Les toxines formeuses de pore (PFTs) sont des protéines exogènes responsables d’un grand nombre de maladies infectieuses qui perméabilisent les membranes cellulaires de leur hôte. La formation des pores ou l’introduction d’une enzyme dans le cytoplasme peut entrainer l’apparition de symptômes de maladies connues (l’anthrax, le botulisme) et, dans le pire des cas, la mort. Les mécanismes d’infection et de destruction des cellules infectées sont bien caractérisés. Toutefois, l’aspect dynamique des changements de conformation durant le processus de perméabilisation reste à découvrir pour la majorité des toxines formeuses de pore. Le but de cette thèse est d’étudier les mécanismes d’oligomérisation des PFTs, ainsi que la formation des pores à la membrane lipidique grâce à la spectroscopie de fluorescence. Nous avons choisi la toxine Cry1Aa, un bio pesticide produit par le bacille de Thuringe et qui a été rigoureusement caractérisé, en tant que modèle d’étude. La topologie de la Cry1Aa à l’état actif et inactif a pu être résolue grâce à l’utilisation d’une technique de spectroscopie de fluorescence, le FRET ou transfert d’énergie par résonance entre un fluorophore greffé au domaine formeur de pore (D1) et un accepteur non fluorescent (le DPA ou dipicrylamine) localisé dans la membrane et qui bouge selon le potentiel membranaire. Le courant électrique, ainsi que la fluorescence provenant de la bicouche lipidique membranaire horizontale ont été enregistrés simultanément. De cette manière, nous avons pu localiser toutes les boucles reliant les hélices de D1 avant et après la formation des pores. Dans la forme inactive de la toxine, toutes ces boucles se trouvent du côté interne de la bicouche lipidique, mais dans sa forme active l’épingle α3-α4 traverse du côté externe, alors que toutes les autres hélices demeurent du côté interne. Ces résultats suggèrent que α3-α4 forment le pore. Nous avons découvert que la toxine change significativement de conformation une fois qu’elle se trouve dans la bicouche lipidique, et que la Cry1Aa attaque la membrane lipidique de l’extérieur, mais en formant le pore de l’intérieur. Dans le but de caractériser la distribution de toxines à chaque extrémité de la bicouche, nous avons utilisé une technique de double FRET avec deux accepteurs ayant des vitesses de translocation différentes (le DPA et l’oxonol) dans la membrane lipidique. De cette manière, nous avons déterminé que la toxine était présente des deux côtés de la bicouche lipidique durant le processus de perméabilisation. La dynamique d’oligomérisation de la toxine dans une bicouche lipidique sans récepteurs a été étudiée avec une technique permettant le compte des sauts de fluorescence après le photoblanchiment des fluorophore liés aux sous unités composant un oligomère présent dans la bicouche lipidique supportée. Nous avons confirmé de cette manière que la protéine formait ultimement des tétramères, et que cet état résultait de la diffusion des monomères de toxine dans la bicouche et de leur assemblage subséquent. Enfin nous avons voulu étudier le « gating » de la colicine Ia, provenant de la bactérie E.Coli, dans le but d’observer les mouvements que font deux positions supposées traverser la bicouche lipidique selon le voltage imposé aux bornes de la bicouche. Nos résultats préliminaires nous permettent d’observer un mouvement partiel (et non total) de ces positions, tel que le suggèrent les études de conductances du canal. / Pore forming toxins (PFTs) are exogenous often pathogenic proteins that permeabilize the host membrane. Permeabilization or subsequent introduction of an enzyme leads to health disorders and sometimes death. Although the fundamental infection and destruction mechanisms are known, the underlying molecular basis and their link to the structural information remains undetermined for many pore forming toxins. The purpose of this thesis was to study the mechanisms of oligomerization on the membrane and pore formation of PFTs using fluorescence spectroscopy in planar lipid bilayer. We chose Cry1Aa as the most intensively studied member of Bacillus thuringiensis’s toxins. In order to probe the topology both in inactive and active congformation, we used Förster resonance energy transfer (FRET) between a fluorophore site-directedly attached to different positions in the pore forming domain (D1) of Cry1Aa toxin and an acceptor compound dipicrylamine (DPA) in the membrane, which moves in response to the membrane potential. Electrical current and fluorescence emission from planar lipid bilayers in a horizontal configuration were simultaneously recorded. We probed all loops between the seven α helices of D1. All of them were located on the inner leaflet of the bilayer prior to pore