Recent Submissions

  • Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

    Rodriguez Buitrago, Jhon Alexander; Klünemann, Thomas; Blankenfeldt, Wulf; Schallmey, Anett; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley & Sons, 2020-07-28)
    he ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.
  • Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high: In vivo efficacy

    Testolin, Giambattista; Cirnski, Katarina; Rox, Katharina; Prochnow, Hans; Fetz, Verena; Grandclaudon, Charlotte; Mollner, Tim; Baiyoumy, Alain; Ritter, Antje; Leitner, Christian; et al. (RSC, 2020-01-01)
    There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL−1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL−1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
  • Protein-Templated Hit Identification through an Ugi Four-Component Reaction.

    Mancini, Federica; Unver, M Yagiz; Elgaher, Walid A M; Jumde, Varsha R; Alhayek, Alaa; Lukat, Peer; Herrmann, Jennifer; Witte, Martin D; Köck, Matthias; Blankenfeldt, Wulf; et al. (Wiley-VCH, 2020-05-19)
  • Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

    Theobald, Sebastian J; Kreer, Christoph; Khailaie, Sahamoddin; Bonifacius, Agnes; Eiz-Vesper, Britta; Figueiredo, Constanca; Mach, Michael; Backovic, Marija; Ballmaier, Matthias; Koenig, Johannes; et al. (2020-07-15)
    Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.
  • Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies

    Moreira, Gustavo Marçal Schmidt Garcia; Köllner, Sarah Mara Stella; Helmsing, Saskia; Jänsch, Lothar; Meier, Anja; Gronow, Sabine; Boedeker, Christian; Dübel, Stefan; Mendonça, Marcelo; Moreira, Ângela Nunes; et al. (Springer Science and Business Media LLC, 2020-09-17)
    The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.
  • Development of a Social Network for People Without a Diagnosis (RarePairs): Evaluation Study.

    Kühnle, Lara; Mücke, Urs; Lechner, Werner M; Klawonn, Frank; Grigull, Lorenz; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (JMIR publications, 2020-09-29)
    Background: Diagnostic delay in rare disease (RD) is common, occasionally lasting up to more than 20 years. In attempting to reduce it, diagnostic support tools have been studied extensively. However, social platforms have not yet been used for systematic diagnostic support. This paper illustrates the development and prototypic application of a social network using scientifically developed questions to match individuals without a diagnosis. Objective: The study aimed to outline, create, and evaluate a prototype tool (a social network platform named RarePairs), helping patients with undiagnosed RDs to find individuals with similar symptoms. The prototype includes a matching algorithm, bringing together individuals with similar disease burden in the lead-up to diagnosis. Methods: We divided our project into 4 phases. In phase 1, we used known data and findings in the literature to understand and specify the context of use. In phase 2, we specified the user requirements. In phase 3, we designed a prototype based on the results of phases 1 and 2, as well as incorporating a state-of-the-art questionnaire with 53 items for recognizing an RD. Lastly, we evaluated this prototype with a data set of 973 questionnaires from individuals suffering from different RDs using 24 distance calculating methods. Results: Based on a step-by-step construction process, the digital patient platform prototype, RarePairs, was developed. In order to match individuals with similar experiences, it uses answer patterns generated by a specifically designed questionnaire (Q53). A total of 973 questionnaires answered by patients with RDs were used to construct and test an artificial intelligence (AI) algorithm like the k-nearest neighbor search. With this, we found matches for every single one of the 973 records. The cross-validation of those matches showed that the algorithm outperforms random matching significantly. Statistically, for every data set the algorithm found at least one other record (match) with the same diagnosis. Conclusions: Diagnostic delay is torturous for patients without a diagnosis. Shortening the delay is important for both doctors and patients. Diagnostic support using AI can be promoted differently. The prototype of the social media platform RarePairs might be a low-threshold patient platform, and proved suitable to match and connect different individuals with comparable symptoms. This exchange promoted through RarePairs might be used to speed up the diagnostic process. Further studies include its evaluation in a prospective setting and implementation of RarePairs as a mobile phone app.
  • Gastrointestinal stress as innate defence against microbial attack.

