Publications by authors named "Steffen Rupp"

76 Publications

Comparison of Different Lactobacilli Regarding Substrate Utilization and Their Tolerance Towards Lignocellulose Degradation Products.

Curr Microbiol 2020 Oct 29;77(10):3136-3146. Epub 2020 Jul 29.

Institute of Interfacial Process Engineering and Plasma Technology, University Stuttgart, Nobelstraße 12, 70569, Stuttgart, Germany.

Fermentative lactic acid production is currently impeded by low pH tolerance of the production organisms, the successive substrate consumption of the strains and/or the requirement to apply purified substrate streams. We identified Lactobacillus brevis IGB 1.29 in compost, which is capable of producing lactic acid at low pH values from lignocellulose hydrolysates, simultaneously consuming glucose and xylose. In this study, we compared Lactobacillus brevis IGB 1.29 with the reference strains Lactobacillus brevis ATCC 367, Lactobacillus plantarum NCIMB 8826 and Lactococcus lactis JCM 7638 with regard to the consumption of C5- and C6-sugars. Simultaneous conversion of C5- and C6-monosaccharides was confirmed for L. brevis IGB 1.29 with consumption rates of 1.6 g/(L h) for glucose and 1.0 g/(L h) for xylose. Consumption rates were lower for L. brevis ATCC 367 with 0.6 g/(L h) for glucose and 0.2 g/(L h) for xylose. Further trials were carried out to determine the sensitivity towards common toxic degradation products in lignocellulose hydrolysates: acetate, hydroxymethylfurfural, furfural, formate, levulinic acid and phenolic compounds from hemicellulose fraction. L. lactis was the least tolerant strain towards the inhibitors, whereas L. brevis IGB 1.29 showed the highest tolerance. L. brevis IGB 1.29 exhibited only 10% growth reduction at concentrations of 26.0 g/L acetate, 1.2 g/L furfural, 5.0 g/L formate, 6.6 g/L hydroxymethylfurfural, 9.2 g/L levulinic acid or 2.2 g/L phenolic compounds. This study describes a new strain L. brevis IGB 1.29, that enables efficient lactic acid production with a lignocellulose-derived C5- and C6-sugar fraction.
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http://dx.doi.org/10.1007/s00284-020-02131-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452873PMC
October 2020

Confocal Raman microscopy to identify bacteria in oral subgingival biofilm models.

PLoS One 2020 11;15(5):e0232912. Epub 2020 May 11.

Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.

The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the 'Zürich Model' were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213720PMC
July 2020

Influence of microorganism and plant oils on the structure of mannosylerythritol lipid (MEL) biosurfactants revealed by a novel thin layer chromatography mass spectrometry method.

J Ind Microbiol Biotechnol 2019 Aug 7;46(8):1191-1204. Epub 2019 Jun 7.

Institute of Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Nobelstraße 12, 70569, Stuttgart, Germany.

Mannosylerythritol lipids (MEL) are microbial glycolipid biosurfactants with great potential for application in cosmetics and household detergents. In current biotechnological processes, they are produced by basidiomycetous fungi, the Ustilaginaceae, as a complex mixture of different chemical structures. It was the aim of this paper to study the influence of producer organisms and substrates on the resulting MEL structures with a novel high-resolution HPTLC-MALDI-TOF method. Given the seven different microbes and four plant oils, our analysis revealed that the product concentrations varied strongly between organisms, while they were similar for the different substrates. Coconut oil presented an exception, since only one organism was able to synthesize MEL from this substrate in considerable yields. Analysis by GC-FID further showed that the chain length pattern of hydrophobic fatty acid side-chains was very specific for individual organisms, while substrates had only a minor influence on the chain length. Our novel HPTLC-MALDI-TOF combination method finally demonstrated the presence of multiple MEL sub-variants with differing acetylation and fatty acid chain lengths. It also revealed the production of a more hydrophilic biosurfactant mannosylmannitol lipid (MML) as a side-product in certain fungi. Overall, it was concluded that the pattern of produced biosurfactant structures are mainly governed by producer organisms rather than substrates.
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http://dx.doi.org/10.1007/s10295-019-02194-2DOI Listing
August 2019

Computationally Designed Bispecific MD2/CD14 Binding Peptides Show TLR4 Agonist Activity.

