• Triazole-based cross-linkers in radical polymerization processes: tuning mechanical properties of poly(acrylamide) and poly( -dimethylacrylamide) hydrogels

      Götz, Tobias; Schädel, Nicole; Petri, Nadja; Kirchhof, Manuel; Bilitewski, Ursula; Tovar, Günter E. M.; Laschat, Sabine; Southan, Alexander; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
      Triazole-based cross-linkers with different spacer lengths and different functional end groups (acrylamides, methacrylamides, maleimides and vinylsulfonamides) were synthesized, investigated for cytotoxic and antibacterial activity, and incorporated into poly(acrylamide) (PAAm) and poly(N,N-dimethylacrylamide) (PDMAAm) hydrogels by free-radical polymerization. Hydrogels prepared with different cross-linkers and cross-linker contents between 0.2% and 1.0% were compared by gel yields, equilibrium degrees of swelling (S) and storage moduli (G0). Generally with increasing cross-linker content, G0 values of the hydrogels increased, while S values decreased. The different polymerizable cross-linker end groups resulted in a decrease of G0 in the following order for cross-linkers with C4 spacers: acrylamide > maleimide > methacrylamide > vinylsulfonamide. Longer cross-linker alkyl spacer lengths caused an increase in G0 and a decrease in S. Independent of the cross-linker used, a universal correlation between G0 and equilibrium polymer volume fraction f was found. For PAAm hydrogels, G0 ranged between 4 kPa and 23 kPa and f between 0.07 and 0.14. For PDMAAm hydrogels, G0 ranged between 0.1 kPa and 4.9 kPa and f between 0.02 and 0.06. The collected data were used to establish an empirical model to predict G0 depending on f. G0 of PAAm and PDMAAm hydrogels is given by G0 ¼ 4034 kPa f2.66 and G0 ¼ 4297 kPa f2.46, respectively.
    • Self-assembly and biological activities of ionic liquid crystals derived from aromatic amino acids.

      Neidhardt, Manuel M; Schmitt, Katharina; Baro, Angelika; Schneider, Carmen; Bilitewski, Ursula; Laschat, Sabine; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-08)
      The self-assembly of amino acid-derived ionic liquid crystals (ILCs) into lamellar or micellar-like aggregates suggests that they might interact with biological membranes. To get some insight, guanidinium chlorides derived from the natural l-amino acids phenylalanine (Phe), tyrosine (Tyr) and 3,4-dihydroxyphenylalanine (DOPA) were synthesized and their mesomorphic properties were investigated via polarizing optical microscopy (POM), differential scanning calorimetry (DSC) and X-ray diffraction (SAXS, WAXS). Mesophase types depended on the number of alkoxy side chains. Phe- and Tyr-based ILCs with one and two side chains, respectively, self-assembled into smectic A bilayers (SmA2), while Dopa-derived ILCs with three side chains formed columnar (Colh) mesophases. The mesophase ranges for Phe ILCs increased steadily with side chain length, for Tyr- and Dopa-based ILCs, however, size matching effects were observed. To clarify whether the mesomorphic behaviour has an impact on biological properties, cytotoxic and antibacterial activities of the ILCs were studied. Phe and Tyr ILCs exhibited much higher cytotoxicities (against the L-929 mouse fibroblast cell line) and/or antibacterial activities (against Staphylococcus aureus) than Dopa ILCs, which were mostly inactive. Furthermore, within each series, the side chain length largely influenced the biological activity. Thus, the bulk mesophase behaviour appeared to correlate with the biological properties, in particular, the interactions with membranes, as shown by measuring the intracellular Ca2+ concentration in human monocytic U937 cells after treatment with the amino acid-based ILCs.
    • Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen

      Lünsdorf, Heinrich; Gurramkonda, Chandrasekhar; Adnan, Ahmad; Khanna, Navin; Rinas, Ursula (2011-06-26)
      Abstract Background A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. Results High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. Conclusions It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.
    • Deletion of the HAMP domains from the histidine kinase CaNik1p of Candida albicans or treatment with fungicides activates the MAP kinase Hog1p in S. cerevisiae transformants

