Institut für Mikrobiologie - Abteilung für Genetik & Biochemie
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- Streptococcus pneumoniae (7)
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Currently, plastic materials are an integral part of our lives, but their production mostly bases on fossil fuels or derivatives, which resources are decreasing. Extraction and processing of non-renewable resources have also negative impact on environment. One of the most promising and environmentally friendly approaches is use of microorganism. This PhD dissertation presents the non-conventional yeast Arxula adeninivorans as a host for production of bio-based and biodegradable poly(hydroxyalkanoates) plastics poly(hydroxybutyrate) and co-polymer poly(hydroxybutyrate-co-hydroxyvalerate). Additionally, the constructed yeast strain was able to secrete enantiomerically pure (R)-3-hydroxybutyric acid.
The production of PHAs requires three enzymes: β-ketothiolase, acetoacetyl-CoA reductase and PHA synthase. The strategy followed in this project was divided into two parts. While all three enzymes are responsible for intracellular production of PHA polymer, first two only lead to secretion of (R)-3-HB into culture media, which was used in a first stage of work to establish and optimize polymer production. Both, different bacterial strains and yeast A. adeninivorans were taken into account in screening of the genes encoding aforementioned enzymes. Bacterial genes were chemically synthesized using codon optimization pattern and endogenous genes were obtained using PCR and genomic DNA template from A. adeninivorans LS3 wild-type strain. Each gene was cloned into Xplor2 vector between TEF1 constitutive promoter and PHO5 terminator. Vector containing both thiolase and reductase genes was used for A. adeninivorans transformation.
The best combination of heterologous genes was overexpression of β-ketothiolase gene from Clostridium acetobutylicum and acetoacetyl-CoA reductase gene from Cupriavidus necator which led to secretion of 4.84 g L−1 (R)-3-HB, at a rate of 0.023 g L−1 h−1 over 214 h in shaking flask cultivation. Further optimization by fed-batch culturing with glucose as a carbon source did not improve (R)-3-HB secretion, but the rate of production was doubled to 0.043 g L−1 h−1 [3.78 g L−1 of (R)-3-HB at 89 h].
The product of acetoacetyl-CoA reductase is (R)-3-HB-CoA and further removing of CoA moiety is needed for acid secretion into culture media. A. adeninivorans is able to conduct this process without any additional modification but the conversion rate is unknown. Two thioesterases, cytosolic TesBp encoded by TesB gene from E. coli and mitochondrial ATes1p encoded by ATES1 gene from A. adeninivorans, were analysed to enhance secretion process. Additionally, a cytosolic version of ATES1 gene (ATES1cyt) was tested. All three genes were expressed in A. adeninivorans cells under TEF1 constitutive promoter together with thiolase and reductase genes. Despite detected enzymatic activity the yield of (R)-3-HB synthesis and secretion was not increased. Moreover, overexpressed thioesterases negatively influenced cell growth, indicating that they act on other metabolic components. The results provided two sets of information, first, the endogenous secretion system is sufficient for (R)-3-HB production; second, further screening of suitable genes needs to be performed.
Based on optimization of (R)-3-HB synthesis, thiolase gene (thl) from C. acetobutylicum and reductase gene (phaB) from C. necator were chosen to combine with PHA synthase gene (phaC) for creating the PHB-V producing strain. The PHA synthase expression module, containing TEF1 promoter and PHO5 terminator, was cloned into Xplor2 vector together with thiolase and reductase expression modules and used for A. adeninivorans transformation. The engineered strain accumulated up to 7.47% PHB of dcw. During the set of cells passaging A. adeninivorans lost the ability to accumulate polymer with maximal 23.1 % of primary accumulation level. Additionally, use of a vector including hygromycin B antibiotic resistance marker (instead of auxotrophic marker in Xplor2) did not improve polymer accumulation and stability.
To counteract the effect of loss of accumulation stability, phasin gene (phaP1), originated from C. necator, was introduce together with PHA pathway genes. First screening cultivations resulted in stabilizing of polymer production reaching 9.58 % PHB of dcw and only 12.0 % loss of production ability. Further experiments increased PHB content with 19.9% PHB of dcw (3.85 g L-1) after 180 h of cultivation using rich medium. Use of another thiolase gene, the second thiolase from C. necator (bktB), which theoretically should induce production of PHBV copolymer, led to accumulation only 11.4% PHB of dcw after 139 h and no PHV fraction was detected.
Variation of the ratio between flask volume and amount of media influences the level of aeration. Importantly, decrease of aeration level significantly increased polymer synthesis. Additionally, PHB-V copolymer accumulation has been induced by use of different carbon source co-substrates. Use of rich media supplemented with ethanol allow the strain with thl thiolase to accumulate up to 42.9 % PHB of dcw without PHV fraction and with bktB thiolase to 30.5 % PHB of dcw. Nevertheless, despite of lower total amount of polymer, supplementation with 1-propanol allow both strains to accumulate PHB-V copolymer with 7.30 %mol and 22.5 %mol of PHV for thl and bktB strains, respectively.
Optimization based on genetic engineering further enhanced polymer production yield led to exceeding of 50 % PHB-V of dcw. For doubling the gene dosage, PHA synthesizing strains of A. adeninivorans were again transformed with Xplor2 vector containing PHA pathway genes. Resulting strains exhibited twice the level of enzymatic activities of thiolase and reductase compared with strains transformed once with expression vector. In a shaking flask experiment the strain transformed twice with vector containing bktB thiolase reached after 240 h 52.1% PHB-V of dcw (10.8 g L-1) with 12.3 %mol of PHV fraction which is the highest level found in yeast. As another genetic approach, a fusion strain has been created. Two different strains have been established and merged using protoplast fusion technique. Doubling of genetic material resulted in similar level of copolymer produced by Arxula as in former experiments (50.2% of dcw, 10.7 g L-1).
Culture conditions were optimized in controllable cultivation using fed-batch mode. Although optimal oxygen and pH level and continuous carbon source and nitrogen feeding were maintained, final polymer level in % of dry mass was around three times lower than for shaking flask experiment. Nevertheless, efficient growth of Arxula in fed-batch mode led to increase of total copolymer level in g L-1 (16.5 g L-1 compare to 10.8 g L-1 for shaking flasks) showing the feasibility of using Arxula strain for up-scaling production of copolymer.
Acetyl-CoA is a main precursor in synthesis of PHB-V copolymer and change of its pool was investigated. ATP citrate lyase is a cytosolic enzyme converting citrate into oxaloacetate and acetyl-CoA, supporting the biosynthesis of fatty acids. Two genes encoding Acl subunits from Aspergillus nidulans (AnAcl1 and AnAcl2) were again cloned into Xplor2 vector and transformed into A. adeninivorans PHA producing strain. Despite of higher enzymatic activity of AnAclp, accumulation of polymer was around three times higher for control without expression of lyase genes. Expectedly, the strain expressing AnAcl1/2 genes accumulated larger amount of each stearic, palmitic and oleic acid in both standard and fatty acid inducing conditions (lower nitrogen level). Thus, overexpression of AnAcl1/2 genes in A. adeninevorans cells may improve biosynthesis of fatty acids but is ineffective for PHB polymer accumulation.