J Immunol 2018 12 22;201(11):3383-3391. Epub 2018 Oct 22.

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, 70569 Stuttgart, Germany;

Toll-like receptor 4 plays an important role in the regulation of the innate and adaptive immune response. The majority of TLR4 activators currently in clinical use are derivatives of its prototypic ligand LPS. The discovery of innovative TLR4 activators has the potential of providing new therapeutic immunomodulators and adjuvants. We used computational design methods to predict and optimize a total of 53 cyclic and linear peptides targeting myeloid differentiation 2 (MD2) and cluster of differentiation 14 (CD14), both coreceptors of human TLR4. Activity of the designed peptides was first assessed using NF-κB reporter cell lines expressing either TLR4/MD2 or TLR4/CD14 receptors, then binding to CD14 and MD2 confirmed and quantified using MicroScale Thermophoresis. Finally, we incubated select peptides in human whole blood and observed their ability to induce cytokine production, either alone or in synergy with LPS. Our data demonstrate the advantage of computational design for the discovery of new TLR4 peptide activators with little structural resemblance to known ligands and indicate an efficient strategy with which to identify TLR4 targeting peptides that could be used as easy-to-produce alternatives to LPS-derived molecules in a variety of settings.
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http://dx.doi.org/10.4049/jimmunol.1800380DOI Listing
December 2018

Localization and functional characterization of the pathogenesis-related proteins Rbe1p and Rbt4p in Candida albicans.

PLoS One 2018 6;13(8):e0201932. Epub 2018 Aug 6.

Department of Molecular Biotechnology, Fraunhofer IGB, Stuttgart, Germany.

Members of the Cysteine-rich secretory protein, Antigen 5 and Pathogenesis-related 1 (CAP) protein superfamily are important virulence factors in fungi but remain poorly characterized on molecular level. Here, we investigate the cellular localization and molecular function of Rbe1p and Rbt4p, two CAP family members from the human pathogen Candida albicans. We unexpectedly found that Rbe1p localizes to budding sites of yeast cells in a disulfide bond-dependent manner. Furthermore, we show that Rbe1p and Rbt4p bind free cholesterol in vitro and export cholesteryl acetate in vivo. These findings suggest a previously undescribed role for Rbe1p in cell wall-associated processes and a possible connection between the virulence attributes of fungal CAP proteins and sterol binding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201932PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078311PMC
February 2019

The E. coli S30 lysate proteome: A prototype for cell-free protein production.

N Biotechnol 2018 Jan 21;40(Pt B):245-260. Epub 2017 Sep 21.

Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt am Main, Germany. Electronic address:

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.
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http://dx.doi.org/10.1016/j.nbt.2017.09.005DOI Listing
January 2018

Interaction of Candida Species with the Skin.

Microorganisms 2017 Jun 7;5(2). Epub 2017 Jun 7.

Department of Molecular Biotechnology, Fraunhofer Institute for Interfacial Engineering and Biotechnology, 70569 Stuttgart, Germany.

The human skin is commonly colonized by diverse fungal species. Some species, especially , do not only reside on the skin surface as commensals, but also cause infections by growing into the colonized tissue. However, defense mechanisms at the skin barrier level are very efficient, involving residential non-immune and immune cells as well as immune cells specifically recruited to the site of infection. Therefore, the skin is an effective barrier against fungal infection. While most studies about commensal and pathogenic interaction of species with host epithelia focus on the interaction with mucosal surfaces such as the vaginal and gastrointestinal epithelia, less is known about the mechanisms underlying interaction with the skin. In this review, we focus on the ecology and molecular pathogenesis of species on the skin and give an overview of defense mechanisms against in this context. We also discuss new research avenues in dermal infection, including the involvement of neurons, fibroblasts, and commensal bacteria in both mouse and human model systems.
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http://dx.doi.org/10.3390/microorganisms5020032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488103PMC
June 2017

Central Role for Dermal Fibroblasts in Skin Model Protection against Candida albicans.

