Applications of bacterial enterotoxins, ribosome-inactivating proteins and viral cytotoxins

A toxin can be described as a foreign substance that inflicts damages to living organisms. Naturally occurring proteinaceous toxins can derive from bacteria, fungi, plants, animal venoms and even viruses. Identifying the toxins’ underlying mechanisms of action has been a major research interest in order to develop inhibitors against their effects. Nonetheless, various findings have sparked the use of toxic moieties for the medical benefit resulting in treatment options as for example for cancers. To gain novel insights into the structure and function of a toxin, the toxin itself has to be synthesized. In vivo production can involve high laboratory safety standards as well as a low total protein amount since the toxin might harm the overexpressing cell. An alternative to circumvent these drawbacks is cell-free protein synthesis (CFPS). Within this doctoral thesis CFPS was established as a platform technology for the production and application of proteinaceous toxins in diagnostic and medical fields. As a first step, various bacterial toxins were analyzed. The mechanisms of action of the tripartite pore-forming toxins (PFT) Hbl and Nhe were studied by hemolytic activity assays, cell-based toxicity assessments and electrophysiological recordings. Next, the PFT CytK was analyzed to identify its potential as a biological nanopore that can be used as a diagnostic tool. This thesis identified the CytK1 variant as a candidate for a nanopore development. Further, two AB5 toxins, namely the cholera toxin and the heat-labile enterotoxin, were modified. These modified toxins could be fluorescently labeled and tested for their functional activity. These data are a proof-of-concept for using CPFS for intracellular trafficking of toxins and coupling of payloads for drug delivery. In a second step, a targeted toxin combining the plant-derived toxin Dianthin and the epidermal growth factor (EGF) was assessed for its potency as a potential cancer therapeutic. The medical benefit of this Dianthin-EGF targeted toxin was demonstrated on human squamous cell carcinoma samples. 0.1 nM Dianthin-EGF in combination with an endosomal escape enhancer suppressed the growth of carcinoma colonies by almost 50%. As a third and last step, CFPS was assessed for its potential as a rapid response system against novel viral pathogens using SARS-CoV2 viral proteins. All SARS-CoV2 proteins could be synthesized and analyzed. The cytotoxic behaviors of the nsp1 and envelope protein were determined. The nucleocapsid protein was quantitatively detected by specific antibodies thereby facilitating cell-free systems for the validation of available antibodies. All in all, this thesis successfully developed a platform technology for the cell-free synthesis, functional characterization and application of toxic proteins in clinical and diagnostic fields

Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins

The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural, structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-free protein synthesis in a fast and efficient manner. The inhibitory effect of the non-structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for the validation of available antibodies. The cytotoxic envelope protein was characterized in electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates the potential of eukaryotic cell-free systems as a rapid response mechanism for the synthesis, functional characterization and antibody validation against a viral pathogen.BMBF, L1FHG42421, Unterstützung anwendungsorientierte Forschung für Fraunhofer 202

Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles

Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.</p

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. Key points • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin&rsquo;s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl&rsquo;s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions

Studierende in 'Maennerfaechern' Eine Sonderauswertung der Konstanzer Studierendensurveys zu Aspekten der Sozio- und Bildungsbiografie

In dem Beitrag wird untersucht, ob bzw. wie sich Frauen, die Faecher studieren, in denen der Frauenanteil laut Hochschulstatistik des Statistischen Bundesamtes unter 30 Prozent liegt, in verschiedenen sozialen und bildungsbiographischen Merkmalen von ihren maennlichen Kommilitonen bzw. von Frauen in anderen Studienfaechern unterscheiden. Datengrundlage sind sechs Erhebungen, die die AG Hochschulforschung zwischen 1985 und 1998 durchgefuehrt hat. Es wird festgestellt, dass Frauen bei der Studienfachwahl tradierte Geschlechtergrenzen eher ueberschreiten koennen, wenn sie ueber vergleichsweise gute, privilegierte und 'ueberschuessige' Voraussetzungen verfuegen. Dies zeigt sich etwa in der tendenziell hoeheren sozialen Herkunft der Studentinnen, die in Maennerfaecher eingemuendet sind, in ihren vor allem an den Fachhochschulen 'ueberwertigen' Hochschulbefaehigungen, in ihrem hoeheren Anteil bei den Notenbesten im Abitur und moeglicherweise auch an ihren an den Universitaeten gerade angesichts ihrer sozialen Herkunft erstaunlich hohen Anteilen an absolvierten Berufsausbildungen. (IAB2)Available from http://www.iab.de/ftproot/wb0602.pdf / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions

Cell-Free Systems Enable the Production of AB₅ Toxins for Diagnostic Applications

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA’s active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin’s mechanism of action

Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA&rsquo;s active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin&rsquo;s mechanism of action