The aim of the project was use of starch-based media, manufactured as by-products, for polymer production. Genetically engineered Arxula strains were cultivated using these media instead of glucose-based media. Although yeast cells were both able to secrete (R)-3-HB and to accumulate PHB, the yield was lower than for previous media. Additionally, only trace of PHV was found at the end of cultivation time when 1-propanol was supplemented. Obtained results showed that use of cheaper media is a promising approach to decrease production costs but further optimization needs to be performed especially for extended scale of production.
Determination of produced copolymer has been done based on microscopic analysis and studies of physical and chemical properties. Results revealed that Arxula accumulated PHA polymer in cytosolic granules with a similar size range compared to the ones produced by bacteria. The physicochemical study showed that produced polymer exhibited slightly different properties in comparison to bacterial polymer with similar content of PHV, i.e. very-low molecular mass, higher melting and glass transition temperature.
All above results showed that A. adeninivorans is a promising host for PHB-V production. Expression of phasin greatly increased production and stability of polymer, which led to an accumulation level never found before in yeast. Further optimization in higher production scale using cheap starch-based media may establish Arxula strain as a valuable tool for industrial production of PHB-V copolymer.
Bacteria are exposed to oxidative stress as an unavoidable consequence of their aerobic lifestyle. Reactive oxygen species (ROS) are generated in the stepwise one-electron reduction of molecular oxygen during the respiration. Pathogens encounter ROS during the oxidative burst of macrophages as part of the host immune defense. Besides ROS, bacteria also have to cope with reactive chlorine, electrophilic and nitrogen species (RCS, RES, RNS). To cope with these reactive species, bacteria have evolved different defense and repair mechanisms. To maintain the reduced state of the cytoplasm, they utilize low molecular weight (LMW) thiols. LMW thiols are small thiol-containing compounds that can undergo post-translational thiolmodifications with protein thiols, termed as S-thiolations. S-thiolations function as major redox regulatory and thiol-protection mechanism under oxidative stress conditions. In eukaryotes and Gram-negative bacteria, the tripeptide glutathione (GSH) functions as major LMW thiol, which is present in millimolar concentrations. The Actinomycetes, such as Mycobacterium and Corynebacterium species do not produce GSH and utilize instead mycothiol (MSH) as their alternative LMW thiol. In Firmicutes, including Bacillus and Staphylococcus species, bacillithiol (BSH) functions as the major LMW thiol. LMW thiols protect protein thiols against the irreversible overoxidation of cystein residues to sulfinic and sulfonic acids. In addition, LMW thiols contribute to the virulence and survival of pathogens, function in metal homeostasis and serve as enzyme cofactors for detoxification of xenobiotics and antibiotics. In this doctoral thesis, we aimed to investigate the roles of MSH and BSH in redox regulation of main metabolic enzymes under oxidative stress in the pathogens Corynebacterium diphtheriae and Staphylococcus aureus. Previous redox proteomics studies identified the glyceraldehyde-3-phosphate dehydrogenase GapDH and the aldehyde dehydrogenase AldA as S-thiolated in S. aureus and C. diphtheriae. Thus, we aimed to study the redox regulation of the metabolic enzyme GapDH in C. diphtheriae in response to NaOCl and H2O2 stress by S-mycothiolation, which is described in chapter 1. Moreover, we studied the involvement of the mycoredoxin-1 (Mrx1) and thioredoxin (Trx) pathways in reactivation of S-mycothiolated GapDH in vitro. Using shotgun proteomics, 26 S-mycothiolated proteins were identified under NaOCl stress in C. diphtheriae. These are involved in energy metabolism (Ndh, GlpD) and in the biosynthesis of amino acids (ThrA, LeuB), purines (PurA) and cell wall metabolites (GlmS). The glycolytic GapDH was identified as conserved target for S-thiolation across Gram-positive bacteria. GapDH was the most abundant protein, contributing with 0.75 % to the total cystein proteome. Moreover, GapDH is a conserved target for redox regulation and S-glutathionylation in response to oxidative stress in several prokaryotic and eukaryotic organisms. Treatment of GapDH with NaOCl and H2O2 in the absence of MSH resulted in irreversible enzyme inactivation due to overoxidation. Pretreatment of GapDH with MSH prior to H2O2 or NaOCl exposure resulted in reversible inactivation due to S-mycothiolation of the active site Cys153. Since S-mycothiolation is faster compared to overoxidation, S-mycothiolation efficiently protects the GapDH active site against overoxidation. The activity of S-mycothiolated GapDH could be restored by both, the Mrx1 and Trx pathway in vitro. Interestingly, the recovery of Smycothiolated GapDH by Mrx1 was faster compared to its reduction by the Trx pathway. In previous studies, the reactivation of S-mycothiolated Mpx and MrsA by the mycoredoxin pathway occurred also faster compared to the Trx pathway, which is consistent with our results. We were further interested to analyze the redox regulation of the glyceraldehyde-3phosphate dehydrogenase Gap of S. aureus under NaOCl and H2O2 stress, which is described in chapter 2. Using the quantitative redox proteomic approach OxICAT, 58 NaOCl-sensitive cystein residues with >10% thiol oxidation under NaOCl stress were identified. Gap and AldA showed the highest oxidation increase of 29% under NaOCl stress at their active site cystein residues. Using shotgun proteomics, five S-bacillithiolated proteins were identified, including Gap, AldA, GuaB, RpmJ and PpaC. Gap contributed with 4 % as most abundant cystein protein to the total cystein proteome. Our activity assays demonstrated that Gap of S. aureus is highly sensitive to overoxidation by H2O2 and NaOCl in vitro in the absence of BSH. The active site Cys151 of Gap was oxidized to the BSH mixed disulfide under H2O2 and NaOCl stress in the presence of BSH in vitro, which resulted in the reversible Gap inactivation. Moreover, inactivation of Gap by NaOCl and H2O2 due to S-bacillithiolation was faster compared to overoxidation, indicating that S-bacillithiolation protects the Gap active site against overoxidation in vitro. We further showed that the bacilliredoxin Brx catalyzes the reduction of S-bacillithiolated Gap in vitro. Molecular docking of BSH into the Gap active site revealed that S-bacillithiolation does not require major structural changes. Apart from Gap, the aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus previously. Thus, the expression, function, redox regulation and structural changes of AldA were analysed under NaOCl and aldehyde stress in S. aureus as summarized in chapter 3. AldA was S-bacillithiolated in the presence of H2O2 and BSH as demonstrated in BSH-specific Western blots in vitro. The expression of aldA was previously shown to be regulated by the alternative sigma factor SigmaB in S. aureus. Transcription of aldA was strongly increased in a SigmaB-independent manner under formaldehyde, NaOCl and diamide stress in S. aureus. Using an aldA deletion mutant, we demonstrated that aldA is required for growth and survival under NaOCl stress in S. aureus. The purified AldA enzyme was shown to catalyze the oxidation of various aldehyde substrates, including formaldehyde, methylglyoxal, glycolaldehyde and acetaldehyde in vitro. In addition, the function of the conserved Cys279 for AldA activity was investigated in vivo and in vitro. The purified AldAC279S mutant was shown to be inactive for aldehyde oxidation in vitro. Moreover, the aldAC279S mutant was very sensitive under NaOCl stress in vivo, and this phenotype could be reversed using the aldA complemented strain. These experiments demonstrate the function of Cys279 for AldA activity both in vitro and in vivo. AldA activity assays showed that AldA is sensitive to overoxidation and irreversible inactivation by H2O2 alone in vitro. In the presence of BSH, AldA is protected against overoxidation by reversible Sbacillithiolation in vitro. Molecular docking and molecular dynamics simulations revealed that BSH occupies two different positions in the Cys279 active site, which depend on the NAD+ cofactor. In the apoenzyme, BSH forms the disulfide with Cys279 in the “resting” state position, while Cys279 is S-bacillithiolated in the “attacking” state position in the holoenzyme in the presence of the NAD+ cofactor.