J Infect Dis 2017 06;215(11):1742-1752

Department of Molecular Biotechnology, Fraunhofer Institute for Interfacial Engineering and Biotechnology.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces, including the skin. Under certain conditions, such as immunosuppression, invasion of the epithelia occurs. Not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis. Using immune cell-supplemented 3D skin models we defined a new role for fibroblasts in the dermis and identified a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA sequencing of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift toward an antimicrobial phenotype with broad specificity that is dependent on Toll-like receptor 2 and interleukin 1β. These results highlight a central function of dermal fibroblasts for skin protection, opening new possibilities for treatment of infectious diseases.
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http://dx.doi.org/10.1093/infdis/jix153DOI Listing
June 2017

Draft Genome Sequence of Pseudonocardia autotrophica Strain DSM 43083, an Efficient Producer of Peroxidases for Lignin Modification.

Genome Announc 2017 Feb 2;5(5). Epub 2017 Feb 2.

Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany

Pseudonocardia autotrophica strain DSM 43083 is a filamentous actinobacterium and was described to degrade or modify lignin. Here, we present its draft genome sequence, with a size of 5.8 Mb, to unravel the gene set coding for promising monooxygenases, dioxygenases, and DyP-type peroxidases associated with aromatic metabolism and lignin modification.
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http://dx.doi.org/10.1128/genomeA.01562-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289690PMC
February 2017

Candida guilliermondii as a potential biocatalyst for the production of long-chain α,ω-dicarboxylic acids.

Biotechnol Lett 2017 Mar 30;39(3):429-438. Epub 2016 Nov 30.

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany.

Objectives: To explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway.

Results: We identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene.

Conclusion: C. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary.
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http://dx.doi.org/10.1007/s10529-016-2264-3DOI Listing
March 2017

Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses.

Viruses 2016 11 23;8(11). Epub 2016 Nov 23.

Fraunhofer-Institute for Cell Therapy and Immunology IZI, Perlickstrasse 1, 04103 Leipzig, Germany.

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria () and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.
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http://dx.doi.org/10.3390/v8110319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127033PMC
November 2016

Immune Cell-Supplemented Human Skin Model for Studying Fungal Infections.

Methods Mol Biol 2017 ;1508:439-449

Fraunhofer Institut für Grenzflächenund Bioverfahrenstechnik, Nobelstr. 12, Stuttgart, 70569, Germany.

Human skin is a niche for various fungal species which either colonize the surface of this tissue as commensals or, primarily under conditions of immunosuppression, invade the skin and cause infection. Here we present a method for generation of a human in vitro skin model supplemented with immune cells of choice. This model represents a complex yet amenable tool to study molecular mechanisms of host-fungi interactions at human skin.
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http://dx.doi.org/10.1007/978-1-4939-6515-1_25DOI Listing
January 2018

Microarray Technologies in Fungal Diagnostics.

Authors:
Steffen Rupp

Methods Mol Biol 2017 ;1508:385-409

Fraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik, Nobelstr. 12, Stuttgart, 70569, Germany.

Microarray technologies have been a major research tool in the last decades. In addition they have been introduced into several fields of diagnostics including diagnostics of infectious diseases. Microarrays are highly parallelized assay systems that initially were developed for multiparametric nucleic acid detection. From there on they rapidly developed towards a tool for the detection of all kind of biological compounds (DNA, RNA, proteins, cells, nucleic acids, carbohydrates, etc.) or their modifications (methylation, phosphorylation, etc.). The combination of closed-tube systems and lab on chip devices with microarrays further enabled a higher automation degree with a reduced contamination risk. Microarray-based diagnostic applications currently complement and may in the future replace classical methods in clinical microbiology like blood cultures, resistance determination, microscopic and metabolic analyses as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel nucleic acid based biomarkers. Here I focus an microarray technologies in diagnostics and as research tools, based on nucleic acid-based arrays.
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http://dx.doi.org/10.1007/978-1-4939-6515-1_22DOI Listing
January 2018

Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3.

