henrivin

• Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare; Sørensen, Henrik Vinther; Kommedal, Eirik; Cordara, Gabriele & Vaaje-Kolstad, Gustav [Show all 7 contributors for this article] (2024). Calcium-binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environmental survival and infection. QRB Discovery. 5. doi: 10.1017/qrd.2024.14. Full text in Research Archive Show summary

  Despite major efforts toward its eradication, cholera remains a major health threat and economic burden in many low- and middle-income countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, for example, on zooplankton. N-acetyl glucosamine-binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides V. cholerae with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca2+, Mg2+, or K+ binding close to its carbohydrate-binding surface. In addition to the X-ray crystal structures of cation-LPMO complexes (to 1.5 Å resolution), we explored how the presence of ions affects the stability and activity of the protein. Calcium and magnesium ions were found to bind to GbpA specifically, with calcium ions – abundant in natural sources of chitin – having the strongest effect on protein stability. When the cation-binding site was rendered non-functional, a decrease in activity was observed, highlighting the importance of the structural elements stabilized by calcium. Our findings suggest a cation-binding site specific to GbpA and related LPMOs that may fine-tune binding and activity for its substrates during environmental survival and host infection.

• Askarian, Fatemeh; Tsai, Chih-Ming; Cordara, Gabriele; Zurich, Raymond H.; Bjånes, Elisabet & Golten, Ole [Show all 22 contributors for this article] (2023). Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 120(30). doi: 10.1073/pnas.2301538120. Full text in Research Archive Show summary

  Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

• Sørensen, Henrik Vinther; Canals, Mateu Montserrat; Loose, Jennifer Sarah Maria; Fisher, S. Zoë; Moulin, Martine & Blakeley, Matthew P. [Show all 9 contributors for this article] (2023). Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase. ACS Omega. 8(32), p. 29101–29112. doi: 10.1021/acsomega.3c02168. Full text in Research Archive Show summary

  Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

• Sørensen, Henrik Vinther; Montserrat-Canals, Mateu; Prévost, Sylvain François; Vaaje-Kolstad, Gustav; Bjerregaard-Andersen, Kaare & Lund, Reidar [Show all 7 contributors for this article] (2023). Tangled up in fibers: How a lytic polysaccharide monooxygenase binds its chitin substrate. BioRxiv. ISSN 0362-4331. doi: 10.1101/2023.09.21.558757. Full text in Research Archive
• Askarian, Fatemeh; Uchiyama, Satoshi; Masson, Helen; Sørensen, Henrik Vinther; Golten, Ole & Bunæs, Anne Cathrine [Show all 20 contributors for this article] (2021). The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection. Nature Communications. 12, p. 1–19. doi: 10.1038/s41467-021-21473-0. Full text in Research Archive Show summary

  The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of <i>Pseudomonas aeruginosa</i>, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by <i>P. aeruginosa</i>. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of <i>cbpD</i> rendered <i>P. aeruginosa</i> unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

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• Canals, Mateu Montserrat; Bjerregaard-Andersen, Kaare; Sørensen, Henrik Vinther; Kommedal, Eirik; Cordara, Gabriele & Vaaje-Kolstad, Gustav [Show all 7 contributors for this article] (2024). Calcium binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environmental survival and infection                                               . Full text in Research Archive
• Canals, Mateu Montserrat; Sørensen, Henrik Vinther; Oksanen, Esko & Krengel, Ute (2022). Neutron studies of the bacterial colonization factor GbpA. Full text in Research Archive
• Sørensen, Henrik Vinther; Bjerregaard-Andersen, Kaare; Vaaje-Kolstad, Gustav; Krengel, Ute & Lund, Reidar (2020). Applying soft matter techniques to understand bacterial colonization. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Lund, Reidar; Browning, Kathryn & Bjerregaard-Andersen, Kaare (2019). Bacterial colonization investigated with X-ray and neutron techniques. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Bjerregaard-Andersen, Kaare; Lund, Reidar & Krengel, Ute (2019). Applying X-rays and neutrons to understand a bacterial virulence factor. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Vaaje-Kolstad, Gustav; Lund, Reidar; Browning, Kathryn & Bjerregaard-Andersen, Kaare [Show all 7 contributors for this article] (2019). Using X-rays and neutrons to understand a bacterial colonization factor. Full text in Research Archive
• Sørensen, Henrik Vinther; Krengel, Ute; Browning, Kathryn; Bjerregaard-Andersen, Kaare; Cardenas, Marite & Lund, Reidar [Show all 7 contributors for this article] (2019). Study of bacterial colonization with X-ray and neutron scattering techniques. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Bjerregaard-Andersen, Kaare; Lund, Reidar & Krengel, Ute (2018). Borrowing material science techniques to study biological problems. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Lund, Reidar; Bjerregaard-Andersen, Kaare & Krengel, Ute (2018). A surface active enzyme and colonization factor investigated by x-ray and neutron techniques. Full text in Research Archive
• Sørensen, Henrik Vinther; Bjerregaard-Andersen, Kaare; Lund, Reidar & Krengel, Ute (2018). A bacterial colonization factor studied by X-ray and neutron techniques. Full text in Research Archive
• Sørensen, Henrik Vinther; Browning, Kathryn; Lund, Reidar; Bjerregaard-Andersen, Kaare & Krengel, Ute (2017). A surface active enzyme and colonization factor investigated by x-ray and neutron scattering - Hercules. Full text in Research Archive

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