Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischemic tissues become contaminated with C. perfringens. C. perfringens quickly multiplies in ischemic tissues and spreads to healthy areas, leading to high levels of morbidity and mortality. As a species, the bacterium can synthesize thirteen different toxins. The alpha toxin (PLC) and perfringolysin O (PFO) are thought to be important virulence factors in gangrene. We wished to understand how C. perfringens is capable of avoiding killing by the host immune system, and determine if PLC and PFO play a role in this avoidance. We found C. perfringens was not killed by J774-33 cells or mouse peritoneal macrophages under aerobic or anaerobic conditions. Using electron microscopy, we showed that C. perfringens could escape the phagosome of J774-33 and mouse peritoneal macrophages. We believe the ability of C. perfringens to survive in the presence of macrophages is due to its ability to escape the phagosome. Using a variety of inhibitors of specific receptors, we identified those used by J774-33 cells to phagocytose C. perfringens. The scavenger receptor, mannose receptor(s), and complement receptor (CR3) were involved in the phagocytosis of C. perfringens. To determine if PFO or PLC were involved in the ability of C. perfringens to survive in the presence of macrophages, we constructed C. perfringens strains lacking these toxins. The ability of C. perfringens to survive in the presence of J774-33 cells is dependent on PFO, while survival in mouse peritoneal macrophages is dependent on PFO and PLC. The ability of C. perfringens to escape the phagosome of J774-33 cells and mouse peritoneal macrophages is mediated by either PFO or PLC. Using a mouse model, we found that PFO and PLC were necessary for C. perfringens to survive in vivo using infectious doses 1000 times lower than those required to initiate a gangrene infection. We propose that PFO and PLC play a critical role in the survival of C. perfringens during the early stages of gangrene infections, when phagocytic cells are present and bacterial numbers are low. / Ph. D.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

Proteins modularity enhances the multi-functionality and versatility of proteins by providing such properties as multiple and various ligand-binding sites, increased ligand affinity through the avidity effect, and the juxtaposition of ligand-binding modules near catalytic domains. An NMR-based "dissect-and-build" approach to studying modular protein structure and function has proven very successful, whereby modules are initially characterized individually and then correlated with the overall function of a protein. We have used the dissect-and-build approach and NMR to study two modular protein systems. Chapter 2 details the NMR solution structure of the weak-lysine-binding kringle IV type 8 (KIV8) module from the apolipoprotein(a) (apo(a)) component of lipoprotein(a) was determined and its ligand-binding properties assessed. In vitro studies have demonstrated the importance of the apo(a) KIV7 and KIV8 modules in mediating specific lysine-dependent interactions with the apolipoproteinB-100 (apoB-100) component of LDL in the initial non-covalent step of lipoprotein assembly. Notable differences identified in the lysine binding site (LBS) of the KIV8 were deemed responsible for the differential modes of apoB-100 recognition by KIV7 and KIV8. In addition, the KIV8 structure has brought to light the importance of an RGD sequence at the N-terminus of the apo(a) KIV8 module, which may mediate important apo(a)-integrin interactions. In Chapters 3-6, structure-function studies of the CpGH84C X82 and the CpGH84A dockerin-containing modular pair were conducted to understand how the varying modularity unique to the C-terminal regions of the secreted multi-modular family 84 glycoside hydrolases influences the spreading of Clostridium perfringens. Identification of a CpGH84C cohesin module (X82), and the structural characterization of a dockerin-containing modular pair provides the first evidence for multi-enzyme complex formation mediated by non-cellulosomal cohesin-dockerin interactions. The formation of large hydrolytic enzyme complexes introduces a novel mechanism by which C. perfringens may enhance its role in pathogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-09-27 11:46:38.753

