Obesity and type 2 diabetes are associated with low-grade inflammation and specific 34 changes in gut microbiota composition [1][2][3][4][5][6][7] . We previously demonstrated that administration 35 of Akkermansia muciniphila prevents the development of obesity and associated 36 complications 8 . However, its mechanisms of action remain unclear, whilst the sensitivity of 37 A. muciniphila to oxygen and the presence of animal-derived compounds in its growth 38 medium currently limit the development of translational approaches for human medicine 9 . 39Here we addressed these issues by showing that A. muciniphila retains its efficacy when Akkermansia muciniphila is one of the most abundant members of the human gut 53 microbiota, representing between 1 and 5% of our intestinal microbes 10,11 to improve glucose intolerance and insulin resistance regardless of the growth medium used and 71 independently of food intake ( Fig. 1a-g). 72 We previously showed that autoclaving A. muciniphila abolished its beneficial effects 8 . (Fig. 1a-c and Supplemental Fig. 1a-c). In both sets of 81 experiments, we found that mice treated with pasteurized A. muciniphila displayed a much lower 82 glucose intolerance and insulin concentration when compared to the HFD group, resulting in a 83 lower insulin resistance (IR) index (Fig. 1d-g and Supplemental Fig. 1d-g). Treatment with 84 pasteurized A. muciniphila also led to greater goblet cell density in the ileum when compared to 85 ND-fed mice (Fig. 1h), suggesting a higher mucus production, while normalizing the mean 86 adipocyte diameter (Fig. 2a-b) and significantly lowering plasma leptin when compared to HFD-87 fed mice (Fig. 2c). These effects were not observed in mice treated with live A. muciniphila. A 88 similar trend could be observed for plasma resistin (Supplemental Fig. 1h), thereby suggesting 89 improved insulin sensitivity, while plasma adiponectin remained unaffected in all conditions 90 (Supplemental Fig. 1i). We found that mice treated with pasteurized A. muciniphila had a higher 91 fecal caloric content when compared to all other groups (Fig. 2d), suggesting a lower energy (Fig. 2e-g). This resulted in a normalization of the HFD-induced shift of 37% with the 104 pasteurized bacterium, and 17% with the live bacterium ( Fig. 2f). 105By comparing the metabolic profiles of the different groups, we found that the shift 106 induced by pasteurized A. muciniphila was mainly associated with trimethylamine (TMA) and TMA to TMAO, a metabolite associated with atherosclerosis 19,20 . While exposure to a HFD led 114 to a two-fold higher Fmo3 expression when compared to ND-fed mice, treatment with 115 pasteurized A. muciniphila reversed this effect (Fig. 2j) Fmo3 expression were not associated with a modification of plasma TMA and TMAO, as all 121 HFD-fed group displayed similar concentrations for both metabolites (Fig. 2k,l) (Fig. 3a), but not cells expressing TLR5, TLR9 or the NOD2 receptor (Fig. 3b-131 d). 132Genomic and proteomic analyses of A. muciniphila identified p...

The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19-20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19-20:C3d complex at 2.3-Å resolution shows that FH19-20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19-20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces. structure and function | X-ray crystallography | hemolysis | kidney diseases | human mutations P reviously unencountered microbes invading a human body must be rapidly recognized and eliminated. This is the function of innate immunity, which includes the alternative pathway (AP) of complement. AP components can attack targets with hydroxyl or amine groups (i.e., all biological surfaces). This is a powerful defense mechanism, because there is rapid amplification leading to efficient opsonization or target lysis by the membrane attack complex (MAC). The AP attack is, therefore, also potentially dangerous for the host if one's cells and acellular structures are not protected.The AP activation is based on spontaneous hydrolysis of C3 in plasma leading to production of C3b, which then randomly attaches onto any surface hydroxyl or amine group through a reactive thioester located on the C3d part [i.e., thioester domain (TED)] of C3b. If these surface-attached C3b molecules are not quickly inactivated to iC3b and C3d, C3b deposition is rapidly amplified by a positive enzymatic feedback loop, leading to opsonophagocytosis and formation of the lytic membrane attack complex. On host surfaces, which are naturally nonactivators of the AP, efficient down-regulation of bound C3b occurs in three ways: factor I-mediated cleavage of C3b to inactive iC3b, acceleration of the decay of the preformed C3 convertases, or inhibition of factor B binding to C3b. Factor H (FH) is required for all these. It also down-regulates C3b deposition on noncellular surfaces, such as the heparan sulfate-rich glomerular basement membrane. FH is, thus, essential for restricting AP attack against host surfaces while allowing AP attack against foreign surfaces (i.e., for target discrimination) (1). A long-standing central question in complemen...

This year marks the 50th anniversary of the coiled-coil triple helical structure of collagen, first proposed by Ramachandran's group from Madras. The structure is unique among the protein secondary structures in that it requires a very specific tripeptide sequence repeat, with glycine being mandatory at every third position and readily accommodates the imino acids proline/hydroxyproline, at the other two positions. The original structure was postulated to be stabilized by two interchain hydrogen bonds, per tripeptide. Subsequent modeling studies suggested that the triple helix is stabilized by one direct inter chain hydrogen bond as well as water mediated hydrogen bonds. The hydroxyproline residues were also implicated to play an important role in stabilizing the collagen fibres. Several high resolution crystal structures of oligopeptides related to collagen have been determined in the last ten years. Stability of synthetic mimics of collagen has also been extensively studied. These have confirmed the essential correctness of the coiled-coil triple helical structure of collagen, as well as the role of water and hydroxyproline residues, but also indicated additional sequence-dependent features. This review discusses some of these recent results and their implications for collagen fiber formation.

To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19–20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a “superevasion site.”

Background: It is unknown why patients with autoantibodies against complement factor H (CFH) lack homologous CFHR1 protein.Results: The autoantibody epitope on CFH was identified, and the structure of the corresponding part of CFHR1 was solved.Conclusion: The autoantigenic epitope of CFH and its homologous site in CFHR1 are structurally different.Significance: A plausible explanation for formation of autoantibodies due to CFHR1 deficiency in autoimmune atypical hemolytic uremic syndrome was obtained.