The highly oncogenic alphaherpesvirus Marek’s disease virus (MDV) causes immense economic losses in the poultry industry. The main targets of in vivo MDV infection are primary B and T lymphocytes. The cytolytic infection of B cells leads to depletion of lymphoid cells results in severe immunosuppression. Infected B cells recruit and activate T cells. The close interaction between B cells and T cells enables efficient intercellular transfer of MDV. During infection of T cells, the virus enters a latent state. Infection of T cells can lead to transformation of these cells and formation of lymphoma, which manifest in various visceral organs. This study aimed at the characterization of the proteomes of MDV-infected lymphocytes during the lytic and latent phases of infection.
Previous in vitro studies concerning the MDV pathogenesis and host-virus interactions have been mainly conducted with primary fibroblasts or kidney cells, due to the short lifespan of primary lymphocytes in cell culture. Recently, a cultivation system has been established that extents the lifespan of primary lymphocytes through the addition of cytokines to the growth medium. This allowed the infection of B cells in vitro and to conduct quantitative proteomic analysis of primary lymphocytes. Infection with GFP labelled virus recombinants allowed the isolation of infected cells by FACS for the proteome analysis of MDV infected B lymphocytes. An efficient quantitative proteomic workflow was developed, which consisted of a filter-aided (FASP) digest of the extracted proteins, followed by differential dimethyl chemical labeling of the peptides for quantitative evaluation prior to LC-MALDI TOF/TOF mass spectrometry. Only few alterations of the protein and transcript expression profiles were observed after infection of primary B cells with the very virulent RB-1B and the live-attenuated vaccine strain CVI988/Rispens. Relevant changes in relative protein levels were found for only twelve and six interesting host proteins after RB1B and CVI988 infection, respectively. However, the regulations were confirmed by inspection of the spectra from all experiments. The identified candidates play a role in immune response, translation and inflammatory response.
To confirm the potential infection markers, RNA-seq analysis of three biological replicates of each RB-1B -, CVI988- and mock-infected B cells was performed. Eighty expressed MDV transcripts could be identified, which were associated with lytic infection. The same MDV proteins were identified after infection with RB-1B or CVI988. However, transcriptome and proteome analysis of MDV-infected primary B cells showed only poor correlation. This indicates that the changes in protein expression profiles are mostly due to posttranscriptional events. Infection marker candidates were identified by the RNA-seq analysis, for which the gene expression was altered by MDV infection. Although almost 12,000 transcripts were identified, only few transcript levels changed markedly after MDV infection. The biological processes immune response, apoptotic process, signal transduction, cell migration and response to virus were enriched after MDV infection. The RNA-seq results confirm the observation that alterations of protein levels early after MDV infection are rare.
Most notably, MDV induces transformation of lymphocytes leading to malignant T-cell lymphomas in visceral organs with mortalities of up to 100 %. While several factors involved in MDV tumorigenesis have been identified, the transformation process is not fully understood. Therefore, we set out to fill this knowledge gap using proteome analysis of transformed T-cells ex vivo. In addition, the role of the viral telomerase RNA during transformation was assessed by comparison of tumors that had formed after infection with WT-virus or a telomerase RNA negative mutant. A major obstacle for tumor proteome analyses is the preparation of sufficient amounts of homogenous tumor tissue, as tumors appear with a dispersed morphology in the affected organs. The quantitation of cell types within the tumors indicated varying portions of hepatocytes, connective tissue, and CD3+ lymphocytes even with the same virus strain in different animals. However, the ∆vTR-induced tumors contained lower levels of hepatocytes and higher levels of CD3+ lymphocytes compared to WT tumors in all tested tumor samples. Thus, ∆vTR tumors were chosen for determination of differences in protein expression profiles of tumors and naïve T cells for their lower content of liver cells. We developed a workflow for the proteome analysis of T cell tumors from livers of MDV-infected chickens. Samples included laser capture micro-dissected tissue cuts from tumors and surrounding healthy liver tissue as well as naïve T-cells prepared from thymus. To enable quantitative proteome analysis, samples were digested using the FASP protocol and peptides were isotope-coded by differential dimethyl labeling. To improve proteome analysis peptides were fractionated by preparative isoelectric focusing prior to nano-HPLC MALDI/TOF-TOF mass- spectrometric analysis.
Proteomic analyses of LCM dissected ΔvTR tumor compared to naïve T cells, the main targets of transformation, identified nineteen potential transformation markers but again only minor changes in relative levels were observed. Several of the identified markers could also be verified by RT-qPCR on transcript level. The identified transformation candidates were associated with nucleosome assembly, regulation of transcription, inflammatory response, immune response and oxidation-reduction process.
However, further functional analyses are necessary to fully elucidate the role of the identified markers during MDV infection and transformation.
