Now showing items 1-20 of 3330

    • How to Study the Metabolism of New Psychoactive Substances for the Purpose of Toxicological Screenings-A Follow-Up Study Comparing Pooled Human Liver S9, HepaRG Cells, and Zebrafish Larvae.

      Wagmann, Lea; Frankenfeld, Fabian; Park, Yu Mi; Herrmann, Jennifer; Fischmann, Svenja; Westphal, Folker; Müller, Rolf; Flockerzi, Veit; Meyer, Markus R; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Frontiers, 2020-07-17)
      The new psychoactive substances (NPS) market continues to be very dynamic. A large number of compounds belonging to diverse chemical groups continue to emerge. This makes their detection in biological samples challenging for clinical and forensic toxicologists. Knowledge of the metabolic fate of NPS is crucial for developing comprehensive screening procedures. As human studies are not feasible due to ethical concerns, the current study aimed to compare the NPS' metabolic pattern in incubations with pooled human liver S9 fraction (pHLS9), human liver HepaRG cells, and zebrafish larvae. The latter model was recently shown to be a promising preclinical surrogate for human hepatic metabolism of a synthetic cannabinoid. However, studies concerning other NPS classes are still missing and therefore an amphetamine-based N-methoxybenzyl (NBOMe) compound, a synthetic cathinone, a pyrrolidinophenone analog, a lysergamide, as well as another synthetic cannabinoid were included in the current study. Liquid chromatography coupled to Orbitrap-based high-resolution tandem mass spectrometry was used to analyze metabolic data. Zebrafish larvae were found to produce the highest number of phase I but also phase II metabolites (79 metabolites in total), followed by HepaRG cells (66 metabolites). Incubations with pHLS9 produced the least metabolites (57 metabolites). Furthermore, the involvement of monooxygenases and esterases in the metabolic phase I transformations of 4F-MDMB-BINACA was elucidated using single-enzyme incubations. Several cytochrome P450 (CYP) isozymes were shown to contribute, and CYP3A5 was involved in all CYP-catalyzed reactions, while amide and ester hydrolysis were catalyzed by the human carboxylesterase (hCES) isoforms hCES1b and/or hCES1c. Finally, metabolites were compared to those present in human biosamples if data were available. Overall, the metabolic patterns in HepaRG cells provided the worst overlap with that in human biosamples. Zebrafish larvae experiments agreed best with data found in human plasma and urine analysis. The current study underlines the potential of zebrafish larvae as a tool for elucidating the toxicokinetics of NPS in the future.
    • Comparative Target Analysis of Chlorinated Biphenyl Antimicrobials Highlights MenG as a Molecular Target of Triclocarban.

      Macsics, Robert; Hackl, Mathias W; Fetzer, Christian; Mostert, Dietrich; Bender, Jennifer; Layer, Franziska; Sieber, Stephan A; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (2020-08-03)
      Triclocarban (TCC), a formerly used disinfectant, kills bacteria via an unknown mechanism of action. A structural hallmark is its N,N'-diaryl urea motif, which is also present in other antibiotics, including the recently reported small molecule PK150. We show here that, like PK150, TCC exhibits an inhibitory effect on Staphylococcus aureus menaquinone metabolism via inhibition of the biosynthesis protein demethylmenaquinone methyltransferase (MenG). However, the activity spectrum (MIC90) of TCC across a broad range of multidrug-resistant staphylococcus and enterococcus strains was much narrower than that of PK150. Accordingly, TCC did not cause an overactivation of signal peptidase SpsB, a hallmark of the PK150 mode of action. Furthermore, we were able to rule out inhibition of FabI, a confirmed target of the diaryl ether antibiotic triclosan (TCS). Differences in the target profiles of TCC and TCS were further investigated by proteomic analysis, showing complex but rather distinct changes in the protein expression profile of S. aureus Downregulation of the arginine deiminase pathway provided additional evidence for an effect on bacterial energy metabolism by TCC.IMPORTANCE TCC's widespread use as an antimicrobial agent has made it a ubiquitous environmental pollutant despite its withdrawal due to ecological and toxicological concerns. With its antibacterial mechanism of action still being unknown, we undertook a comparative target analysis between TCC, PK150 (a recently discovered antibacterial compound with structural resemblance to TCC), and TCS (another widely employed chlorinated biphenyl antimicrobial) in the bacterium Staphylococcus aureus We show that there are distinct differences in each compound's mode of action, but also identify a shared target between TCC and PK150, the interference with menaquinone metabolism by inhibition of MenG. The prevailing differences, however, which also manifest in a remarkably better broad-spectrum activity of PK150, suggest that even high levels of TCC or TCS resistance observed by continuous environmental exposure may not affect the potential of PK150 or related N,N'-diaryl urea compounds as new antibiotic drug candidates against multidrug-resistant infections.
    • ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli.