J Biotechnol 2017 Jan 9;241:69-75. Epub 2016 Nov 9.

University of Stuttgart, Institute of Interfacial Process Engineering and Plasma Technology, Stuttgart, Germany; Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany. Electronic address:

Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling.
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http://dx.doi.org/10.1016/j.jbiotec.2016.11.007DOI Listing
January 2017

Computational Discovery and Experimental Confirmation of TLR9 Receptor Antagonist Leads.

J Chem Inf Model 2016 09 1;56(9):1835-46. Epub 2016 Sep 1.

Molecular Modeling and Drug Design, Institute for Drug Research, The Hebrew University of Jerusalem , Jerusalem, 91120, Israel.

Toll-like receptors (TLR) are receptors of innate immunity that recognize pathogen associated molecular patterns. They play a critical role in many pathological states, in acute and chronic inflammatory processes. TLR9 is a promising target for drug discovery, since it has been implicated in several pathologies, including defense against viral infections and psoriasis. Immune-modulators are promising molecules for therapeutic intervention in these indications. TLR9 is located in the endosome and activated by dsDNA with CpG motives encountered in microbial DNA. Here we report on a combined approach to discover new TLR9 antagonists by computational chemistry and cell based assays. We used our in-house iterative stochastic elimination (ISE) algorithm to create models that distinguish between TLR9 antagonists ("actives") and other molecules ("inactives"), based on molecular physicochemical properties. Subsequent screening and scoring of a data set of 1.8 million commercially available molecules led to the purchasing of top scored molecules, which were tested in a new cell based system based on human pattern recognition receptors (PRRs) stably expressed in NIH3T3 fibroblasts. As described previously, this cell line shows a very low endogenous PRR-activity and contains a reporter gene which is selectively activated by the integrated human PRR enabling rapid screening of potential ligands. IC50 values of each of these top scored molecules were determined. Out of 60 molecules tested, 56 showed antagonistic activity. We discovered 21 new highly potential antagonists with IC50 values lower than 10 μM, with 5 of them having IC50 values under 1 μM.
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http://dx.doi.org/10.1021/acs.jcim.6b00070DOI Listing
September 2016

Optimization and Scale-up of Inulin Extraction from Taraxacum kok-saghyz roots.

Nat Prod Commun 2016 May;11(5):689-92

The optimization and scale-up of inulin extraction from Taraxacum kok-saghyz Rodin was successfully performed. Evaluating solubility investigations, the extraction temperature was fixed at 85 degrees C. The inulin stability regarding degradation or hydrolysis could be confirmed by extraction in the presence of model inulin. Confirming stability at the given conditions the isolation procedure was transferred from a 1 L- to a 1 m3-reactor. The Reynolds number was selected as the relevant dimensionless number that has to remain constant in both scales. The stirrer speed in the large scale was adjusted to 3.25 rpm regarding a 300 rpm stirrer speed in the 1 L-scale and relevant physical and process engineering parameters. Assumptions were confirmed by approximately homologous extraction kinetics in both scales. Since T. kok-saghyz is in the focus of research due to its rubber content side-product isolation from residual biomass it is of great economic interest. Inulin is one of these additional side-products that can be isolated in high quantity (- 35% of dry mass) and with a high average degree of polymerization (15.5) in large scale with a purity of 77%.
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May 2016

Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

Mar Drugs 2016 Apr 15;14(4). Epub 2016 Apr 15.