Clostridium perfringens is a gram positive spore forming anaerobe and a causative agent of gas gangrene, necrotic enteritis (pig-bel) and food poisoning in humans and other animals. This organism secretes a battery of exotoxins during the course of infection as well as a variety of virulence factors which may help to potentiate the activities of the toxins. Among these virulence factors is the μ-toxin, a family 84 glycoside hydrolase which acts to degrade hyaluronan, a component of human connective tissue. C. perfringens has 53 open reading frames encoding glycoside hydrolases. About half of these glycoside hydrolases are predicted to be secreted. Among these are CpGH84C, a paralogue of the μ-toxin, and CpGH89. CpGH89 shares sequence similarity to the human α-N-acetylglucosaminidase, NAGLU, in which mutations can cause a devastating genetic disease called mucopolysaccharidosis IIIB. One striking feature of the secreted glycoside hydrolase enzymes of C. perfringens is their modularity, with modules predicted to be dedicated to catalysis, carbohydrate-binding, protein-protein interactions and cell wall attachment. The extent of the modularity is remarkable, with some enzymes containing up to eight ancillary modules. In order to help understand the role of carbohydrate-active enzymes produced by bacterial pathogens, this thesis will focus on the structure and function of the modular extracellular glycoside hydrolase enzymes secreted by the disease causing bacterium, C. perfringens. These structure function studies examine two family 32 CBMs (carbohydrate-binding modules), one from the μ-toxin and the other from CpGH84C. As well we examine the complete structure of CpGH84C in order to help further our understanding of the structure of carbohydrate-active enzymes as a whole. Finally, the catalytic module of CpGH89 is characterized and its relationship to the human NAGLU enzyme is discussed.

Thesis addresses the physiological role of formyl tetrahydrofolate synthetase (Fhs) and bifunctional folate dehydrogenase (FolD) in folate mediated one carbon metabolism in bacteria. Thesis consists of 5 chapters. First chapter provides the details of the literature on folate metabolism, enzymes involved the synthesis and physiological roles various folate co-factors. Second chapter discusses the study of Clostridium perfringens Fhs generation of folD deletion in the support of fhs. Third chapter explores the characterization of the folD deletion strain. Fourth chapter presents the characterization of monofunctional versions of FolD from Clostridium perfringens. Fifth chapters talks about anti-correlation existence of Fhs and PurT (phosphoribosyl glycinamide formyl transferase II) The detailed experimental study is discussed below i. Characterization of Clostridium perfringens Formyl Tetrahydrofolate Synthetase (Fhs) In this chapter we have characterized Fhs from pathogenic Clostridium perfringens. Fhs catalyzes the formation of N10-formyl THF from THF and formate. Previously Fhs has been characterized from various non-pathogenic species of Clostridium. In addition, the detailed kinetic parameters are not known. In this report we have characterized the Fhs Clostridium perfringens and detailed kinetic parameters were determined. We have also shown the biological function by rescue of UV photorepair sensitive strain. ii. One-carbon metabolic pathway rewiring in Escherichia coli reveals an evolutionary advantage of 10-formyltetrahydrofolate synthetase (Fhs) in survival under hypoxia In cells, N10-formyltetrahydrofolate (N10-formyl THF) required for formylation of eubacterial/organeller initiator tRNA and purine biosynthesis is produced by methylene- tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). folD is present in all organisms, where as fhs shows mixed distribution. We show that in E. coli, which naturally lacks fhs, essential function of folD could be replaced with fhs of Clostridium perfringens when provided on a medium copy plasmid or integrated as single copy gene in the chromosome of the ∆folD strains, for their growth in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N10-formyl THF in the ∆folD strains (harboring fhs) were limiting despite their high enzymatic capacity to synthesize the same. Auxotrophy for purines could be alleviated by adding formate to the medium, and that for glycine by engineering THF import into the cells. The ∆folD strains showed high NADP+/NADPH ratio and were hypersensitive to trimethoprim (TMP). Further, the presence of fhs was disadvantageous to E. coli under aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. And, the computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria. We also propose that inhibitors aimed at folD could potentiate the effect TMP drugs. iii. 5, 10-methylene-THF dehydrogenase (DH) and 5, 10-methenyl-THF cyclohydrolase (CH) activities of FolD are essential to maintain folate homeostasis and anti-folate resistance While E. coli and many other organisms have folD alone or folD and fhs, Clostridium species possess an annotated bi-functional FolD and an annotated methenyl tetrahydrofolate cyclohydrolase (FchA). Simultaneous presence of 3 enzymes for the synthesis of N10-formyl THF was intriguing. To understand this unusual feature we have cloned Clostridium perfringens CpeFolD and