      Wallenstein, Alexander; Rehm, Nadine; Brinkmann, Marina; Selle, Martina; Bossuet-Greif, Nadège; Sauer, Daniel; Bunk, Boyke; Spröer, Cathrin; Wami, Haleluya Tesfaye; Homburg, Stefan; et al. (ASM, 2020-07-15)
      Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the Enterobacteriaceae which can be correlated with induction of DNA double-strand breaks and interference with cell cycle progression in eukaryotes. Regulatory features of colibactin expression are only incompletely understood. We used Escherichia coli strain M1/5 as a model to investigate regulation of expression of the colibactin determinant at the transcriptional level and to characterize regulatory elements located within the colibactin pathogenicity island itself. We measured clbR transcription in vitro and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of clbR directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbR-dependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of clbR We investigated the impact of clbR overexpression or deletion at the transcriptome and proteome levels. Moreover, we compared global gene regulation under these conditions with that occurring upon overexpression or deletion of clbQ, which affects the flux of colibactin production. Combining the results of the transcriptome and proteome analyses with indirect measurements of colibactin levels by cell culture assays and an approximate quantification of colibactin via the second product of colibactin cleavage from precolibactin, N-myristoyl-d-asparagine, we demonstrate that the variable number of tandem repeats plays a significant regulatory role in colibactin expression. We identify ClbR as the only transcriptional activator known so far that is specific and essential for efficient regulation of colibactin production.IMPORTANCE The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.
    • Complete Genome Sequence and Manual Reannotation of Mycobacterium avium subsp. Strain DSM 44135.

      Goethe, Ralph; Basler, Tina; Meissner, Thorsten; Goethe, Elke; Spröer, Cathrin; Swiderski, Jolantha; Gerlach, Gerald-F; Weiss, Siegfried; Jarek, Michael; Bunk, Boyke; et al. (ASM, 2020-08-13)
      Here, we report the complete genome sequence of the Mycobacterium avium subsp. paratuberculosis reference strain DSM 44135, amended with a manual genome reannotation. The strain was originally described as M. paratuberculosis strain 6783. It was isolated from feces from a dairy cow in northern Germany.
    • Identification of a Biosynthetic Gene Cluster Responsible for the Production of a New Pyrrolopyrimidine Natural Product-Huimycin.

      Shuai, Hui; Myronovskyi, Maksym; Nadmid, Suvd; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2020-07-18)
      Pyrrolopyrimidines are an important class of natural products with a broad spectrum of biological activities, including antibacterial, antifungal, antiviral, anticancer or anti-inflammatory. Here, we present the identification of a biosynthetic gene cluster from the rare actinomycete strain Kutzneria albida DSM 43870, which leads to the production of huimycin, a new member of the pyrrolopyrimidine family of compounds. The huimycin gene cluster was successfully expressed in the heterologous host strain Streptomyces albus Del14. The compound was purified, and its structure was elucidated by means of nuclear magnetic resonance spectroscopy. The minimal huimycin gene cluster was identified through sequence analysis and a series of gene deletion experiments. A model for huimycin biosynthesis is also proposed in this paper.
    • Identification of a Novel LysR-Type Transcriptional Regulator in Staphylococcus aureus That Is Crucial for Secondary Tissue Colonization during Metastatic Bloodstream Infection.