    Panwar, H; Rokana, N; Aparna, S V; Kaur, J; Singh, A; Singh, J; Singh, K S; Chaudhary, V; Puniya, A K; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-08-31)
    A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.
  • Hepatic Transcriptome Analysis Identifies Divergent Pathogen-Specific Targeting-Strategies to Modulate the Innate Immune System in Response to Intramammary Infection.

    Heimes, Annika; Brodhagen, Johanna; Weikard, Rosemarie; Seyfert, Hans-Martin; Becker, Doreen; Meyerholz, Marie M; Petzl, Wolfram; Zerbe, Holm; Hoedemaker, Martina; Rohmeier, Laura; et al. (Frontiers, 2020-04-29)
    Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.
  • CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease.

    Slabik, Constanze; Kalbarczyk, Maja; Danisch, Simon; Zeidler, Reinhard; Klawonn, Frank; Volk, Valery; Krönke, Nicole; Feuerhake, Friedrich; Ferreira de Figueiredo, Constanca; Blasczyk, Rainer; et al. (Elsevier (Cell Press), 2020-08-08)
    Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350+ 293T cells in vitro. The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV+ B95-8 cell line, showing selectivity against gp350+ cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34+ cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8+gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER+ B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8+gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV+ lymphoproliferation and lymphomagenesis.
  • Results of the first pilot external quality assessment (EQA) scheme for anti-SARS-CoV2-antibody testing.

    Haselmann, Verena; Özçürümez, Mustafa K; Klawonn, Frank; Ast, Volker; Gerhards, Catharina; Eichner, Romy; Costina, Victor; Dobler, Gerhard; Geilenkeuser, Wolf-Jochen; Wölfel, Roman; et al. (DeGruyter, 2020-08-27)
  • Clarithromycin Exerts an Antibiofilm Effect against rdar Biofilm Formation, and Transforms the Physiology towards an Apparent Oxygen-depleted Energy and Carbon Metabolism.

    Zafar, Munira; Jahan, Humera; Shafeeq, Sulman; Nimtz, Manfred; Jänsch, Lothar; Römling, Ute; Choudhary, M Iqbal; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2020-08-24)
    Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection towards various forms of environmental stresses to individual cells. Thus, bacterial cells become tolerant against antimicrobials and the immune system within biofilms. In the current study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry and rough) biofilm formation of the gastrointestinal pathogen Salmonella typhimurium ATCC14028 Nalr at 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be consistently downregulated. While fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways, but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifts the cells towards an apparent oxygen- and energy- depleted status, whereby the metabolism that channels into oxidative phosphorylation is downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and L-arginine catabolism, potentially also to prevent cytosolic, is upregulated. This analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.
  • Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.

    Mahieu, Emilie; Covès, Jacques; Krüger, Georg; Martel, Anne; Moulin, Martine; Carl, Nico; Härtlein, Michael; Carlomagno, Teresa; Franzetti, Bruno; Gabel, Frank; et al. (Elsevier, 2020-06-24)
    The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.
  • Heparin 2.0: A New Approach to the Infection Crisis.

    Seffer, Malin-Theres; Cottam, Daniel; Forni, Lui G; Kielstein, Jan T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Karger AG, 2020-07-02)
    In April 2020, the US Food and Drug Administration granted emergency use authorization for certain medical devices to be used in patients with coronavirus disease 2019 (CO-VID-19). This included extracorporeal blood purification devices. This narrative review will give a brief overview regarding some of the extracorporeal devices that could be used to treat COVID-19 patients, including the Seraph® 100 Microbind® Affinity Blood Filter, produced by ExThera Medical (Martinez, CA, USA), first licensed in the European Economic Area in 2019. The Seraph® 100 contains ultrahigh molecular weight polyethylene beads with end point-attached heparin and is approved for the reduction of pathogens from the bloodstream either as a single agent or as an adjunct to conventional anti-infective agents. Bacteria, viruses, fungi, and toxins have been shown to bind to the immobilized heparin in a similar way to the interaction with heparan sulfate on the cell surface. This binding is nonreversible and as such, the pathogens are removed from the bloodstream. In this review, we describe the pathophysiological basis and rationale for using heparin for pathogen removal from the blood as well as exploring the technology behind the adaptation of heparin to deprive it of its systemic anticoagulant activity. In addition, we summarize the in vitro data as well as the available preclinical testing and published clinical reports. Finally, we discuss the enormous potential of this technology in an era of increasing antibiotic resistance and high mortality associated with sepsis and consider the application of this as a possible treatment option for COVID-19.
  • Clearance of chloroquine and hydroxychloroquine by the Seraph® 100 Microbind® Affinity blood filter - approved for the treatment of COVID-19 patients.