Streptococcus pneumoniae (pneumococci) and Staphylococcus aureus (S. aureus) are human-specific commensals of the upper respiratory tract. Every individual is asymptomatically colonized with both bacteria at least once in their life-time. The opportunistic pathogens can affect further organs and invade into deeper tissue. The occupation of normally sterile niches of the human body with the bacteria can lead to local infections such as sinusitis, otitis media and abscesses, or to life-threatening diseases like pneumonia, meningitis or sepsis. A strong interaction between the bacterium and the respiratory epithelial cells is a prerequisite for a successful colonization. This interaction is ensured by bacterial surface proteins, so called adhesins. The binding of the adhesins to the epithelial lineage occurs predominantly indirectly via components of the extracellular matrix (ECM), but also directly to cellular receptors. Pneumococci and S. aureus bind to various ECM glycoproteins, amongst others: fibronectin, fibrinogen, vitronectin, and collagen. Also binding of both pathogens to human thrombospondin-1 has been described. Thrombospondin-1 is mainly stored in the α-granula of thrombocytes (platelets) and released into the circulation upon activation. However, thrombospondin-1 is also produced and secreted by other cell types like endothelial cells, macrophages, and fibroblasts, which gets subsequently incorporated as component into the ECM. So far, no thrombosponin-1-binding adhesins of pneumococci were identified. PspC, Hic, and PavB are important surface-localized virulence factors, which were shown to interact with human ECM and plasma proteins. PspC and Hic bind to vitronectin and factor H, which inhibits the complement cascade of the human immune system. PavB interacts with fibronectin and plasminogen, and a pavB-deficient mutant of S. pneumoniae showed diminished capacity in colonization in a mouse model. Among the surface proteins of S. aureus, only Eap was identified as thrombospondin-1-binding adhesin. Beyond colonization, pneumococci and S. aureus can enter the blood circulation, interact with platelets, and cause their activation. The aggregation of platelets, especially initiated by S. aureus, plays an important role in the clinic, because most of the septic patients develop thrombocytopenia. Surface localized factors of
S. pneumoniae triggering platelet activation are unknown to date. In contrast, few proteins of S. aureus with potential to activate platelets, including Eap, were identified previously.
This study identified the surface proteins PavB, PspC, and Hic of S. pneumoniae as specific ligands of the human thrombospondin-1. Flow cytometric, surface plasmon resonance spectroscopic and immunological analyses revealed interactions between the pneumococcal proteins and soluble as well as immobilized thrombospondin-1. The use of specific pneumococcal deletion mutants verified the importance of the three virulence factors as binding partners of soluble thrombospondin-1. The results suggest that pneumococci are capable of acquiring soluble thrombospondin-1 from blood as well as utilizing immobilized glycoprotein of the ECM as substrate for adhesion. Furthermore, the thrombospondin-1-binding domain within the pneumococcal proteins was analyzed by use of recombinant fragments of PavB, PspC, and Hic. The binding capacity of thrombospondin-1 increased proportionally with the amount of repetitive sequences in PavB and PspC, and the length of the α-helical region within the Hic molecule. The binding behavior of thrombospondin-1 towards PavB and PspC is comparable with that of the ECM proteins vitronectin and fibronectin, but is unique towards Hic.
The localization of the binding domain of the adhesins within the thrompospondin-1 molecule occurred via use of glycosaminoglycans as competitive inhibitors for the interaction. The results suggest that the pneumococcal proteins Hic and PspC target the identical binding region within thrombospondin-1, which differs from the binding domain for PavB. However, all three virulence factors seem to bind in the N-terminal part of thrombospondin-1.
Two-dimensional gel electrophoresis, thrombospondin-1 overlay assay and subsequent mass spectrometric analysis identified AtlA of S. aureus as a surface localized interaction partner of human thrombospondin-1. Moreover, a vitronectin binding activity for AtlA was determined. Immunological and surface plasmon resonance binding studies with recombinant AtlA fragments revealed that interactions with both matrix proteins is mediated via the C-terminal located repeats R1R2 of the AtlA amidase domain. Binding of thrombospondin-1 and vitronectin occurred not simultaneously, due to a competitive inhibition.
The second part of the study focused on the activation of human platelets by recombinant pneumococcal and staphylococcal proteins. In total, 28 proteins of S. pneumoniae and 52 proteins of S. aureus were incubated with human platelets. The activation of the cells was detected by flow cytometry using the activation markers P-selectin and the dimerization of the integrin αIIbβIII. The proteins CbpL, PsaA, PavA, and SP_0899 of S. pneumoniae induced platelet activation, however, the detailed mechanism has to be deciphered in further studies. Furthermore, the secreted proteins CHIPS, FLIPr, and AtlA of S. aureus were discovered as inductors for the activation of platelets. In addition, the domains of AtlA and Eap, crucial for platelet activation, were narrowed down. Interestingly, CHIPS, FLIPr, and Eap were described as inhibitors of neutrophil recruitment. Platelets are recently recognized as immune cells, due to the expression of immune receptors. The data obtained in this study highlight a comprehensive spectrum of effects of the S. aureus proteins towards different type of immune cells. Besides the activation of platelets in suspension buffer and plasma, the aggregation of platelets in whole blood was triggered by the proteins CHIPS, AtlA, and Eap. These results suggest a contribution of the proteins during the S. aureus-induced infectious endocarditis. Secretion of the platelet activating virulence factors, which were identified within this study, might represent a pathogenic strategy during S. aureus infection in which a direct contact between S. aureus and platelets is not required or even avoided.
In conclusion, PavB, PspC, and Hic of S. pneumoniae and AtlA of S. aureus were identified as interaction partners of human thrombospondin-1. Furthermore, CHIPS, FLIPr, AtlA, and Eap were characterized as platelet activators. This study provides candidates for the development of protein-based vaccines, to prevent bacterial colonization and to neutralize secreted pathogenic factors.