Institute of Bioprocess Engineering, University of Kaiserslautern, Gottlieb-Daimler-Straße 49, 67663 Kaiserslautern, Germany.

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.
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http://dx.doi.org/10.3390/md14040079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849083PMC
April 2016

Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV.

J Control Release 2016 05 21;229:163-171. Epub 2016 Mar 21.

Fraunhofer IGB, Stuttgart, Germany.

Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8±10.1nm, shelf-life stability of >1year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20μM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400μM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy.
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http://dx.doi.org/10.1016/j.jconrel.2016.03.025DOI Listing
May 2016

Draft Genome Sequence of Amantichitinum ursilacus IGB-41, a New Chitin-Degrading Bacterium.

Genome Announc 2015 Nov 19;3(6). Epub 2015 Nov 19.

Fraunhofer IGB, Stuttgart, Germany

Amantichitinum ursilacus IGB-41 is a new species of chitin-degrading bacterium isolated from soil, which secretes potential industrial enzymes. The genome of A. ursilacus was sequenced, and the gene set encoding chitinases was identified. Here, we present the draft genome of 4.9 Mb, comprising 38 contigs, and the corresponding annotation.
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http://dx.doi.org/10.1128/genomeA.01309-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653779PMC
November 2015

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols.

Antimicrob Agents Chemother 2015 Oct 27;59(10):6296-307. Epub 2015 Jul 27.

Universität Stuttgart, Institut für Grenzflächenverfahrenstechnik und Plasmatechnologie, Stuttgart, Germany Fraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik, Stuttgart, Germany

Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3β,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3β,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.
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http://dx.doi.org/10.1128/AAC.00640-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4576113PMC
October 2015

Structural Hot Spots Determine Functional Diversity of the Candida glabrata Epithelial Adhesin Family.

J Biol Chem 2015 Aug 23;290(32):19597-613. Epub 2015 Jun 23.

From the Departments of Genetics and

For host colonization, the human fungal pathogen Candida glabrata is known to utilize a large family of highly related surface-exposed cell wall proteins, the lectin-like epithelial adhesins (Epas). To reveal the structure-function relationships within the entire Epa family, we have performed a large scale functional analysis of the adhesion (A) domains of 17 Epa paralogs in combination with three-dimensional structural studies of selected members with cognate ligands. Our study shows that most EpaA domains exert lectin-like functions and together recognize a wide variety of glycans with terminal galactosides for conferring epithelial cell adhesion. We further identify several conserved and variable structural features within the diverse Epa ligand binding pockets, which affect affinity and specificity. These features rationalize why mere phylogenetic relationships within the Epa family are weak indicators for functional classification and explain how Epa-like adhesins have evolved in C. glabrata and related fungal species.
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http://dx.doi.org/10.1074/jbc.M115.655654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528126PMC
August 2015

Of mice, flies--and men? Comparing fungal infection models for large-scale screening efforts.

Dis Model Mech 2015 May 18;8(5):473-86. Epub 2015 Mar 18.

Equipe Fondation Recherche Médicale, Unité Propre de Recherche 9022 du Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Moléculaire et Cellulaire (IBMC), Université de Strasbourg, 67084 Strasbourg, France.

Studying infectious diseases requires suitable hosts for experimental in vivo infections. Recent years have seen the advent of many alternatives to murine infection models. However, the use of non-mammalian models is still controversial because it is often unclear how well findings from these systems predict virulence potential in humans or other mammals. Here, we compare the commonly used models, fruit fly and mouse (representing invertebrate and mammalian hosts), for their similarities and degree of correlation upon infection with a library of mutants of an important fungal pathogen, the yeast Candida glabrata. Using two indices, for fly survival time and for mouse fungal burden in specific organs, we show a good agreement between the models. We provide a suitable predictive model for estimating the virulence potential of C. glabrata mutants in the mouse from fly survival data. As examples, we found cell wall integrity mutants attenuated in flies, and mutants of a MAP kinase pathway had defective virulence in flies and reduced relative pathogen fitness in mice. In addition, mutants with strongly reduced in vitro growth generally, but not always, had reduced virulence in flies. Overall, we demonstrate that surveying Drosophila survival after infection is a suitable model to predict the outcome of murine infections, especially for severely attenuated C. glabrata mutants. Pre-screening of mutants in an invertebrate Drosophila model can, thus, provide a good estimate of the probability of finding a strain with reduced microbial burden in the mouse host.
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http://dx.doi.org/10.1242/dmm.019901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415897PMC
May 2015

The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids.