    Seffer, Malin-Theres; Martens-Lobenhoffer, Jens; Schmidt, Julius J; Eden, Gabriele; Bode-Böger, Stefanie M; Kielstein, Jan T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-06-19)
    On April 17th 2020 the US Food and Drug Administration granted Coronavirus Disease 2019 (COVID-19) emergency use authorizations for the Seraph® 100 Microbind® Affinity Blood Filter. The medical device is aimed to treat critically ill COVID-19 patients with confirmed or imminent respiratory failure. The aim of this life size in vitro pharmacokinetic study was to investigate the in-vitro adsorption of chloroquine and hydroxychloroquine from human plasma using equipment that is also used at the bedside. After start of the hemoperfusion Pre (Cpre ) Seraph® plasma levels were obtained at 5 (C5 ), 10 (C10 ), 15 (C15 ), 30 (C30 ), 60 (C60 ) and 120 (C120 ) minutes into the procedure. At two timepoints (5 min and 120 min) post (Cpost ) Seraph® plasma levels were determined that were used to calculate the plasma clearance. Both drugs were determined using a validated HPLC method Median [IQR] plasma clearance of the Seraph for chloroquine / hydroxychloroquine was 1.71 [0.51-4.38] ml/min / 1.79 [0.21-3.68] ml/min respectively. The lack of elimination was also confirmed by the fact that plasma levels did not change over the 120 min treatment. As neither chloroquine nor hydroxychloroquine were removed by the treatment with the Seraph dose adjustments in COVID-19 patients undergoing this treatment are not necessary. This article is protected by copyright. All rights reserved.
  • The crystal structure of the heme d biosynthesis-associated small c-type cytochrome NirC reveals mixed oligomeric states in crystallo.

    Klünemann, Thomas; Henke, Steffi; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (: International Union of Crystallography, 2020-03-25)
    Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.
  • Structure of heme d-free cd nitrite reductase NirS.

    Klünemann, Thomas; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (International Union of Crystallography, 2020-05-29)
    A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d1-bound form. The heme d1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.
  • Responsiveness to Influenza Vaccination Correlates with NKG2C-Expression on NK Cells.

    Riese, Peggy; Trittel, Stephanie; Pathirana, Rishi D; Klawonn, Frank; Cox, Rebecca J; Guzmán, Carlos A; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-06-05)
    Influenza vaccination often results in a large percentage of low responders, especially in high-risk groups. As a first line of defense, natural killer (NK) cells play a crucial role in the fight against infections. However, their implication with regard to vaccine responsiveness is insufficiently assessed. Therefore, this study aimed at the validation of essential NK cell features potentially associated with differential vaccine responsiveness with a special focus on NKG2C- and/or CD57-expressing NK cells considered to harbor memory-like functions. To this end, 16 healthy volunteers were vaccinated with an adjuvanted pandemic influenza vaccine. Vaccine responders and low responders were classified according to their hemagglutination inhibition antibody titers. A majority of responders displayed enhanced frequencies of NKG2C-expressing NK cells 7- or 14-days post-vaccination as compared to low responders, whereas the expression of CD57 was not differentially modulated. The NK cell cytotoxic potential was found to be confined to CD56dimCD16+ NKG2C-expressing NK cells in the responders but not in the low responders, which was further confirmed by stochastic neighbor embedding analysis. The presented study is the first of its kind that ascribes CD56dimCD16+ NKG2C-expressing NK cells a crucial role in biasing adaptive immune responses upon influenza vaccination and suggests NKG2C as a potential biomarker in predicting pandemic influenza vaccine responsiveness.
  • Biocatalysts from Biosynthetic Pathways: Enabling Stereoselective, Enzymatic Cycloether Formation on a Gram Scale