Natürliche Hormone und Substanzen mit einer hormonellen Wirkung werden als organischen Spurenstoffen oder Mikroschadstoffe bezeichnet und werden über verschiedene Quellen in die Umwelt eingetragen. Dies führt insbesondere bei aquatischen Lebewesen zu Veränderungen im endokrinen System. Um die Belastung der Gewässer mit hormonell aktiven Substanzen zu verringern und einen guten chemischen und ökologischen Status nach europäischer Wasserrahmenrichtlinie zu erreichen, wird eine Reduktion des Eintrags hormonell aktiver Substanzen angestrebt. Die meisten Abwässer werden in Kläranlagen gesammelt, die somit Punktquellen für den Eintrag von hormonell aktiven Substanzen in die Umwelt darstellen. Zur Untersuchung neuer Methoden zur Abwasserreinigung sind zuverlässige und sensitive analytische Messtechniken notwendig. Da aktuelle instrumentelle Messmethoden nicht in der Lage sind hormonell aktive Substanzen im wirkungsrelevanten Konzentrationsbereich zu messen, wurden Hefezellenassays zur Detektion der östrogenen (A-YES) und androgenen (A-YAS) Aktivitäten für eine Anwendung in Oberflächengewässern und Abwässern evaluiert. Im Anschluss wurden diese Assays zur Beurteilung und Optimierung der Eliminationsleistung einer großtechnischen Ozonung auf einer kommunalen und einer Krankenhaus Kläranlage eingesetzt. Die untersuchten Abwassermatrices zeigten keine Effekte auf die Enzym Substrat Reaktion und die optische Dichte der A-YES Hefezellensuspension. Proben eines Oberflächengewässers sowie von Kläranlagen Zuläufen verursachten im A-YAS eine erhöhte optische Dichte der Zellsuspension im Vergleich zur Referenz. Eine verringerte optische Dichte der A-YAS Hefezellsuspension konnte in Extrakten von Zulaufproben bestimmt werden. Durch die Dotierung unterschiedlicher Konzentrationen der Referenzsubstanzen zu Oberflächengewässer- und Abwasserproben konnten Dosis Wirkungskurven mittels A-YES und A-YAS Assays abgebildet werden. Dabei konnte gezeigt werden, dass insbesondere in Kläranlagen-Zulaufproben sowohl eine östrogene als auch eine androgene Aktivität bereits in der undotierten Ausgangsprobe vorhanden war. Des Weiteren konnten inhibierende Effekte in den Proben detektiert werden, die auf antagonistische Substanzen hindeuten. Die Analyse von Kläranlagen Abläufen zeigte östrogene Aktivitäten zwischen 0,035 und 5,5 ng EEQ/L sowie androgene Aktivitäten zwischen < 0,31 und 6,1 ng DHTEQ/L. Während der großtechnischen Ozonung konnte die östrogene Aktivität in einer kommunalen sowie einer Krankenhaus Kläranlage um bis zu 97% bzw. 83% reduziert werden. Die Reduktion der androgenen Aktivität lag bei 80% und 66%. Für zwei Verfahren zur bedarfsabhängigen Steuerung der Ozonung basierend auf der östrogenen Aktivität und auf dem DOC Gehalt konnte die Machbarkeit gezeigt werden. Allerdings stellten sich beide Methoden zum jetzigen Zeitpunkt als nicht wirtschaftlich heraus. Antagonistische Aktivitäten konnten in einem Konzentrationsbereich von 330 - 2.700 µg OHTEQ/L (anti-östrogene Aktivität) und 550 - 730 µg FEQ/L (anti-androgene Aktivität) mittels anti A-YES und anti A-YAS detektiert werden. Während der einzelnen Reinigungsstufen konnte keine Reduktion der antagonistischen Aktivitäten nachgewiesen werden. Sowohl A-YES als auch A-YAS sind für die Analyse von Abwasserproben geeignet und ermöglichen so erstmals die Beurteilung von Verfahren zur Abwasserreinigung im wirkungsrelevanten Konzentrationsbereich.
Streptococcus pneumoniae (pneumococci), a human pathobiont, express and expose several proteinaceous colonization and virulence factors on its surface to facilitate on the one hand colonization of the upper respiratory tract and on the other hand pathogenesis in the host. In this study the interaction of two of such factors referred to as pneumococcal virulence factor A (PavA) and pneumococcal virulence factor B (PavB) and acting as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), was delineated with the two host matricellular proteins fibronectin (Fn) and vitronectin (Vn). Despite similarity in nomenclature, PavA and PavB represent two diverse pneumococcal proteins with respect to their structure and association with the pneumococcal surface. PavA is a non-classical surface protein (NCSP) with an ambiguous mode of secretion and anchorage while PavB is a characteristic MSCRAMM, anchored via sortase A to pneumococcal peptidoglycan. PavB has a signature of repetitive modules termed as streptococcal surface repeats (SSURE). Pneumococci preferentially interact with immobilized human Fn. In vitro cell culture adherence assays demonstrated that cell bound Fn facilitates the adherence of pneumococci to the host cells and this particular interaction is indifferent to host cell type and is species non-specific. Flow cytometry and immunoblot analyses further indicated the ability of pneumococci to interact with the soluble form of Fn in a dose-dependent but species non-specific manner. The molecular interaction of PavA and PavB (via its SSURE domains) with Fn was delineated further in detail via several direct protein-protein interaction approaches. Ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays demonstrated that PavA and PavB target at least 13 out of the 15 type III fibronectin domains located in the C-terminal part of Fn. Strikingly, both pneumococcal fibronectin-binding proteins (FnBPs) recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III epitopes cluster on the inner strands of both β-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1, FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB, respectively. Thus, analysis of interaction of pneumococcal FnBPs PavA and PavB revealed a probable conserved and/or common pattern of molecular interaction with human Fn. In addition to Fn, pneumococcal PavB interacts with other host matricellular proteins such as human plasminogen (Plg) and human thrombospondin-1 (hTSP-1). Pneumococcal proteins such as PspC and PspC-like Hic have earlier been demonstrated to interact with hTSP-1 as well as human Vn, thereby depicting a redundant function as MSCRAMMs. In this study the role of PavB as a pneumococcal vitronectin binding protein (VnBP) was assessed. Flow cytometric analysis suggested PavB as VnBP, because strains deficient for PavB exhibited a significantly decreased ability to acquire vitronectin compared to wild-type pneumococci. When using a double knockout, deficient in expression of PavB and the VnBP PspC, the pneumococcal interaction with vitronectin was completely abolished. The direct protein-protein interaction assays such as far western ligand overlay, ELISA, and surface plasmon resonance indicated the interaction of SSURE domains with both soluble and immobilized Vn. However, the binding activity depends on the number of SSURE domains with five SSURE showing the highest binding activity to Vn. The interaction of PavB with Vn was charge dependent and heparin sensitive as analyzed by ELISA. The importance of the heparin binding domains of Vn in this interaction was further analyzed via direct protein-protein interaction approaches. Binding studies (far western ligand overlay, ELISA, and surface plasmon resonance) with truncated recombinant Vn fragments indicated that PavB targets the C-terminal heparin-binding domain (HBD3) of vitronectin, a characteristic shared with PspC, hence, suggesting a conserved molecular interaction of pneumococci with Vn. In addition to its function as an MSCRAMM, PavB has the capability to interact directly with host epithelial cells via an unknown cellular receptor. Thus, this study aimed to identify cellular receptor(s) for PavB. In vitro cell culture adherence and invasion assays confirmed that pneumococcal PavB is involved in promoting pneumococcal adherence to respiratory epithelial cells without employing any molecular bridge. The direct interaction between PavB and host epithelial cells was further confirmed via direct binding assays when using Cy5-labeled PavB and flow cytometric analysis. Strikingly, exogenously added human vitronectin competitively inhibited binding of PavB to respiratory epithelial cells. This observation led us to hypothesize that the major vitronectin receptor αvβ3 integrin acts as a potential receptor for PavB. This hypothesis was supported by functional blocking assays with monoclonal antibodies recognizing specific integrin subunits. The results revealed reduced binding of PavB in the presence of bound antibodies recognizing αv integrin indicating that PavB employs αvβ3 integrin as its direct receptor on eukaryotic cells. This was further confirmed via a direct binding assay of PavB to mouse embryonic fibroblasts (MEFs) where cells lacking αvβ3 demonstrated a marked decrease in binding to PavB. Although functional blocking assay and direct binding assay with MEFs supported the role of αvβ3 integrin as a direct adhesin for PavB, RNA interference of αv integrin in epithelial cells did not impair the binding of PavB in αv-knocked down cells in comparison to non-transfected cells. Finally, surface plasmon resonance (SPR) analysis indicated the direct interaction between pneumococcal PavB and recombinant αvβ3 integrin. In this study we report for the first time the interaction of a Gram-positive extracellular pathogen, namely Streptococcus pneumoniae, with one of the host ICAMs, namely the αvβ3 integrin. In conclusion, the present study analysed some of the aspects of molecular interaction of pneumococcal MSCRAMMs PavA and PavB with hFn and hVn. The hot spots of interaction on C-terminal FnIII repeats were delineated for PavA and PavB. HBD3 was revealed to be pivotal for PavB-Vn interaction. In addition the redundant role of pneumococcal PavB as an MSCRAMM was demonstrated. Furthermore this study successfully identifies a direct receptor for pneumococcal PavB, namely αvβ3 integrin. The mechanism and biological rationale of this newly identified interaction is a matter of debate and awaits further scientific analyses.
