent with proinflammatory Siglec-16 signaling, levels of TAM positive for the M2 marker CD163 were reduced, whereas the M1 marker CD74 and TNF expression were increased, and CD8+ T cells enhanced in SIGLEC16/polySia double-positive tumors. Correspondingly, TNF production was elevated in heterotypic spheroid cultures with Siglec-16–expressing macrophages. Furthermore, a higher, mainly M1-like cytokine release and activating immune signaling was observed in SIGLEC16-positive as compared with SIGLEC16-negative macrophages confronted with glioblastoma cell–derived membranes. Conclusions: Collectively, these results strongly suggest that proinflammatory TAM activation causes the better outcome in patients with glioblastoma with a functional polySia-Siglec-16 axis.

Polymeric sialic acid (polysialic acid, polySia) is a remarkable posttranslational modification of only few select proteins. The major, and most prominent polySia protein carrier is the neural cell adhesion molecule NCAM. Here, the key functions of polySia are to regulate interactions of NCAM and to balance cellular interactions in brain development and plasticity. During recent years, however, increasing evidence points towards a role of polySia in the modulation of immune responses. These immunomodulatory functions can be mediated by polySia on proteins other than NCAM, presented either on the cell surface or released into the extracellular space. This perspective review summarizes our current knowledge and addresses major open questions on polySia and polySia receptors in modulating innate immune responses in the brain.

Vascular endothelial growth factor A (VEGF-A) is a key signalling protein that stimulates blood vessel development and repair. Its tight control is essential for organ development and tissue homeostasis. However, the complex regulatory network for balanced bioavailability of VEGF-A is not fully understood. Here, we assessed the role of the glycocalyx component polysialic acid (polySia) for kidney development and its potential interactions with VEGF-A isoforms, in vitro and in vivo, using mouse models of polySia deficiency. PolySia acts as negative regulator of cell adhesion, but also may interact with extracellular components. In murine kidney, polySia was identified on nephron progenitor and endothelial cell subsets in developing nephrons with declining expression during maturation. Loss of polySia in Ncam ^−/− mice revealed the neural cell adhesion molecule NCAM as major protein carrier. Both polysialyltransferase-negative and Ncam ^−/− mice displayed impaired glomerular microvasculature development with reduced endothelial cell numbers, reminiscent to the phenotype of mice with impaired VEGF-A signalling. In vitro, immobilized polySia specifically interacted with the VEGF-A188 isoform demonstrating an isoform-specific direct interaction. Single cell RNA sequencing data analysis of newborn mouse kidneys implicated activation of VEGF-A-signalling in polysialyltransferase-positive endothelial cells. Consistently, loss of polySia resulted in diminished VEGFR2 activation in perinatal kidney and human endothelial cells. At transcriptional level, the expression of polysialyltransferases and known polySia carrier proteins is conserved in human developing kidney. Together, these data demonstrate a direct impact of polySia on VEGF-A signalling with the perspective that polysialylation could be a therapeutic target to ameliorate microvasculature repair after renal injury. Graphical abstract

Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III, and VII, which are associated with lysosomal accumulation of heparan sulphate (HS), manifest with neurological deterioration and currently lack effective treatments. We report that neuraminidase 1 (NEU1) activity is drastically reduced in brain tissues of patients with neurological MPS and mouse models but not in neurological lysosomal disorders without HS storage. Accumulated HS disrupts the lysosomal multienzyme complex of NEU1 with cathepsin A, β-galactosidase (GLB1), and glucosamine-6-sulfate sulfatase (GALNS), leading to NEU1 deficiency and partial GLB1 and GALNS deficiencies in cortical tissues and induced pluripotent stem cell–derived (iPSC-derived) cortical neurons of patients with neurological MPS. Increased sialylation of N-linked glycans in brains of patients with MPS and mice implicated insufficient processing of sialylated glycans, except for polysialic acid. Correction of NEU1 activity in MPS IIIC mice by lentiviral (LV) gene transfer ameliorated previously identified hallmarks of the disease, including memory impairment, behavioral traits, and reduced levels of excitatory synapse markers VGLUT1 and PSD95. Overexpression of NEU1 also restored levels of VGLUT1/PSD95–positive puncta in cortical iPSC-derived MPS IIIA neurons. Our results demonstrate that HS-induced secondary NEU1 deficiency and aberrant sialylation of brain glycoproteins constitute what we believe is a novel pathological pathway in the neurological MPS spectrum crucially contributing to CNS pathology.

Supplementary Table S1.Demographic and clinical parameters of GB patient cohorts.Supplementary Table S2.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for the presence of a functional SIGLEC16 allele (A) or for the occurrence of polySia on tumor cells (B).Supplementary Table S3.Cox proportional hazards regression analysis of covariates for SIGLEC16 and polySia status in relation to OS (MHH and UMG cohorts combined).Supplementary Table S4.Median age of GB cases stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S5.OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined).Supplementary Table S6.OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH cohort only).Supplementary Table S7.OS analysis of GB cases receiving gross total resection and chemotherapy stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined, n=101).Supplementary Table S8.OS analysis of IDH-wildtype GB cases (UMG cohort) stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S9.OS analysis of UMG cohort stratified for MGMT gene promoter methylation and SIGLEC16 genotype, polySia on tumor cells, CD163 and CD74 status.Supplementary Table S10.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for CD74low and CD74high.Supplementary Table S11. Median age at resection of GB cases stratified for CD163 to CD68 (A) or CD74 to IBA1 ratios (B) alone or in combination with the SIGLEC16 genotype.Supplementary Table S12. OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined).Supplementary Table S13. OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH cohort only).Supplementary Table S14.OS analysis of GB cases receiving both gross total resection and chemotherapy stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined, n=101).