Appl Microbiol Biotechnol 2015 Feb 15;99(3):1375-88. Epub 2015 Jan 15.

Department of Molecular Biotechnology, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstr. 12, 70569, Stuttgart, Germany.

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.
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http://dx.doi.org/10.1007/s00253-014-6359-2DOI Listing
February 2015

Single-stranded DNA catalyzes hybridization of PCR-products to microarray capture probes.

PLoS One 2014 15;9(7):e102338. Epub 2014 Jul 15.

Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Bad Oeynhausen, Germany.

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102338PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099319PMC
December 2015

Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.

PLoS Pathog 2014 Jun 19;10(6):e1004211. Epub 2014 Jun 19.

Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Vienna, Austria.

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.
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http://dx.doi.org/10.1371/journal.ppat.1004211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063973PMC
June 2014

Control of morphogenesis, protease secretion and gene expression in Candida albicans by the preferred nitrogen source ammonium.

Microbiology (Reading) 2014 Aug 19;160(Pt 8):1599-1608. Epub 2014 May 19.

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany.

Micro-organisms sense the availability of nutrients in their environment to control cellular behaviour and the expression of transporters and enzymes that are required for the utilization of these nutrients. In the pathogenic yeast Candida albicans, the preferred nitrogen source ammonium suppresses the switch from yeast to filamentous growth in response to certain stimuli, and it also represses the secretion of proteases, which are required for the utilization of proteins as an alternative nitrogen source. To investigate whether C. albicans senses the availability of ammonium in the extracellular environment or if ammonium uptake into the cell is required to regulate morphogenesis and gene expression, we compared the behaviour of wild-type cells and ammonium uptake-deficient mutants in the presence and absence of extracellular ammonium. Arginine-induced filamentous growth was suppressed by ammonium in the wild-type, but not in mutants lacking the ammonium permeases Mep1 and Mep2. Similarly, ammonium suppressed protease secretion and extracellular protein degradation in the wild-type, but not in mutants lacking the ammonium transporters. By comparing the gene expression profiles of C. albicans grown in the presence of low or high ammonium concentrations, we identified a set of genes whose expression is controlled by nitrogen availability. The repression of genes involved in the utilization of alternative nitrogen sources, which occurred under ammonium-replete conditions in the wild-type, was abrogated in mep1Δ mep2Δ mutants. These results demonstrate that C. albicans does not respond to the presence of sufficient amounts of the preferred nitrogen source ammonium by sensing its availability in the environment. Instead, ammonium has to be taken up into the cell to control morphogenesis, protease secretion and gene expression.
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http://dx.doi.org/10.1099/mic.0.078238-0DOI Listing
August 2014

An in vivo photo-cross-linking approach reveals a homodimerization domain of Aha1 in S. cerevisiae.

PLoS One 2014 10;9(3):e89436. Epub 2014 Mar 10.

Fraunhofer IGB, Stuttgart, Germany.