      El-Mowafy, Mohammed; Bahgat, Mahmoud M; Bilitewski, Ursula (2013-09-17)
      Abstract Background Microorganisms use two-component signal transduction (TCST) systems to regulate the response of the organism to changes of environmental conditions. Such systems are absent from mammalian cells and are thus of interest as drug targets. Fungal TCST systems are usually composed of a hybrid histidine kinase, comprising the histidine kinase (HisKA) domain and a receiver domain, a histidine phosphotransfer protein and a response regulator. Among the 11 groups of fungal histidine kinases, group III histidine kinases are of particular relevance as they are essential for the activity of different groups of fungicides. A characteristic feature is the N-terminal amino acid repeat domain comprising multiple HAMP domains, of which the function is still largely unknown. In Candida albicans, a fungal human pathogen, three histidine kinases were identified, of which CaNik1p is a group III histidine kinase. Heterologous expression of this protein in Sacchromyces cerevisiae conferred susceptibility to different fungicides. Fungicide activity was associated with phosphorylation of the mitogen activated protein kinase Hog1p. Results We have constructed mutated versions of CaNik1p, from which either all HAMP domains were deleted (CaNik1pΔHAMP) or in which the histidine kinase or the receiver domains were not-functional. Expression of CaNIK1ΔHAMP in S. cerevisiae led to severe growth inhibition. Normal growth could be restored by either replacing the phosphate-accepting histidine residue in CaNik1pΔHAMP or by expressing CaNIK1ΔHAMP in S. cerevisiae mutants, in which single genes encoding several components of the HOG pathway were deleted. Expression of proteins with non-functional histidine kinase or receiver domains resulted in complete loss of susceptibility to antifungals, such as fludioxonil. Conditions leading to growth inhibition of transformants also led to phosphorylation of the MAP kinase Hog1p. Conclusion Our results show that functional histidine kinase and receiver domains of CaNik1p were essential for antifungal susceptibility and for activation of the Hog1p. Moreover, for the first time we show that deletion of all HAMP domains from CaNik1p led to activation of Hog1p without an external stimulus. This phenotype was similar to the effects obtained upon treatment with fungicides, as in both cases growth inhibition correlated with Hog1p activation and was dependent on the functionality of the conserved phosphate-accepting histidine residue.
    • Regulation of Candida albicans Interaction with Macrophages through the Activation of HOG Pathway by Genistein

      Cui, Shuna; Hassan, Rabeay; Heintz-Buschart, Anna; Bilitewski, Ursula; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2016-01-28)
    • Deletion of the HAMP domains from the histidine kinase CaNik1p of Candida albicans or treatment with fungicides activates the MAP kinase Hog1p in S. cerevisiae transformants.

      El-Mowafy, Mohammed; Bahgat, Mahmoud M; Bilitewski, Ursula (2013-09-17)
      Microorganisms use two-component signal transduction (TCST) systems to regulate the response of the organism to changes of environmental conditions. Such systems are absent from mammalian cells and are thus of interest as drug targets. Fungal TCST systems are usually composed of a hybrid histidine kinase, comprising the histidine kinase (HisKA) domain and a receiver domain, a histidine phosphotransfer protein and a response regulator. Among the 11 groups of fungal histidine kinases, group III histidine kinases are of particular relevance as they are essential for the activity of different groups of fungicides. A characteristic feature is the N-terminal amino acid repeat domain comprising multiple HAMP domains, of which the function is still largely unknown. In Candida albicans, a fungal human pathogen, three histidine kinases were identified, of which CaNik1p is a group III histidine kinase. Heterologous expression of this protein in Sacchromyces cerevisiae conferred susceptibility to different fungicides. Fungicide activity was associated with phosphorylation of the mitogen activated protein kinase Hog1p.
    • Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay.

      Heintz-Buschart, Anna; Eickhoff, Holger; Hohn, Erwin; Bilitewski, Ursula; Department of Biological Systems Analysis, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2013-03-10)
      Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast–hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model. We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37 °C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to β-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzo[d]isoxazolones and thiazolo[4,5-e]benzoisoxazoles. In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans.
    • Direct electrochemical determination of Candida albicans activity.

      Hassan, Rabeay Y A; Bilitewski, Ursula; Helmholtz Centre for Infection Research, Working Group BiSA, Inhoffenstr. 7, 38124 Braunschweig, Germany; Microanalysis Lab, Applied Organic Chemistry Department, National Research Centre (NRC), Eltahrir Street, 12311-Dokki, Cairo, Egypt. Electronic address: rabeayy@yhaoo.com. (2013-05-15)
      Despite advances made in the field, rapid detection methods for the human pathogen Candida albicans are still missing. In this regard, bio-electrochemical systems including electrochemical sensors and biosensors satisfy the increasing demand for rapid, reliable, and direct microbial analyses. In this study, the bioelectrochemical characteristics of C. albicans were investigated for use in an analytical system that determines the viability of the organisms. The electrochemical responses of viable and non-viable cells of C. albicans and Saccharomyces cerevisiae were monitored. Cyclic voltammograms (CV) showed an irreversible oxidation peak at about 750mV that accounts for viable cells. The peak current increased at viable cell numbers ranging from 3×10(5) to 1.6×10(7)cells/ml, indicating that the amount of viable cells can be accurately quantified. To elucidate the underlying electron transfer processes, the influence of electron transfer chain (ETC) - inhibitors on the electrochemical behavior of the two organisms were investigated. Inhibition of the function of classical respiratory chain (CRC) led to a decrease in the electrochemical response, whereas the oxidation current increased when the alternative oxidase (AOX) pathway was blocked by salicylhydroxamic acid (SHA). Blocking the AOX pathway improved the electrochemical performance, suggesting an involvement in the CRC, with cytochrome c oxidase (COX) as a relevant protein complex. Mutants, in which components of COX were deleted, showed a lower electro-activity than the wild-type strain. Particularly, deletion of subunit COX5a almost completely abolished the electrochemical signal. We believe that this work can be utilized for the development of early detection assays and opens the door for new technological developments in the field of C. albicans.
    • Involvement of the mitogen activated protein kinase Hog1p in the response of Candida albicans to iron availability.