      Groma, Michaela; Horst, Sarah A; Das, Sudip; Huettel, Bruno; Klepsch, Maximilian; Rudel, Thomas; Medina, Eva; Fraunholz, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2020-08-25)
      Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment.IMPORTANCEStaphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites.
    • 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.
    • Alpha-Toxin Limits Type 1 While Fostering Type 3 Immune Responses.

      Bonifacius, Agnes; Goldmann, Oliver; Floess, Stefan; Holtfreter, Silva; Robert, Philippe A; Nordengrün, Maria; Kruse, Friederike; Lochner, Matthias; Falk, Christine S; Schmitz, Ingo; et al. (Frontiers, 2020-08-07)
      Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.
    • Next Generation Influenza Vaccines: Looking into the Crystal Ball.

      Guzmán, Carlos Alberto; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-08-21)
      Influenza infections are responsible for significant number of deaths and overwhelming costs worldwide every year. Vaccination represents the only cost-efficient alternative to address this major problem in human health. However, current vaccines are fraught by many limitations, being far from optimal. Among them, the need to upgrade vaccines every year through a time-consuming process open to different caveats, and the critical fact that they exhibit poorer efficacy in individuals who are at high risk for severe infections. Where are we? How can knowledge and technologies contribute towards removing current roadblocks? What does the future offer in terms of next generation vaccines?
    • 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)
    • Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin.

      Müller, Laura; Murgia, Xabier; Siebenbürger, Lorenz; Börger, Carsten; Schwarzkopf, Konrad; Sewald, Katherina; Häussler, Susanne; Braun, Armin; Lehr, Claus-Michael; Hittinger, Marius; et al.
      Objectives: In the context of cystic fibrosis, Pseudomonas aeruginosa biofilms often develop in the vicinity of airway mucus, which acts as a protective physical barrier to inhaled matter. However, mucus can also adsorb small drug molecules administered as aerosols, including antibiotics, thereby reducing their bioavailability. The efficacy of antibiotics is typically assessed by determining the MIC using in vitro assays. This widespread technique, however, does not consider either bacterial biofilm formation or the influence of mucus, both of which may act as diffusion barriers, potentially limiting antibiotic efficacy. Methods: We grew P. aeruginosa biofilms in the presence or absence of human tracheal mucus and tested their susceptibility to tobramycin and colistin. Results: A significant reduction of tobramycin efficacy was observed when P. aeruginosa biofilms were grown in the presence of mucus compared with those grown in the absence of mucus. Diffusion of tobramycin through mucus was reduced; however, this reduction was more pronounced in biofilm/mucus mixtures, suggesting that biofilms in the presence of mucus respond differently to antibiotic treatment. In contrast, the influence of mucus on colistin efficacy was almost negligible and no differences in mucus permeability were observed. Conclusions: These findings underline the important role of mucus in the efficacy of anti-infective drugs.
    • Letter by Cochain et al Regarding Article, "Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine Models".

      Cochain, Clément; Saliba, Antoine-Emmanuel; Zernecke, Alma; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
    • User Evaluation Indicates High Quality of the Surveillance Outbreak Response Management and Analysis System (SORMAS) After Field Deployment in Nigeria in 2015 and 2018.

      Tom-Aba, Daniel; Toikkanen, Salla E; Glöckner, Stephan; Adeoye, Olawunmi; Mall, Sabine; Fähnrich, Cindy; Denecke, Kerstin; Benzler, Justus; Kirchner, Göran; Schwarz, Norbert; et al.
      During the West African Ebola virus disease outbreak in 2014-15, health agencies had severe challenges with case notification and contact tracing. To overcome these, we developed the Surveillance, Outbreak Response Management and Analysis System (SORMAS). The objective of this study was to measure perceived quality of SORMAS and its change over time. We ran a 4-week-pilot and 8-week-implementation of SORMAS among hospital informants in Kano state, Nigeria in 2015 and 2018 respectively. We carried out surveys after the pilot and implementation asking about usefulness and acceptability. We calculated the proportions of users per answer together with their 95% confidence intervals (CI) and compared whether the 2015 response distributions differed from those from 2018. Total of 31 and 74 hospital informants participated in the survey in 2015 and 2018, respectively. In 2018, 94% (CI: 89-100%) of users indicated that the tool was useful, 92% (CI: 86-98%) would recommend SORMAS to colleagues and 18% (CI: 10-28%) had login difficulties. In 2015, the proportions were 74% (CI: 59-90%), 90% (CI: 80-100%), and 87% (CI: 75-99%) respectively. Results indicate high usefulness and acceptability of SORMAS. We recommend mHealth tools to be evaluated to allow repeated measurements and comparisons between different versions and users.
    • Lactoferrin-Hexon Interactions Mediate CAR-Independent Adenovirus Infection of Human Respiratory Cells.