formation. In the active form, the α3-α4 hairpin were found to translocate back to the outer leaflet of the bilayer, whereas all other positions remained in the inner leaflet, suggesting that α3-α4 are the pore lining helices. The toxins undergo significant conformational changes once they enter the host membrane, and we found Cry1Aa to attack from the exterior but translocate to the interior. To estimate the distribution of the toxins on either side of the membrane, we used the double-FRET technique. Here, two different acceptors (DPA and oxonol) with different dynamics (time constants) allowed us to determine that approximately equal amounts of the toxin were present on either leaflet during the permeabilization process. We also studied the oligomerization mechanism of Cry1Aa toxins inserted into supported lipid bilayers using a single subunit counting technique based on the step-wise photodestruction (bleaching) of the attached fluorophores. This system allowed determining the number of subunits composing each oligomer. We found that oligomerization is a highly dynamic process which occurs after insertion into the bilayer by lateral diffusion. The final (likely the pore forming) entity of the toxin is tetrameric. Finally, we used the same FRET approach to investigate the gating process of two positions of the pore forming domain of colicin Ia, an antibiotic toxin produced by E. coli. These positions were suspected to translocate reversibly from the outer to the inner leaflet during the gating process. In preliminary results, we found that these positions are moving between the two leaflets of the bilayer during pore formation.

This thesis is an investigation of novel mono-ADP-ribosylating toxins. In the current data-rich era, making the leap from sequence data to knowledge is a task that requires an elegant bioinformatics toolset to pinpoint questions. A strategy to expand important protein-family knowledge is required, particularly in cases in which primary sequence identity is low but structural conservation is high. For example, the mono-ADP-ribosylating toxins fit these criteria and several approaches have been used to accelerate the discovery of new family members. A newly developed tactic for detecting remote members of this family -- in which fold recognition dominates -- reduces reliance on sequence similarity and advances us toward a true structure-based protein-family expansion methodology. Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins identified and characterized using in silico and cell-based techniques. Medically relevant toxins from Mycobacterium avium and Enterococcus faecalis were also uncovered. Agriculturally relevant toxins were found in Photorhabdus luminescens and Vibrio splendidus. Computer software was used to build models and analyze each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. Yeast-based activity tests have since confirmed activity. Vibrio cholerae produces cholix – a potent protein toxin of particular interest that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 2.1Å apo X-ray structure as well as a 1.8Å X-ray structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD+). Hallmark catalytic residues were substituted and analyzed both for NAD+ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These new toxins serve as a reference for ongoing inhibitor development for this important class of virulence factors. In addition to using toxins as targets for antivirulence compounds, they can be used to make vaccines and new cancer therapies. / Natural Sciences and Engineering Research Council (CGS-D), Canadian Institutes of Health Research, Cystic Fibrosis Canada, Human Frontier Science Program, Ontario government (OGSST), University of Guelph (Graduate Research Scholarship)

Les toxines de l’anthrax font partie de la famille des toxines A-B dans laquelle la moitié B se fixe à la membrane de la cellule permettant par la suite la translocation de la moitié A. Dans le cas de l’anthrax, la moitié B est représentée par le Protective Antigen (PA) et la moitié A par les deux protéines Edema Factor (EF) et Lethal Factor (LF). Après le recrutement par les récepteurs cellulaires (CMG2 et TEM8), PA s’organise en heptamère. Il peut fixer jusqu'à 3 ligands (EF et LF) avant d'être endocyté. Les modèles actuels de PA suggèrent que la baisse de pH à l’intérieur des endosomes permet un changement de conformation de la forme pré-pore vers la forme pore et que les ligands EF et LF passeraient au travers le pore pour entrer dans le cytoplasme. Cependant, le diamètre du pore est environ dix fois inférieur à celui des ligands (10 Å contre 100 Å). Un processus de folding/unfolding a été proposé mais demeure controversé. Afin d'identifier le