Das Genus Pestivirus gehört zur Familie der Flaviviridae und enthält eine Reihe von tierpathogenen Erregern, welche (fast) ausschließlich Paarhufer befallen. Das bei Pestiviren vorkommende Strukturprotein ERNS ist einzigartig in der Familie Flaviviridae, es finden sich keine homologen Proteine in den anderen Genera dieser Familie. ERNS ist ein sehr ungewöhnliches Protein, da es für ein virales Strukturprotein verschiedene untypische Eigenschaften aufweist. Neben einer intrinsischen RNase-Aktivität findet sich am C Terminus eine sehr ungewöhnliche Signalpeptidase-Spaltstelle. Während die RNase Aktivität einen wichtigen Virulenzfaktor darstellt, sorgt die ungewöhnliche Spaltstelle mutmaßlich für die verlangsamte Prozessierung des ERNS-E1-Vorläuferproteins. Inwieweit die verlangsamte Spaltung des Vorläuferproteins für das Virus wichtig sein könnte, ist bis dato noch ungeklärt. Auch ist die Ausbildung von Dimeren wichtig für die Virulenz von ERNS. Darüber hinaus erfolgt eine partielle Sekretion von ERNS in den extrazellulären Raum, während ein Großteil in der Zelle verbleibt. Zusätzlich verfügt ERNS über eine untypische Membranverankerung, die durch eine lange, C-terminale amphipathische Helix vermittelt wird. Innerhalb dieser amphipathischen Helix findet sich eine Reihe geladener Aminosäuren, deren Lokalisation und Anordnung zu zwei spiegelsymmetrisch komplementären Gruppen bei Pestiviren konserviert ist. Es stellte sich die Frage, welche biologische Relevanz dieses Muster an geladenen Aminosäuren haben könnte. Ausgehend von der vorgeschlagenen Ausbildung eines „Charge Zippers“ – durch Rückfaltung und Ausbildung von Salzbrücken zwischen den komplementären Ladungen –, wurden mittels transienten Expressionsexperimenten die sechs hoch konservierten Ladungen im „Inneren“ des möglichen „Reißverschlusses“ untersucht, und es zeigte sich, dass der postulierte Charge-Zipper-Mechanismus bei ERNS vermutlich keine Rolle spielt. Für einige der betrachteten Aminosäuren konnten Hinweise erhalten werden, dass sie eine Rolle bei der Prozessierung, der Retention und bei der Dimerisierung von ERNS spielen. Vor allem ein Austausch der Ladung an der Position 194 im ERNS zeigte einen signifikanten Einfluss auf die Prozessierung und Retention von ERNS. Auch bei der Dimerisierung stach diese Position hervor, da entgegen anderer Mutationen ein Austausch hier zu einer vermehrten Dimerbildung führte. Weiterführend wurden diese Mutationen in rekombinante Viren eingeführt, und es zeigte sich, dass vor allem die spezifischen Ladungen an den Positionen 184 und 191 im ERNS wichtig für die effiziente Virusvermehrung sind. Ladungsaustausche an diesen Positionen sorgten für nicht lebensfähige Virusmutanten, während Alaninsubstitutionen im Lauf von Passagen zur ursprünglichen Ladung revertierten. Diese Ergebnisse zeigen die elementare Bedeutung der Ladungen für die Generierung von infektiösen Viren. Die molekularen Mechanismen, in denen diese Reste von Bedeutung sind, müssen in weiteren Arbeiten noch aufgeklärt werden.
Alcohol dehydrogenases as biocatalysts for the production of enantiomerically pure chiral alcohols
(2016)
Summary Enantiomerically pure chiral alcohols are key compounds in the production of certain chemicals including pharmaceuticals. Chemical synthesis allows to obtain maximal yield of 50% for one enantiomer ( >50% yield is achievable with chiral catalysts used in chemical synthesis), whereas biosynthesis leads to nearly 100% yield. Hence, expensive and time consuming resolution of racemic mixture can be avoided. Alcohol dehydrogenases are the most popular enzymes used in the chiral alcohols synthesis due to high activity with appropriate aldehydes or ketones. ADHs require a cofactor which has to be regenerated after the conversion of aldehyde/ketone to the respective alcohol. Thereby, different regeneration methods were used in the practical work to compare and choose the better one. R. erythropolis and C. hydrogenoformans alcohol dehydrogenases were chosen based on the literature screening. Each gene was cloned into Xplor2 vector and pFPMT vector. Xplor2 vector was used for the transformation of A. adeninivorans and pFPMT vector was used for the transformation of H. polymorpha. Chemically synthesized alcohol dehydrogenase sequences from R. erythropolis (ReADH) and C. hydrogenoformans (ChADH) were cloned between TEF1 promoter and PHO5 terminator which are components of Xplor2 vector or between FMD promoter and MOX terminator which are genetic elements of pFPMT vector. Moreover, ChADH and ReADH sequences with His-tag encoding sequence at the 5’ or 3’ end were constructed and the most active form of the protein was selected for further studies. ReADH-6H was used for the synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate whereas ChADH-6H was used for the production of ethyl (R)-mandelate. ReADH-6H synthesized in A. adeninivorans and H. polymorpha was fully biochemically characterized. The enzymes from the two yeast species showed some differences in their pH and temperature optima, thermostability and activity levels. A-ReADH (A. adeninivorans) and H-ReADH (H. polymorpha) were highly active with the same substrates which were: acetophenone, 4-hydroxy-3-butanone and ethyl 4-chloroacetoacetate for reduction reaction along with 1-phenylethanol and 1,6-hexanediol for oxidation reaction. Recombinant A-ReADH-6H and H-ReADH-6H were synthesized in A. adeninivorans and H. polymorpha, respectively. Both enzymes were used for the synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate with the use of substrate-coupled cofactor regeneration system. The enantiopurity of the products was >99%. Moreover, A. adeninivorans whole cell catalyst was also used for the synthesis of both chiral alcohols. BmGDH (Bacillus megaterium glucose dehydrogenase) was co-expressed with ReADH-6H for NADH cofactor regeneration. Comparison between isolated enzymes and permeabilized whole cell catalysts indicate that cell biocatalysts are more suitable for the production of 1-(S)-phenylethanol with 92% of acetophenone being converted in 60 min. However, cells did not show any significant advantage over isolated enzymes in the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate although the velocity of the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate was slightly improved using whole-cell catalysts, giving an 80% substrate conversion in 120 min. Recombinant C. hydrogenoformans alcohol dehydrogenase was synthesized in A. adeninivorans and biochemically characterized. Enzyme showed high activity only with one substrate, ethyl benzoylformate. The A. adeninivorans and H. polymorpha cell catalysts synthesizing ChADH and BmGDH (Bacillus megaterium glucose dehydrogenase) were constructed and used in the synthesis of ethyl (R)-mandelate (reduction product of ethyl benzoylformate) with the enantiopurity of the reaction product being >98%. H. polymorpha catalysts were more effective in the synthesis than A. adeninivorans cells. The first were able to convert 93% of ethyl benzoylformate within 180 min and the latter were converting 94% of the substrate within 360 min. Re-use of non-immobilized cells and catalysts entrapped in Lentikat® was performed and the improvement of the stability of immobilized catalysts was reported. Space time yield of 3.07 mmol l-1 h-1 and 6.07 mmol l-1 h-1 was achieved with A. adeninivorans and H. polymorpha cell catalysts, respectively. Alcohol dehydrogenase 1 from A. adeninivorans was analyzed concerning the synthesis of enantiomerically pure chiral alcohols. The enzyme did not synthesize industrially attractive products. However, based on biochemical characterization enzyme plays a role in the synthesis of 1-butanol or ethanol and thereby it is of biotechnological interest.
