No description available.

No description available.

Clostridium difficile is a Gram positive, anaerobic bacterium which produces two potent protein toxins, A and B. The genes for toxins A and B have been previously cloned and sequenced and lie within 1.4 kb of each other. Upstream and downstream boundaries between sequences shared by both toxigenic and nontoxigenic strains and those sequences which are unique to toxigenic strains were established. A toxigenic element was defined in C. difficile strain 10463 which is 19.6 kb in length and is comprised of five open reading frames, including the toxin A and B genes. One of these open reading frames is previously unidentified and is located upstream of toxin B. Products of Polymerase Chain Reaction (PCR) amplification of three regions in the toxigenic element: the upstream boundary, the downstream boundary, and the region between the toxin A and B genes, were all identical in length in six toxigenic strains, indicating that the toxigenic element is conserved among these strains. A short fragment unique to nontoxigenic strains and occupying the same position on the chromosome as the toxigenic element was identified. peR products of this region were identical in length in three nontoxigenic strains. Transcriptional analyses were undertaken using probes to each of the five open reading frames in the toxigenic element. Transcripts were detected for four of the open reading frames which are contiguous and transcribed in the same direction. In addition, a very large transcript, corresponding to the length of the four open reading frames and processing intermediates were detected, indicating that the toxin genes are cotranscribed. A promoter region and processing sites were identified. Sizes were determined for each of the individual transcripts which correspond well with the sizes of the open reading frames. Six toxigenic strains which vary considerably in toxin production were selected for analysis to determine whether DNA sequence variation could account for the observed differences in toxin production. DNA restriction fragment length polymorphisms were examined, toxin-specific transcripts were analyzed, and sequences of regulatory regions were determined and compared. Whereas quantitative differences in toxin-specific transcripts were found among the toxigenic strains, the remaining analyses showed that DNA sequences were conserved among these strains. / Ph. D.

Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i_b), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i_a) antigen, had little to no biological activity. When fractions containing i_a and i_b were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i_a or i_b, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i_a and i_b, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i_a and i_b were mixed. Monospecific antisera against purified i_a and i_b neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i_a and i_b proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i_a protein is heat stable (85° C/15 min) while i_b lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i_a, but not i_b, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor. / Ph. D.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.

Pore forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. PFTs have potential applications in devel-oping novel drug and gene delivery strategies. Although structural aspects of many pore forming toxins have been studied, very little is known about the dynamics and subsequent rupture mechanisms. In the first part of the thesis, a combined experimental and modeling study to understand the lytic action of Cytolysin A (ClyA) toxins on red blood cells (RBCs) has been presented. Lysis experiments are carried out on a 1% suspension of RBCs for different initial toxin concentrations ranging from 100 – 500 ng/ml and the extent of lysis is monitored spectrophotometrically. Using a mean field approach, we propose a non – equilibrium adsorption-reaction model to quantify the rate of pore formation on the cell surface. By analysing the model in a pre-lysis regime, the number of pores per RBC to initiate rupture was found to lie between 400 and 800. The time constants for pore formation are estimated to lie between 1-25 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Using this model, we are able to predict the extent of cell lysis as a function of the initial toxin concentration. Various kinetic models for oligomerization mechanism have been explored. Irreversible sequential kinetic model has the best agreement with the available experimental data. Subsequent to the mean field approach, a population balance model was also formulated. The mechanics of cell rupture due to pore formation is poorly understood. Efforts to address this issue are concerned with understanding the changes in the membrane mechanical properties such as the modulus and tension in the presence of pores. The second part of the thesis is concerned with using atomic force microscopy to measure the mechanical properties of cells. We explore the possibility of employing tapping mode AFM (TM-AFM) to obtain the elastic modulus of soft samples. The dynamics of TM-AFM is modelled to predict the elastic modulus of soft samples, and predict optimal cantilever stiffness for soft biological samples. From experiments using TM-AFM on Nylon-6,6 the elastic modulus is predicted to lie between 2 and 5 GPa. For materials having elastic moduli in the range of 1– 20 GPa, the cantilever stiffness from simulations is found to lie in the range of 1 – 50 N/m. For soft biological samples, whose elastic moduli are in the range of 10-1000 kPa, a narrower range of cantilever stiffness (0.1 – 0.6 N/m), should be used.