processus de passage des facteurs EF et LF dans le cytoplasme, nous avons déterminé par cryo-microscopie électronique combinée avec l’analyse d’image les structures tridimensionnelles des complexes formés par PA et LF aux étapes prépore et pore. Par la suite, une étude complémentaire par dynamique moléculaire nous a permis de modéliser à haute résolution les différentes interactions qui ont lieu au sein du complexe. La structure 3D du complexe prépore combiné à 3 LF a été déterminée à une résolution de 14 Å. Nous avons aussi calculé une structure préliminaire du complexe pore également combiné à 3 LF Celles-ci n’ont jamais été résolues auparavant et leur connaissance permet d’envisager l’étude en profondeur du mécanisme infectieux de l’Anthrax in vivo. / The anthrax toxins are part of the A-B toxin family in which the B moiety binds to the cell membrane allowing subsequent translocation of the A moiety. In the case of anthrax, the B moiety consists of the Protective Antigen (PA), and the A moiety is composed of the two proteins Edema Factor (EF) and the Lethal Factor (LF). After being recruited by the cell receptors (CGM2 or TEM8), PA organizes itself into a heptamer. It can bind up to three ligands (either EF or LF) before being endocytosed. Current models suggest that the decrease of pH inside the endosomes allows a conformational change of PA from a prepore form to a pore form that allows the EF and LF ligands to pass through the pore and enter the cytoplasm. However, the pore diameter is about ten times smaller than the diameter of the ligands (10Å versus 100Å). A process of ligand folding / unfolding has been proposed, but remains controversial. To identify the mechanism by which EF and LF enter the cytoplasm, we have used cryo-electron microscopy and three-dimensional image analysis to determine the 3D structure of the PA-LF complexes in the pre-pore and pore conformations. Then, we used molecular dynamics to modelise at high resolution the different interactions that occur within the complex. The 3D structure of the pre-pore complex bound with three LF ligands has been determined at 14Å resolution. We also calculated a preliminary structure of the LF-bound pore complex. These structures have never been reported before. They provide the necessary information to study in depth the mechanism of anthrax infection in vivo.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Erst kürzlich wurde der Lipolyse-stimulierte Lipoproteinrezeptor (LSR, engl. lipolysis-stimulated lipoprotein receptor) als der zelluläre Oberflächenrezeptor von CDT und Iota-Toxin, zweier Vertreter der Iota-Toxin-Familie der clostridialen Aktin-ADP-ribosylierenden Toxine, identifiziert. In dieser Arbeit sollte geprüft werden, ob CST, ein weiterer Vertreter der Iota-Toxin-Familie, ebenfalls LSR für den Zelleintritt nutzt. Zunächst wurden die Toxinkomponenten CSTa und CSTb erstmals rekombinant hergestellt. Dazu wurden die für CSTa und CSTb codierenden Genabschnitte mittels PCR amplifiziert und anschließend in einen Expressionsvektor kloniert. Als Expressionsvektor wurde in dieser Arbeit der pHis1522-Vektor verwendet. Zur Amplifizierung wurden die Plasmide in E. coli transformiert und anschließend aufgereinigt. Die Proteinexpression erfolgte in B. megaterium, weil dieses Bakterium sich bereits zur Expression anderer clostridialer Toxine bewährt hatte. Zur Aufreinigung der 6xHis-getaggten Proteine wurde die Nickel-Affinitätschromatographie eingesetzt. Als nächstes wurde gezeigt, dass die rekombinant hergestellten Toxinkomponenten CSTa und CSTb biologisch aktiv waren. Dazu wurden CaCo2-Zellen mit CST behandelt und anschließend die Morphologie der Zellen untersucht. CaCo2-Zellen, die mit CSTa und CSTb behandelt wurden, wiesen Vergiftungserscheinungen wie eine typische Zellabrundung auf. Mit dem „Aktin-Nach-ADP-Ribosylierungs-Assay“ und der fluoreszenzmikroskopischen Untersuchung von TRITC-Phalloidin-gefärbtem Aktin wurde gezeigt, dass das rekombinant hergestellte CST Aktin-ADP-ribosylierende Eigenschaften besaß. Nachdem gezeigt war, dass rekombinant hergestelltes CST sich wie ein biologisch aktives, binäres Aktin-ADP-ribosylierendes Toxin verhält, konnte mithilfe der Vergiftung von H1-HeLa(+LSR)-Zellen und nativen H1-HeLa-Zellen, die kein LSR exprimierten, nachgewiesen werden, dass die Wirkung des Toxins LSR-abhängig ist. FACS-Analysen und Kolokalisationsstudien mit Alexa488-gefärbtem CSTb und Antikörper-gefärbtem LSR erbrachten zusätzlich den Beweis, dass CSTb auf der Zelloberfläche an LSR bindet und bei der Aufnahme in die Zellen mit LSR in endozytischen Vesikeln kolokalisiert. Die Ergebnisse dieser Arbeit zeigen, dass das C. spiroforme Toxin (CST) ebenfalls LSR als Rezeptor für den Zelleintritt verwendet.