Protein-protein interactions play an essential role in almost any biological processes. Therefore, there is a particular need for methods which describe the interactions of a defined target protein in its physiological context. Here we report a method to photo-cross-link interacting proteins in S. cerevisiae by using the non-canonical amino acid p-azido-L-phenylalanine (pAzpa). Based on the expanded genetic code the photoreactive non-canonical amino acid pAzpa was site-specifically incorporated at eight positions into a domain of Aha1 that was previously described to bind Hsp90 in vitro to function as a cochaperone of Hsp90 and activates its ATPase activity. In vivo photo-cross-linking to the cognate binding partner of Aha1 was carried out by irradiation of mutant strains with UV light (365 nm) to induce covalent intermolecular bonds. Surprisingly, an interaction between Aha1 and Hsp90 was not detected, although, we could confirm binding of suppressed pAzpa containing Aha1 to Hsp90 by native co-immunoprecipitation. However, a homodimer consisting of two covalently crosslinked Aha1 monomers was identified by mass spectrometry. This homodimer could also be confirmed using p-benzoyl-L-phenylalanine, another photoreactive non-canonical amino acid. Crosslinking was highly specific as it was dependent on irradiation using UV light, the exact position of the non-canonical amino acid in the protein sequence as well as on the addition of the non-canonical amino acid to the growth medium. Therefore it seems possible that an interaction of Aha1 with Hsp90 takes place at different positions than previously described in vitro highlighting the importance of in vivo techniques to study protein-protein interactions. Accordingly, the expanded genetic code can easily be applied to other S. cerevisiae proteins to study their interaction under physiological relevant conditions in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089436PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948627PMC
March 2016

Genome Sequence of the Basidiomycetous Fungus Pseudozyma aphidis DSM70725, an Efficient Producer of Biosurfactant Mannosylerythritol Lipids.

Genome Announc 2014 Feb 13;2(1). Epub 2014 Feb 13.

Fraunhofer IGB, Stuttgart, Germany.

Pseudozyma aphidis is an efficient producer of mannosylerythritol lipids exceeding concentrations of >100 g/liter from renewable feed stocks. Additionally, a biosurfactant cellobiose lipid is also secreted during nitrogen limitation. Here, we describe the sequencing of P. aphidis to unravel the genomic basis of biosurfactant metabolism in P. aphidis.
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http://dx.doi.org/10.1128/genomeA.00053-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924370PMC
February 2014

Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages.

Eukaryot Cell 2014 Jan 20;13(1):170-83. Epub 2013 Dec 20.

Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute Jena (HKI), Jena, Germany.

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.
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http://dx.doi.org/10.1128/EC.00262-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910963PMC
January 2014

High solids enzymatic hydrolysis of pretreated lignocellulosic materials with a powerful stirrer concept.

Appl Biochem Biotechnol 2014 Feb 19;172(3):1699-713. Epub 2013 Nov 19.

Evonik Industries AG, Rodenbacher Chaussee 4, 63457, Hanau-Wolfgang, Germany.

In this study, we present a powerful stirred tank reactor system that can efficiently hydrolyse lignocellulosic material at high solid content to produce hydrolysates with glucose concentration > 100 g/kg. As lignocellulosic substrates alkaline-pretreated wheat straw and organosolv-pretreated beech wood were used. The developed vertical reactor was equipped with a segmented helical stirrer, which was specially designed for high biomass hydrolysis. The stirrer was characterised according to mixing behaviour and power input. To minimise the cellulase dosage, a response surface plan was used. With the empirical relationship between glucose yield, cellulase loading and solid content, the minimal cellulase dosage was calculated to reach at least 70% yield at high glucose and high substrate concentrations within 48 h. The optimisation resulted in a minimal enzyme dosage of 30 FPU/g dry matter (DM) for the hydrolysis of wheat straw and 20 FPU/g DM for the hydrolysis of beech wood. By transferring the hydrolysis reaction from shaking flasks to the stirred tank reactor, the glucose yields could be increased. Using the developed stirred tank reactor system, alkaline-pretreated wheat straw could be converted to 110 g/kg glucose (76%) at a solid content of 20% (w/w) after 48 h. Organosolv-pretreated beech wood could be efficiently hydrolysed even at 30% (w/w) DM, giving 150 g/kg glucose (72%).
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http://dx.doi.org/10.1007/s12010-013-0607-2DOI Listing
February 2014