      Kaba, Hani Ej; Nimtz, Manfred; Müller, Peter P; Bilitewski, Ursula (2013-01-24)
      ABSTRACT: BACKGROUND: Iron is an essential nutrient for almost all organisms, and generating iron limiting conditions for pathogens is one of the host defense strategies against microbial infections. Excess of iron can be toxic; therefore, iron uptake is tightly controlled. The high affinity iron uptake system of the opportunistic pathogenic yeast Candida albicans has been shown to be essential for virulence. Several transcription factors and regulators of iron uptake genes were identified, but the knowledge of signaling pathways is still limited. Gene expression profiling of the Deltahog1 deletion mutant indicated an involvement of the mitogen activated protein (MAP) kinase Hog1p. However, the function of Hog1p in the response of C. albicans to iron availability was not studied in detail. Thus, we analyzed phenotypic and molecular responses of C. albicans to different iron concentrations particularly with respect to the activity of the Hog1p MAP kinase module. RESULTS: We observed flocculation of yeast cells, when the iron ion concentration was equal to or higher than 5 muM. This phenotype was dependent on the MAP kinase Hog1p and the corresponding MAP kinase kinase Pbs2p. Moreover, high extracellular iron ion concentrations led to hyper-phosphorylation of Hog1p. We determined lower amounts of multicopper ferroxidase (MCFO) proteins and lower ferric reductase activity, when the iron ion concentration in the medium was increased. This effect was also observed for the Deltahog1 mutant. However, the amounts of MCFO proteins and the cell surface ferric reductase activity were increased in the Deltahog1 in comparison to wild type cells. This effect was independent of iron availability in growth media. CONCLUSIONS: In C. albicans, the MAP kinase Hog1p is part of the network regulating the response of the organism to iron availability. Hog1p was transiently phosphorylated under high iron concentrations and was essential for a flocculent phenotype. Furthermore, deletion of HOG1 led to increased levels of components of the reductive iron uptake system in comparison to the wild-type, independent of iron concentrations in the media. However, the additional induction of this system by low iron concentrations was independent of HOG1.
    • The fungicide fludioxonil antagonizes fluconazole activity in the human fungal pathogen Candida albicans.

      Buschart, Anna; Burakowska, Anna; Bilitewski, Ursula; Biological Systems Analysis, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012-12)
      The fungicide fludioxonil is widely used in agriculture. Residua of this fungicide are occasionally detected in fruits and can therefore be ingested by humans. The human fungal pathogen Candida albicans expresses the target of fludioxonil, Nik1p, a type III histidine kinase involved in stress response. Inhibition of yeast and hyphae growth was hardly observable after treatment of C. albicans SC5314 with fludioxonil. As a side effect, however, we observed a concentration-dependent induction of the expression of the genes CDR1 and CDR2, encoding ATP-binding cassette (ABC) transporters. This was independent of the presence of the target of fludioxonil as induction was also observed in a Δnik1 deletion mutant. Deletion of the CDR1 gene aggravated the inhibition of germ tube formation by fludioxonil, indicating that, in the wild-type, the fungicide was discharged from the cell by Cdr1p. Cdr1p is also known as a resistance factor of C. albicans against the commonly used antimycotic fluconazole. Thus, the effect of concurrent exposure to fludioxonil and known cargoes of ABC transporters on their extrusion and the growth of C. albicans was examined. Pre-incubation with fludioxonil decreased the export rate of rhodamine 6G. The resistance to fluconazole was increased by fludioxonil, independently of Nik1p. Therefore, exposure of C. albicans to fludioxonil may lead to increased resistance to fluconazole treatment.
    • A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol.

      Hassan, Rabeay Y A; Bilitewski, Ursula; Biological Systems Analysis Group, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. (2011-12-01)
      Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S.cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.
    • Improving the prediction of Pseudomonas putida mt-2 growth kinetics with the use of a gene expression regulation model of the TOL plasmid

      Koutinas, Michalis; Kiparissides, Alexandros; Lam, Ming-Chi; Silva-Rocha, Rafael; Godinho, Miguel; de Lorenzo, Victor; Martins dos Santos, Vitor A.P.; Pistikopoulos, Efstratios N.; Mantalaris, Athanasios; Helmholtz Center for Infection Research (HZI), 38124 Braunschweig, Germany. (2011-11-22)
    • The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate.

      Lu, Xin; Sun, Jibin; Nimtz, Manfred; Wissing, Josef; Zeng, An-Ping; Rinas, Ursula; Helmholtz Center for Infection Research, Inhoffenstr, Braunschweig, Germany. (2010)
      The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.