CpeFchA, over expressed and purified to near homogeneity. Biochemical analyses revealed that CpeFolD possess only dehydrogenase activity as opposed to in silico prediction, while CpeFchA possess cyclohydrolase activity as expected. We also show that expression of both proteins together allowed folD deletion in E. coli. From this study we found that presence of dehydrogenase and cyclohydrolase functions are very important in the maintenance of folate homeostasis and anti-folate resistance. iv. Analysis of distribution of fhs and purT genes in the organisms While analysing distribution of fhs across genomes, serendipitously we also found that large number of organism which have fhs lack purT(phosphoribosyl glycinamide formyl transferase II), in short where ever purT was present fhs was absent. This kind of anti-correlation was strictly conserved in Bacillus genes as well. Growth competition experiments were done to address anti-correlation between fhs and purT. Growth competition experiments revealed that simultaneous presence of both purT and fhs is disadvantageous, when compared to presence of either one gene.

L’entérite nécrotique aviaire causée par Clostridium perfringens est une maladie économiquement dévastatrice et celle-ci est en émergence dans les troupeaux de poulets de chair éliminant l’usage des antibiotiques. À ce jour, aucune alternative en élevage ne permet de prévenir efficacement la maladie et un contrôle par une stratégie vaccinale serait des plus prisé. Une approche par génomique comparative jumelée à la vaccinologie réverse soustractive et comparative identifiant des protéines bactériennes de surface immunogènes figure parmi les approches méthodologiques des plus prometteuses pour le développement rapide d’un vaccin efficace. Une étude génomique comparative réalisée sur 48 souches de C. perfringens provenant de poulets de chair en santé ou affectés par l’entérite nécrotique a permis d’établir que les génomes analysés étaient composés de 155 700 protéines distinctes, où 13% étaient extracellulaires, 65% cytoplasmiques et 22% membranaires. L’évaluation du pouvoir immunogène de ces protéines à l’aide de l’outil de prédiction VaxiJen v.2.0 a permis d’identifier 4 catégories de scores pour les protéines identifiées, allant de 0,5 (seuil minimal recommandé) à 1,5. Les protéines présentant les scores les plus élevés ont été majoritairement associées à des localisations extracellulaires. La combinaison du score d’immunogénicité et de la localisation cellulaire des protéines analysées a mené à la sélection de 12 protéines candidates vaccinales, la plupart d’entre elles étant de fonction hypothétique. Une description plus approfondie de ces protéines permettra de mieux définir leur fonction, d’évaluer leur potentiel antigénique réel en caractérisant leur interaction avec le système immunitaire de la volaille et ultimement, d’évaluer leur rôle probable dans la pathogénie de l’entérite nécrotique. / Avian necrotic enteritis caused by Clostridium perfringens is a disease with a major economical impact, generating losses up to 6 billion dollars for the poultry industry worldwide. This disease appears in broiler chicken flocks that no longer employ the use of antibiotics. To date, no alternative method allows for the efficient prevention of necrotic enteritis (NE) and a control by a vaccinal strategy would be mostly prized. A comparative genomics approach as well as comparative and subtractive reverse vaccinology identifying immunogenic bacterial surface proteins is one of the most promising methodologies for the rapid development of an efficient vaccine. A comparative genomic study was performed on 48 C. perfringens strains isolated from healthy broiler chickens and from broilers affected by necrotic enteritis. From this study, it was established that the genomes analyzed were composed of 155 700 distinct proteins where 13% were predicted to have an extracellular expression, 65% at the cytoplasma level and 22% within the plasma membrane. The evaluation of the immunogenic potential of these proteins was established with the prediction software VaxiJen v2.0 for which a 0.5 threshold score allowed for the identification of four score categories among the identified proteins, from 0.5 to 1.5. For the most part, proteins with the highest scores were associated with an extracellular localisation. The combination of the immunogenicity score and localisation of the analysed proteins led to the selection of 12 vaccinal candidate proteins that were mostly identified as hypothetical. A more in-depth description of these proteins would allow the assessment of their function, the evaluation of their true immunogenic potential by characterizing their interaction with the avian immune system and ultimately, evaluate their probable role in the pathogenesis of necrotic enteritis.