    Hollmann, Tim; Berkhan, Gesche; Wagner, Lisa; Sung, Kwang Hoon; Kolb, Simon; Geise, Hendrik; Hahn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Chemical Society (ACS), 2020-03-30)
    Biosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular complexity. Enzymes that form complex structural elements with multiple stereocenters, like chiral saturated oxygen heterocycles (CSOH), are of particular interest for a synthetic application, as their use promises to significantly simplify access to these elements. Here, the biocatalytic characterization of AmbDH3, an enzyme that catalyzes intramolecular oxa-Michael addition (IMOMA) is reported. This reaction essentially gives access to various types of CSOH with adjacent stereocenters, but it is not yet part of the repertoire of preparative biocatalysis. An in-depth study on the synthetic utility of AmbDH3 was performed, which made extensive use of complex synthetic precursor surrogates. The enzyme exhibited stability and broad substrate tolerance in in vitro experiments, which was in agreement with the results of molecular modeling. Its selectivity profile enabled kinetic resolution of chiral tetrahydropyrans (THPs) under control of up to four stereocenters. A systematic optimization of the reaction conditions enabled gram-scale conversions yielding preparative amounts of chiral THP. The synthetic utility of AmbDH3 was finally demonstrated by its successful application in the key step of a chemoenzymatic total synthesis to the THP-containing phenylheptanoid (−)-centrolobine. These results highlight the synthetic potential of AmbDH3 and related IMOMA cyclases as a biocatalytic alternative that further develops the available chemical-synthetic IMOMA methodology.
  • 1H, 13C, 15N chemical shift assignments of SHP2 SH2 domains in complex with PD-1 immune-tyrosine motifs.

    Marasco, Michelangelo; Kirkpatrick, John P; Carlomagno, Teresa; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2020-04-01)
    Inhibition of immune checkpoint receptor Programmed Death-1 (PD-1) via monoclonal antibodies is an established anticancer immunotherapeutic approach. This treatment has been largely successful; however, its high cost demands equally effective, more affordable alternatives. To date, the development of drugs targeting downstream players in the PD-1-dependent signaling pathway has been hampered by our poor understanding of the molecular details of the intermolecular interactions involved in the pathway. Activation of PD-1 leads to phosphorylation of two signaling motifs located in its cytoplasmic domain, the immune tyrosine inhibitory motif (ITIM) and immune tyrosine switch motif (ITSM), which recruit and activate protein tyrosine phosphatase SHP2. This interaction is mediated by the two Src homology 2 (SH2) domains of SHP2, termed N-SH2 and C-SH2, which recognize phosphotyrosines pY223 and pY248 of ITIM and ITSM, respectively. SHP2 then propagates the inhibitory signal, ultimately leading to suppression of T cell functionality. In order to facilitate mechanistic structural studies of this signaling pathway, we report the resonance assignments of the complexes formed by the signaling motifs of PD-1 and the SH2 domains of SHP2.
  • Crystal structure of NirF: insights into its role in heme d biosynthesis.

    Klünemann, Thomas; NIMTZ, MANFRED; Jänsch, Lothar; Layer, Gunhild; Blankenfeldt, Wulf; HZI, Helmholtz Zentrum für Infektionsforschung, GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (Wiley Online Open, 2020-04-07)
    Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes, for example, to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitric oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d1 . While heme d1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d1 , the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom-to-bottom β-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N termini are adjacent to each other and project away from the core structure, which hints at simultaneous membrane anchoring via both N termini. Further, the complex with dihydroheme d1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirF strain of P. aeruginosa. Together, these data suggest that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives. DATABASE: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9.

View more