      Persson, B David; Lenman, Annasara; Frängsmyr, Lars; Schmid, Markus; Ahlm, Clas; Plückthun, Andreas; Jenssen, Håvard; Arnberg, Niklas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (ASM, 2020-07-01)
      Virus entry into host cells is a complex process that is largely regulated by access to specific cellular receptors. Human adenoviruses (HAdVs) and many other viruses use cell adhesion molecules such as the coxsackievirus and adenovirus receptor (CAR) for attachment to and entry into target cells. These molecules are rarely expressed on the apical side of polarized epithelial cells, which raises the question of how adenoviruses-and other viruses that engage cell adhesion molecules-enter polarized cells from the apical side to initiate infection. We have previously shown that species C HAdVs utilize lactoferrin-a common innate immune component secreted to respiratory mucosa-for infection via unknown mechanisms. Using a series of biochemical, cellular, and molecular biology approaches, we mapped this effect to the proteolytically cleavable, positively charged, N-terminal 49 residues of human lactoferrin (hLF) known as human lactoferricin (hLfcin). Lactoferricin (Lfcin) binds to the hexon protein on the viral capsid and anchors the virus to an unknown receptor structure of target cells, resulting in infection. These findings suggest that HAdVs use distinct cell entry mechanisms at different stages of infection. To initiate infection, entry is likely to occur at the apical side of polarized epithelial cells, largely by means of hLF and hLfcin bridging HAdV capsids via hexons to as-yet-unknown receptors; when infection is established, progeny virions released from the basolateral side enter neighboring cells by means of hLF/hLfcin and CAR in parallel.IMPORTANCE Many viruses enter target cells using cell adhesion molecules as receptors. Paradoxically, these molecules are abundant on the lateral and basolateral side of intact, polarized, epithelial target cells, but absent on the apical side that must be penetrated by incoming viruses to initiate infection. Our study provides a model whereby viruses use different mechanisms to infect polarized epithelial cells depending on which side of the cell-apical or lateral/basolateral-is attacked. This study may also be useful to understand the biology of other viruses that use cell adhesion molecules as receptors.
    • 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.
    • IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism.

      Rungelrath, Viktoria; Weiße, Christine; Schütze, Nicole; Müller, Uwe; Meurer, Marita; Rohde, Manfred; Seele, Jana; Valentin-Weigand, Peter; Kirschfink, Michael; Beineke, Andreas; et al. (Taylor & Francis, 2018-08-28)
      Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab')2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.
    • Methylene Blue Treatment of Grafts During Cold Ischemia Time Reduces the Risk of Hepatitis C Virus Transmission.

      Helfritz, Fabian A; Bojkova, Denisa; Wanders, Verena; Kuklinski, Nina; Westhaus, Sandra; von Horn, Charlotte; Rauen, Ursula; Gallinat, Anja; Baba, Hideo A; Skyschally, Andreas; et al. (Oxford Academic, 2018-12-01)
      Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.
    • Ten reasons why a sequence-based nomenclature is not useful for fungi anytime soon.