No description available.

Zum Studium des prophylaktischen Einflusses ausgewählter antibiotischer Leistungsförderer [Avilamycin (10 ppm), Avoparcin (15 ppm), Virginiamycin (20 ppm)] und ionophorer Antikokzidia [Monensin (100 ppm), Narasin (70 ppm)] sowie des metaphylaktischen bzw. therapeutischen Einsatzes von Tylosin [Tylan 0,5 g/l H2O] auf das Auftreten und die Ausprägung der Clostridium (Cl.) perfringens-Enterotoxämie (CPE) wurden Untersuchungen an insgesamt 33 Versuchsgruppen mit 825 Broilerküken durchgeführt. Die Erkrankung konnte mittels intraduodenaler Inokulation einer Vollkultur Cl. perfringens Typ A (ATCC 3624) sicher reproduziert werden. Die Morbiditätsrate betrug in allen infizierten Gruppen 100 %. An klinischer Symptomatik zeigte sich hauptsächlich profuser wässriger Durchfall. Schwerere Störungen, Apathie und Anorexie, waren selten und in allen beobachteten Fällen vom schnellen Tod des betreffenden Tieres begleitet. Allgemein fiel auf, daß die infizierten und nicht medikamentierten Tiere schneller und länger erkrankten. Bei infizierten und unmedikamentierten Tieren ergab sich eine Mortalitätsrate von 16 bis 36 %, in den medikamentierten Gruppen maximal 8 %. Tylosin zeigte eine sehr gute metaphylaktischen bzw. therapeutische Wirkung. Die Lebendmasseentwicklung betrachtend, konnte Avoparcin unter den Leistungsförderern beste Ergebnisse erzielen. Ähnliche Resultate wurden in den Kombinationsgruppen [Avilamycin plus Monensin oder Narasin] bzw. mittels Narasin erreicht. In absteigender Reihenfolge zeigten Avilamycin, Virginiamycin und Monensin eine geringere leistungsfördernde Wirkung. Die Bestimmung fäkaler bzw. ileozäkaler Clostridienkonzentrationen lebender, respektive verendeter Hühner erbrachte nur wenige und relativ unbedeutende statistisch gesicherte Korrelationen zu anderen Ergebnissen. Es konnten keine Zusammenhänge zwischen Erregerzahl und Lebendmassezunahme, bzw. Todesursache, aufgedeckt werden. Die Resultate aus allen Versuchen zusammenfassend, müssen den Kombinationen von Avilamycin mit Narasin bzw. Monensin beste Effekte hinsichtlich einer positiven Beeinflussung CPE-bedingter Morbidität, Mortalität und Lebendmasseverluste bescheinigt werden. Tylosin war in der Lage, die Verlustzahlen durch CPE rasch zu senken. Für die Ausprägung kompensatorischer Effekte hinsichtlich der Lebendmasseverluste unter der Infektion muß mit einer größeren Zeitspanne gerechnet werden. Die Polyether und auch Avilamycin sind als Futtermittelzusatzstoffe für die europäische Geflügelhaltung zugelassen. Durch die ständige Kokzidiosebedrohung in den Hühnerbeständen kann auf einen prophylaktischen Einsatz antikokzidieller Futtermittelzusatzstoffe momentan nicht verzichtet werden. Es ist zu vermuten, daß es durch den simultanen Einsatz von Polyether und Leistungsförderer zu einer positiven Beeinflussung der schädigenden Wechselwirkungen von Kokzidien und Cl. perfringens im Darm kommt. Bei vorhandener Empfindlichkeit der Eimerien sollte dies sowohl die Bekämpfung von CPE als auch von Kokzidiosen begünstigen. Der positive Eindruck von Avoparcin spielt, bedingt durch das europaweite Verbot, momentan für die Praxis keine Rolle. Die Entwicklungstendenzen auf dem Sektor antibiotisch wirksamer Futtermittelzusatzstoffe, eng verknüpft mit der bakteriellen Resistenzproblematik, werden in der Arbeit ausführlich diskutiert. / Investigations with 825 chickens in 33 trials were performed in order to find out the prophylactic effect of selected antibiotic growth promoters [avilamycin (10 ppm), avoparcin (15 ppm) virginiamycin (20 ppm)] and polyether