Dumbliai – mikroskopiniai planktono organizmai – vienas iš pagrindinių hidroekosistemų komponentų, pirminiai organinės medžiagos producentai. Didėjant vandens telkinių trofiškumui, mažėja dumblių rūšių įvairovė, keičiasi vyraujančių rūšių kompleksas. Dažnai eutrofikuotuose vandens telkiniuose ima dominuoti prokariotiniai autotrofiniai mikroorganizmai – melsvabakterės, kurios sukelia intensyvius vandens „žydėjimo“ procesus ežeruose, jūrinėse lagūnose, jūrose bei vandenynuose. Dėl šios priežasties blogėja vandens kokybė. Pastaraisiais dešimtmečiais išsamių tyrimų objektu visame pasaulyje tampa toksiniai fitoplanktono dumbliai ir melsvabakterės. Tyrimais yra nustatyta, kad pusė iš visų vandens „žydėjimo“ atvejų yra toksiški (RAPALA, LAHTI, 2002). Pasaulyje atliekami monitoringiniai tyrimai, siekiant įvertinti toksinių dumblių ir melsvabakterių vystymosi tendencijas, priklausomybę nuo aplinkos sąlygų, toksinio vandens „žydėjimo“ priežastis. Pasitelkiant cheminius bei genetinius metodus, nustatoma toksinių medžiagų cheminė sudėtis, vertinamas jų poveikis gyviems organizmams. Šiaurinės Kuršių marių dalies vasariniame planktone 2004-2006 m. aptiktos 223 dumblių rūšys, priklausančios 5 klasėms. 97 rūšys (43 %), priklauso Chlorophyceae klasei, 71 rūšis (32 %) – Cyanophyceae, 40 rūšių (18 %) – Bacillariophyceae, 9 rūšys (4 %) – Euglenophyceae ir 6 rūšys (3 %) – Dinophyceae klasei, iš jų aptiktos 26 potencialiai toksinės dumblių ir melsvabakterių rūšys, priklausančios 3 klasėms, 14... [toliau žr. visą tekstą] / Curonian Lagoon is a shallow transitional water basin located in the south-eastern part of the Baltic Sea. The southern and central parts of the lagoon contain freshwater due to discharge from the Nemunas River, while the salinity in the northern part varies from 0 to 8 PSU, depending on winds activity affecting brackish water inflow from the Baltic Sea. The investigation was carried out in the fresh-brackish water mixing zone (Influence zone of Baltic Sea), in the central part and Nemunas River influence zone in July-August 2004 - 2006. Changes in physico-chemical parameters, chlorophyll a concentration, phytoplankton and toxic algae cell density were monitored. Totally 223 species and varieties mainly belonging to Chlorophyceae (43 %) and Cyanophyceae (32 %) were found. 26 algae species from 3 algae classes (Cyanophyceae, Chlorophyceae and Dinophyceae) were identified as potential toxic species in the northern part of Curonian Lagoon during 2004 and 2006 summer time. Dominated toxic species Ahpanizomenon flos-aquae, Microcystis aeruginosa, M. viridis, M. wesenbergii, Woronichinia compacta. Phytoplankton biomass in Curonian Lagoon surface ranged from 12,27 to 50,22 mg/l. The peak of phytoplankton (33,11 mg/l) and potential toxic algae (28,67 mg/l) biomass in 2004 summer time was observed near by Klaipeda Strait, were Aphanizomenon flos-aquae contain 36 % from total biomass. In 2005 summer time the highest phytoplankton (50,22 mg/l) and toxic algae (21.46 mg//l) biomass were... [to full text]

Planar electromagnetic sensing system has been used as one of the NDT methods to evaluate the material properties i.e., to evaluate near-surface properties such as conductivity, permeability and dielectric properties. The applications of planar electromagnetic sensors will depend on both the characteristic of the sensor type chosen and also the characteristic of material under test. Conventional planar interdigital sensors and novel planar interdigital sensors have been designed, fabricated and tested for detection of dangerous marine biotoxins in seafood. Our main objective is to sense the presence of dangerous contaminated acid in mussels and other seafoods. Initial studies were conducted with three peptide derivatives namely Sarcosine, Proline and Hydroxylproline. These three chemicals are structurally closely related to our target molecule (domoic acid). The initial results have shown that all sensors respond very well to the chemicals