      Thines, Marco; Crous, Pedro W; Aime, M Catherine; Aoki, Takayuki; Cai, Lei; Hyde, Kevin D; Miller, Andrew N; Zhang, Ning; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2018-05-28)
      The large number of species still to be discovered in fungi, together with an exponentially growing number of environmental sequences that cannot be linked to known taxa, has fuelled the idea that it might be necessary to formally name fungi on the basis of sequence data only. Here we object to this idea due to several shortcomings of the approach, ranging from concerns regarding reproducibility and the violation of general scientific principles to ethical issues. We come to the conclusion that sequence-based nomenclature is potentially harmful for mycology as a discipline. Additionally, a classification based on sequences as types is not within reach anytime soon, because there is a lack of consensus regarding common standards due to the fast pace at which sequencing technologies develop.
    • Validation of an Autoclave Procedure for Sterilization of Mouse (Mus musculus) Carcasses.

      Pils, Marina C; Kränzler, Katrin; Beyer, Petra; Heise, Ulrike; Pasche, Bastian; Riedesel, Hermann; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Association for Laboratory Animal Science, 2018-11-06)
      The sterilization of potentially infectious animal carcasses is an important biologic safety issue in animal facilities operating as infection or quarantine barriers. However, the literature lacks a validated protocol. Here we describe the validation of an autoclave program suitable for daily use in a small rodent biocontainment unit. We evaluated several procedures for processing mouse carcasses in a standard autoclave. Heat sensors and biologic indicators were implanted inside the peritoneal cavity of dead mice, which were loaded at various densities into IVC cages or metal boxes. Heat sensors revealed broad differences in temperature inside carcasses compared with the autoclave chamber. Achieving the appropriate sterilization temperature was considerably prolonged in carcasses compared with typical laboratory waste material. We show that for 5 cadavers placed well separated inside an IVC, a modified program for mouse cage sterilization using 134 °C for 15 min is suitable. To sterilize approximately 1 kg of carcasses in autoclavable boxes, a period of 6 h is required to reach an effective temperature of 121 °C for 60 min at the center of the waste by using an autoclave program for liquids. In conclusion, we here validated 2 protocols for the sterilization of potentially infectious mouse carcasses, to ensure the application of efficacious procedures.
    • Mortality, morbidity, and hospitalisations due to influenza lower respiratory tract infections, 2017: an analysis for the Global Burden of Disease Study 2017.

      Troeger, Christopher E; GBD 2017 Influenza Collaborators; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2018-12-12)
      Background: Although the burden of influenza is often discussed in the context of historical pandemics and the threat of future pandemics, every year a substantial burden of lower respiratory tract infections (LRTIs) and other respiratory conditions (like chronic obstructive pulmonary disease) are attributable to seasonal influenza. The Global Burden of Disease Study (GBD) 2017 is a systematic scientific effort to quantify the health loss associated with a comprehensive set of diseases and disabilities. In this Article, we focus on LRTIs that can be attributed to influenza. Methods: We modelled the LRTI incidence, hospitalisations, and mortality attributable to influenza for every country and selected subnational locations by age and year from 1990 to 2017 as part of GBD 2017. We used a counterfactual approach that first estimated the LRTI incidence, hospitalisations, and mortality and then attributed a fraction of those outcomes to influenza. Findings: Influenza LRTI was responsible for an estimated 145 000 (95% uncertainty interval [UI] 99 000-200 000) deaths among all ages in 2017. The influenza LRTI mortality rate was highest among adults older than 70 years (16·4 deaths per 100 000 [95% UI 11·6-21·9]), and the highest rate among all ages was in eastern Europe (5·2 per 100 000 population [95% UI 3·5-7·2]). We estimated that influenza LRTIs accounted for 9 459 000 (95% UI 3 709 000-22 935 000) hospitalisations due to LRTIs and 81 536 000 hospital days (24 330 000-259 851 000). We estimated that 11·5% (95% UI 10·0-12·9) of LRTI episodes were attributable to influenza, corresponding to 54 481 000 (38 465 000-73 864 000) episodes and 8 172 000 severe episodes (5 000 000-13 296 000). Interpretation: This comprehensive assessment of the burden of influenza LRTIs shows the substantial annual effect of influenza on global health. Although preparedness planning will be important for potential pandemics, health loss due to seasonal influenza LRTIs should not be overlooked, and vaccine use should be considered. Efforts to improve influenza prevention measures are needed.