ionophore antibiotics [monensin (100 ppm), narasin (70 ppm)] on the incidence of Clostridium (Cl.) perfringens enterotoxemia (CPE) in chickens as well as the therapeutic resp. metaphylactic influence of tylosin [Tylan 0,5 g/l H20]. The enterotoxemia could be reproduced regularly by intraduodenal infection with high numbers of vegetative cells of Cl. perfringens type A (ATCC 3624). The morbidity rate always reached 100 %. In spite of a profuse and watery diarrhoea the chickens normally showed no further considerable disturbances of the general status. Apathy or anorexia were rather rare and immediately followed by Exitus letalis of the related chickens. It was striking that the infected and non-medicated broilers contracted the disease more quickly and for a longer time. The mortality rate among the infected and non-medicated animals was 16 to 36 %, among the medicated groups max. 8 %. Tylosin showed a considerable metaphylactic effect in decreasing CPE mortality. The avoparcin group showed the best weight gain among the growth promoters, comparable to the results by means of the combinations [avilamycin + monensin or narasin] or narasin only. Decreasingly avilamycin, virginiamycin and monensin were less successful. Analysing the faecal resp. ileocecal quantities of Cl. perfringens adduced only a few statistically guaranteed correlation with other results. There was no causal connection between numbers of Cl. perfringens and life weight development. It was impossible to discover a numerical threshold of germs responsible for the death of the chickens. Summarising all the results of the entire attempts the combinations of avilamycin and narasin resp. monensin were the most effective concerning the reduction of morbidity, mortality and life weight losses by CPE. By application of tylosin it was possible to stop the mortality rate quickly. But it needs more time to achieve reductions of the CPE related weight losses. The two polyethers and also avilamycin are still admitted in the European Union. Currently an abandonment of anticoccidial feed supplements seems to be impossible due to the present danger of coccidiosis in poultry. By means of monensin/narasin plus avilamycin the adverse health effects of interactions of both pathogens should be reduced. Presupposing susceptibility of the coccida this should be a notable contribution to a better controlling and to the prevention of CPE and coccidiosis, too.

No description available.

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien zeigten eine selektive optCPE vermittelte Verminderung der Tumorvitalität in Verbindung mit Nekrose, die in fast allen Fällen mit einer Reduktion des Tumorvolumens einher ging. Die tierexperimentellen Studien belegen damit die Effektivität der CPE-basierten Gentherapie im Pankreaskarzinom. Mit diesen neu gewonnenen Erkenntnissen zum „Onkoleaking“ Konzept der CPE Suizid-Gentherapie und deren Wirkungsmechanismen sind Kombinationen mit konventionellen Therapien möglich. / Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3  /  4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated the selective CPE mediated tumor growth inhibition, associated with reduced tumor viability and effective induction of tumor necrosis. This further corroborated the advantages of this novel oncoleaking strategy. With this gain of knowledge about our new oncoleaking concept of suicidal gene therapy and its mechanism of action, novel combinations with conventional therapies are possible to further improve therapeutic efficacy and to overcome resistance in pancreas carcinoma.