and it is possible to discriminate the different chemicals from the output of the sensor. Novel interdigital sensors have shown better sensitivity measurement compared to conventional interdigital sensors. The novel interdigital sensors were then being tested with three seafood products. Results from the analysis have shown that novel interdigital sensor with configuration #1 (Sensor_1) has better sensitivity compared to other sensors. Sensor_1 has been chosen for experiment using proline and mussels. The changes in sensor sensitivity were analysed with mussels before and after adding the proline. The presence of proline on the mussel surface and also injected proline to the mussel samples were clearly detected by Sensor_1. Further experiment was conducted with small amount of domoic acid (0.5 µg to 5.0 µg) injected to a mussel and it was found that Sensor_1 was able to detect small amount of domoic acid (1.0 µg) injected into the mussel sample. Sensor_1 was able to detect approximately 12.6 µg/g of domoic acid in mussel meat. Three threshold levels of particular sample thickness have been established for detection of domoic acid. The first prototype of a low cost sensing system known as SIT (Seafood Inspection Tool) has been developed. The outcomes from the experiments provide chances of opportunity for further research in developing a low cost miniature type of sensors for reliable sensing system for commercial use.

The investigation presented in this thesis examined the microbial and functional diversity of the meltwater ponds Fresh, Orange and Salt Ponds on the McMurdo Ice Shelf, near Bratina Island, Antarctica. These sites were chosen because of the ecological importance and absence of detailed characterisations of their diversity and function as part of Antarctica?s largest wetland. Particular focus was on cyanobacterial diversity, nitrogen fixation and secondary metabolite production. Using 16S rRNA gene and morphological analysis a large diversity of cyanobacteria (more than 22 phylotypes) was identified with high phylogenetic similarities (up to 99% sequence identity) to cyanobacteria from mats in other regions of Antarctica. In addition biogeographical distributions were identified including potentially endemic and cosmopolitan cyanobacteria. High salinities were also connected to the change and reduction of diversity. Lipid marker analyses were performed targeting hydrocarbons, ether-linked hydrocarbons, methylated fatty acid esters (FAME), wax esters, hopanols and sterols. Lipid biomarker profiles were similar to typical cyanobacteria dominated mats with major input from microorganisms including oxygenic and anoxygenic phototrophs, obligate aerobic and anaerobic heterotrophs that conduct the metabolic processes of fermentation, sulphate reduction, sulphate and iron-oxidation, methanogeneses. Signature lipids indicative of Chloroflexus and archaea, as well as branched aliphatic alkanes with quaternary substituted carbon atoms (BAQCs), were identified for the first time in Fresh, Orange and Salt Ponds. Based on nifH gene analysis, the nitrogen fixing diversity characterised in Orange Pond consisted of cyanobacterial Nostoc sp. as well as firmicutes, beta-, gamma- and delta-proteobacteria. Acetylene reduction assays and nifH gene RNA transcript diversity identified Nostoc sp. as a main contributor of nitrogenase activity in these ponds. Furthermore, analytical methods were used to identify the cyanobacterial secondary metabolites microcystins, although the genetic basis for this production and the toxin producer could not been identified. However non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) genes were identified which could be the genetic basis for novel bioactives. The use of a multi-disciplinary approach synthesis and subsequent results significantly increased our understanding of the diversity and function of microbial mat communities in the unique meltwater ponds of the McMurdo Ice shelf, Antarctica.