Die Hefe Saccharomyces cerevisiae reagiert auf die sich ständig ändernden Umweltbedingungen durch eine präzise Regulation der Genexpression. Möglich wird dies durch ein komplexes Netzwerk aus spezifischen Regulatoren und pleiotropen Faktoren. Aktivatorproteine binden an Aktivierungssequenzen (UAS-Elemente) in ihren Zielpromotoren und rekrutieren basale Transkriptionsfaktoren sowie Coaktiva¬toren. Dadurch erhöhen sich Wahrscheinlichkeit und Häufigkeit der Transkriptions¬initiation und die DNA im Promotorbereich wird durch die Aktivität von Komplexen der Chromatinremodellierung und -modifizierung für die Transkriptionsmaschinerie zugänglich gemacht. Dagegen binden spezifische Repressor¬proteine an ihre Regula¬tionssequenzen (URS-Elemente) oder an Aktivatorproteine, inhibieren deren Wirkung oder rekrutieren Histondeacetylase-Komplexe wie den Sin3-Corepressor, die eine Verdichtung des Chromatins bewirken. Der Sin3-Corepressorkomplex ist an einer Vielzahl von Regulationsprozessen beteiligt. In Hefe existieren zwei Sin3-Varianten, die als Rpd3L bzw. Rpd3S bezeichnet werden und sich in ihrer Zusammensetzung unterscheiden. Neben Sin3 als zentralem Gerüst¬protein in beiden Komplexen sind im Rpd3L strukturelle Untereinheiten wie Sds3, Sap30 und Pho23 sowie die Histondeacetylase (HDAC) Rpd3 als enzymatische Komponenten enthal¬ten. Durch Funktionsanalysen von Mutanten einzelner Unterein¬heiten wurde festge¬stellt, dass zusätzlich zu Rpd3 weitere HDACs an der Repression ICRE-abhängiger Gene der Phospholipid-biosynthese Gene beteiligt sind. Interaktionsstudien zeigten, dass auch die HDACs Hda1 und Hos1 an Sin3 binden. Die Bindung erfolgt über drei sogenannte HDAC-Interaktionsdomänen (HID1-3), wobei Hda1 und Hos1 an HID2 und HID3 binden, Rpd3 dagegen an HID1 und HID3. In dieser Arbeit konnte gezeigt werden, dass die HDACs direkt an ihre jeweiligen HIDs binden. Außerdem inter¬agieren Hda1 und Hos1 auch in vivo mit Sin3. Die HID1 wurde auf die Aminosäuren 801-950 verkürzt und es wurde nachge¬wiesen, dass eine funktionsfähige katalyti¬sche Domäne von Rpd3 nicht für die Wechselwirkung mit Sin3 notwendig ist. Außerdem wurden die Interaktionsdomänen von Sds3 und Sin3 kartiert. Die erhaltenen Befunde ergänzen die Daten zu Protein-Protein-Inter¬aktionen im Sin3-Corepressorkomplex und komplettieren funktionelle Aspekte der HDAC-Rekrutierung. Eine weitere Zielstellung dieser Arbeit war die Erstellung eines Interaktionsnetzwerks zwischen spezifischen Aktivatoren und allgemeinen Faktoren der Transkription. Eukaryotische Aktivatorproteine sind modular aufgebaut und besitzen voneinander separierbare Funktionsdomänen. Die Erkennung und Bindung von UAS-Elementen in den Zielpromotoren erfolgt über die DNA-Bindedomäne (DBD), während Tran¬skriptions¬¬aktivierungs¬domänen (TADs) basale Transkriptionsfaktoren und Co¬aktiva¬toren rekrutieren und somit die aktivierende Wirkung vermitteln. Im Gegensatz zu den DBDs folgen TADs meist keinen durch Sequenzanalysen vorhersagbaren Strukturmotiven und müssen manuell eingegrenzt werden. Für die Kartierung funktioneller TADs wurden Längenvarianten von über 30 Aktiva-toren aus verschiedenen Familien DNA-bindender Proteine an die Gal4DBD fusioniert und auf ihre Fähigkeit überprüft, ein UASGAL-abhängiges Reportergen zu aktivieren. Dabei konnten 15 neue TADs eingegrenzt werden. Weiterhin wurden die bisher nicht charakterisierten Zinkcluster¬proteine Yjl206c, Yer184c, Yll054c und Ylr278c als Aktivatoren bestätigt. Dadurch stand eine Samm¬lung aus 20 bekannten und neukartierten TADs zur Verfügung, die nach Konstruktion von GST-Fusionen für in vitro-Interaktionsexperimente mit Unter¬einheiten des Mediators, des TFIID- und des SWI/SNF-Komplexes eingesetzt wurden. Es konnten direkte Wechselwirkungen von Aktivatoren (u. a. Aft2, Aro80, Mac1 und Zap1) mit den TFIID-Komponenten TBP, Taf1, Taf4 und Taf5 detektiert werden. Die Bindung an Taf1 erfolgte im Bereich von aa 1-250, der zwei Aktivator¬interaktions-domänen (AID) enthält und in vorangegangenen Experimenten auch mit Ino2 und Adr1 interagierte. Die Rap1-Bindedomäne (RBD) von Taf4 (aa 253-344) interagierte auch mit Mac1, Aft2 und Ino2. Daher wurde dieser Bereich als allgemeine AID klassifiziert. Für die Aktivatorinteraktion essentielle Aminosäuren konnten allerdings nicht identi¬fiziert werden. 17 von 20 TADs interagierten direkt mit der Mediator-Untereinheit Med15, während für Med17 10 Kontakte zu Aktivatoren detektiert wurden, was die Relevanz des Mediators für die Aktivatorfunktion unterstreicht. Die katalytische Untereinheit des SWI/SNF-Komplexes Swi2 zeigte ähnlich viele TAD-Interaktionen wie Med15. Der N-terminale Bereich von Swi2 (aa 1-450) stellte sich als ausreichend für die Bindung der Aktivatoren heraus und enthält demnach eine oder mehrere AIDs. Damit konnte das Interaktionsnetzwerk zwischen Aktivatoren und allgemeinen transkriptionalen Cofaktoren substantiell erweitert werden.