Planar electromagnetic sensing system has been used as one of the NDT methods to evaluate the material properties i.e., to evaluate near-surface properties such as conductivity, permeability and dielectric properties. The applications of planar electromagnetic sensors will depend on both the characteristic of the sensor type chosen and also the characteristic of material under test. Conventional planar interdigital sensors and novel planar interdigital sensors have been designed, fabricated and tested for detection of dangerous marine biotoxins in seafood. Our main objective is to sense the presence of dangerous contaminated acid in mussels and other seafoods. Initial studies were conducted with three peptide derivatives namely Sarcosine, Proline and Hydroxylproline. These three chemicals are structurally closely related to our target molecule (domoic acid). The initial results have shown that all sensors respond very well to the chemicals and it is possible to discriminate the different chemicals from the output of the sensor. Novel interdigital sensors have shown better sensitivity measurement compared to conventional interdigital sensors. The novel interdigital sensors were then being tested with three seafood products. Results from the analysis have shown that novel interdigital sensor with configuration #1 (Sensor_1) has better sensitivity compared to other sensors. Sensor_1 has been chosen for experiment using proline and mussels. The changes in sensor sensitivity were analysed with mussels before and after adding the proline. The presence of proline on the mussel surface and also injected proline to the mussel samples were clearly detected by Sensor_1. Further experiment was conducted with small amount of domoic acid (0.5 µg to 5.0 µg) injected to a mussel and it was found that Sensor_1 was able to detect small amount of domoic acid (1.0 µg) injected into the mussel sample. Sensor_1 was able to detect approximately 12.6 µg/g of domoic acid in mussel meat. Three threshold levels of particular sample thickness have been established for detection of domoic acid. The first prototype of a low cost sensing system known as SIT (Seafood Inspection Tool) has been developed. The outcomes from the experiments provide chances of opportunity for further research in developing a low cost miniature type of sensors for reliable sensing system for commercial use.

Planar electromagnetic sensing system has been used as one of the NDT methods to evaluate the material properties i.e., to evaluate near-surface properties such as conductivity, permeability and dielectric properties. The applications of planar electromagnetic sensors will depend on both the characteristic of the sensor type chosen and also the characteristic of material under test. Conventional planar interdigital sensors and novel planar interdigital sensors have been designed, fabricated and tested for detection of dangerous marine biotoxins in seafood. Our main objective is to sense the presence of dangerous contaminated acid in mussels and other seafoods. Initial studies were conducted with three peptide derivatives namely Sarcosine, Proline and Hydroxylproline. These three chemicals are structurally closely related to our target molecule (domoic acid). The initial results have shown that all sensors respond very well to the chemicals and it is possible to discriminate the different chemicals from the output of the sensor. Novel interdigital sensors have shown better sensitivity measurement compared to conventional interdigital sensors. The novel interdigital sensors were then being tested with three seafood products. Results from the analysis have shown that novel interdigital sensor with configuration #1 (Sensor_1) has better sensitivity compared to other sensors. Sensor_1 has been chosen for experiment using proline and mussels. The changes in sensor sensitivity were analysed with mussels before and after adding the proline. The presence of proline on the mussel surface and also injected proline to the mussel samples were clearly detected by Sensor_1. Further experiment was conducted with small amount of domoic acid (0.5 µg to 5.0 µg) injected to a mussel and it was found that Sensor_1 was able to detect small amount of domoic acid (1.0 µg) injected into the mussel sample. Sensor_1 was able to detect approximately 12.6 µg/g of domoic acid in mussel meat. Three threshold levels of particular sample thickness have been established for detection of domoic acid. The first prototype of a low cost sensing system known as SIT (Seafood Inspection Tool) has been developed. The outcomes from the experiments provide chances of opportunity for further research in developing a low cost miniature type of sensors for reliable sensing system for commercial use.