In der Hefe S. cerevisiae erfolgt die Transkriptionsregulation der Strukturgene der Phospholipid-Biosynthese in Abhängigkeit der intrazellulären Konzentration der beiden Phospholipid¬vorstufen Inositol und Cholin (IC). Bei IC-Mangel kommt es zu einer Akkumulation des Signalmoleküls Phosphatidsäure, wodurch der Repressor Opi1 extranukleär am endoplasmatischen Retikulum (ER) verankert wird. Dadurch kann der heterodimere Aktivator Ino2/Ino4 an eine spezifische „upstream activation site” (UAS) in der Promotorregion, die als ICRE-Motiv („inositol/choline-responsive element“) bezeichnet wird, binden und die Initiation der Transkription vermitteln. Die aktivierende Wirkung geht dabei von zwei Transkriptions¬aktivierungsdomänen (TAD) im N-Terminus von Ino2 aus. Da bisher unbekannt war, wie die Ino2-vermittelte Genaktivierung erfolgt, bestand das Ziel dieser Arbeit in der Identifizierung der Coaktivatoren, die direkt an die TADs von Ino2 binden. Ferner sollten die für die Transkriptionsaktivierung wichtigen Wechselwirkungen innerhalb der Coaktivatoren präzise kartiert werden. Es konnte hier mit Hilfe der affinitätschromatographischen Methode des GST-„Pulldown“ gezeigt werden, dass TAD1 und TAD2 von Ino2 mit den generellen Transkriptionsfaktoren TFIID und TFIIA interagieren. Innerhalb des TFIID wurden die Untereinheiten Taf1, Taf4, Taf6, Taf10 und Taf12 in vitro als direkte Ino2-Interaktionspartner identifiziert. Dabei binden alle identifizierten Taf-Proteine an die starke TAD1, Taf10 zusätzlich an die TAD2. Frühere Untersuchungen hatten gezeigt, dass Mutationen innerhalb der TAD1 von Ino2 (D20K, F21R) zu einem vollständigen Verlust der Aktivierungsleistung führen. In dieser Arbeit wurde nachgewiesen, dass die gerichtete Mutation dieser Aminosäuren zu einem vollständigen Interaktionsverlust mit den Taf-Proteinen führt. Mit Hilfe von Interaktionsexperimenten wurden innerhalb von Taf1 zwei distinkte Aktivatorinteraktionsdomänen (AID1: AS 1-100; AID2: AS 182-250) kartiert, die die Bindung an Ino2 vermitteln. Mutationen hydrophober und basischer Aminosäure-Reste innerhalb der Taf1-AID2 hatten einen vollständigen Verlust der Interaktion mit Ino2 zur Folge. Möglicherweise sind also ionische und hydrophobe Wechselwirkungen an der Interaktion von Ino2 und Taf1 beteiligt. Mit Hilfe der Chromatin-Immunopräzipitation (ChIP) erfolgte der Nachweis, dass Taf1 in Abhängigkeit von Ino2 auch in vivo an den ICRE-haltigen Promotoren INO1 und CHO2 vorhanden ist. Im Folgenden wurden auch die Ino2-Interaktionsbereiche innerhalb der Proteine Taf6, Taf10 und Taf12 durch die Generierung sukzessiver GST-Verkürzungen eingegrenzt. Taf10 und Taf12 besitzen wie Taf1 zwei separate AIDs (Taf10: AID1 AS 1-100; AID2 AS 131-176; Taf12: AID1 AS 50-100; AID2 AS 100-178). Untersuchungen mit mutagenisierten Varianten, bei denen wie zuvor im Fall von Taf1 hydrophobe und basische Aminosäuren innerhalb der Taf12 AID2 ausgetauscht wurden, führten lediglich zu einer Verringerung der Bindungsintensität. Dies lässt vermuten, dass mehrere kleine Domänen innerhalb der AID2 existieren, die funktionell redundant sind. Mit Hilfe weiterer ChIP-Experimente konnte auch nachgewiesen werden, dass Taf6 und Taf12 abhängig von Ino2 an den untersuchten Promotoren INO1 und CHO2 vorhanden sind. Die Proteine Taf1 und Taf6 wurden exemplarisch für Genexpressionsstudien ausgewählt, um ihren Einfluss auf die Transkription des Gens INO1 unter in vivo Bedingungen nachzuweisen. Durch vergleichende Northernblot-Hybridisierungen mit temperatursensitiven (ts) taf-Mutanten wurde gezeigt, dass die INO1-Expression unter nichtpermissiven Bedingungen (37°C) auf 7% (taf1ts) bzw. 4% (taf6ts) abfällt. Diese Befunde belegen, dass INO1 zu den Taf-abhängigen Genen zählt. Der generelle Transkriptionsfaktor TFIIA wurde ebenfalls auf eine Interaktion mit Ino2 untersucht. Bekannt war bereits, dass der Aktivator Rap1, der ähnlich wie Ino2 mit mehreren TFIID-Untereinheiten interagiert, auch TFIIA kontaktiert. Durch GST-„Pulldown“-Studien konnte die Untereinheit Toa1 als direkter Ino2-Interaktionspartner identifiziert werden. Dabei zeigte sich, dass Toa1 sowohl mit der TAD1 als auch der TAD2 von Ino2 interagiert und die TAD1 Aminosäuresubstitutionen D20K und F21R zu einem vollständigen Interaktionsverlust führen. In dieser Arbeit konnte somit gezeigt werden, dass die generellen Transkriptionsfaktoren TFIID und TFIIA als Coaktivatoren des für die Transkription der Strukturgene der Phospholipid-Biosynthese essentiellen Aktivators Ino2 fungieren.