Este trabalho realizou importantes avanços na elucidação do potencial das toxinas termo-lábeis do tipo II (LT-IIs) como adjuvantes por via intradérmica e transcutânea. Os dados gerados indicam que LT-IIb e LT-IIc nativas atuam como potentes adjuvantes vacinais por via intradérmica induzindo respostas imunológicas antígeno específicas, como medido pela produção de anticorpos sistêmicos (IgG) e ativação de linfócitos T CD8+ citotóxicos. Soma-se ao potencial adjuvante demonstrado para as LT-IIs por essa via a baixa reatogenicidade das moléculas, com menores níveis de edema e reduzida migração leucocitária para o sítio de inoculação em comparação a LT-I. Os resultados indicam também que o efeito adjuvante das LTs aplicadas por via transcutânea pode estar relacionado à capacidade de ligação ao gangliosídeo GM1 já que a toxina LT-IIb, incapaz de interagir com este receptor, não apresenta efeito adjuvante por essa via. O conjunto de dados apresentados abre perspectivas para o emprego das LT-IIs nativas como adjuvantes parenterais em vacinas para animais e humanos. / This work has made significant advances in the understanding of the potential of type II heat-labile toxins (LT-IIs) as vaccine adjuvants by intradermal and transcutaneous route. The generated data indicate that native forms of LT-IIb and LT-IIc act as potent vaccine adjuvants when intradermally injected inducing antigen-specific immune responses, as measured by the generation of systemic serum antibody (IgG) and activation of cytotoxic CD8+ T lymphocytes. Besides the adjuvant effects, LT-IIs show reduced side effects, measured by the lesser edema formation and reduced leukocytes migration to the site of injection, in comparison to LT-I. The results also indicate that the adjuvant activity of LTs applied transcutaneously may be related to the the ganglioside GM1 binding property since the LT-IIb toxin, unable to interact with this receptor, has no adjuvant effect by this route. The presented data set opens prospects for the employment of native LT-IIs as parenteral adjuvants in vaccines for animals and humans.

As toxinas do envenenamento paralisante por moluscos (Paralytic Shellfish Poisoning – PSP) são compostos naturais bioativos conhecidos devido ao consumo acidental de frutos do mar contaminados. Estas moléculas, das quais a mais potente é a saxitoxina (STX), são uma classe de alcalóides neurotóxicos que possuem diferentes análogos e diferentes toxicidades, e são produzidas por algumas cianobactérias e algumas espécies de dinoflagelados do gênero Alexandrium, Gymnodinium e Pyrodinium. As toxinas paralisantes são neurotoxinas solúveis em água que agem sobre células nervosas e musculares através do bloqueio dos canais de sódio dependentes de voltagem, desta maneira, impedindo a condução do sinal no neurônio o que leva a uma paralisia muscular. Em casos graves, pode ocorrer morte por insuficiência respiratória. O envenenamento diarréico por moluscos (Diarrhetic Shelfish Poisoning – DSP) é caracterizado por problemas gastrointestinais com sintomas como diarréia, náusea, vômito, dor de cabeça, calafrios e dores abdominais. DSP é conseqüência do consumo de mariscos contaminados que ingeriram dinoflagelados do gênero Dynophysis e Prorocentrun através de sua alimentação por filtração da água. Contaminação de frutos do mar por toxinas PSP ou DSP coloca-se como sério problema para a indústria pesqueira e para a saúde pública. Neste estudo, estabeleceu-se um programa de monitoração para mexilhões (Perna perna) e para peixes (Sardinella brasiliensis, Anchoviella lepidentostole e Brevoortia aurea) coletados em peixarias e entrepostos de pesca no municípios de Caraguatatuba e São Sebastião, São Paulo. Os extratos para PSP foram preparados de duas maneiras: de acordo com a AOAC (Association of Official Analytical Chemists), através do aquecimento por 5 min de uma mistura de 100 g de tecidos homogeneizados com ácido acético 0,1 N; ou a partir da concentração de extratos etanólicos de músculo + pele dos peixes. Os bioensaios com camundongos para PSP consistem na injeção intraperitonial de 1 mL do extrato ácido em cada um dos três camundongos (~ 20 g). O animal é observado quanto aos sintomas clássicos de PSP e o tempo de morte é anotado e então a toxicidade é determinada (em mouse units, MU) pela tabela de Sommer. Para as toxinas causadoras de DSP, os extratos foram preparados pela extração com acetona do homogeneizado das glândulas digestivas, e a determinação da presença destas toxinas é feita através da injeção intraperitonial em camundongos. Nos bioensaios com os extratos preparados segundo o método da AOAC, não houve casos positivos. Para o bioensaio realizado com extratos etanólicos obtiveram-se resultados positivos para 77,8% dos extratos testados. A média de MU de todas as amostras, neste caso, foi de 0,147 MU/g. Nos bioensaios para DSP, três amostras resultaram em sinais que evidenciam a presença destas toxinas, pois os camundongos injetados apresentaram quadro diarréico. Os extratos etanólicos, com positividade para as toxinas de PSP, foram fracionados usando-se colunas Sep-Pak C18. A primeira eluição, com ácido acético 0,1 M, foi analisada usando-se o método de préderivatização e cromatografia líquida de alta eficiência com detecção de fluorescência. As analises em CLAE indicaram a presença de compostos semelhantes às toxinas paralisantes de PSP, confirmando os bioensaios. Portanto, pela primeira vez no Brasil demonstrou-se que as espécies S. brasiliensis, A. lepidentostole e B. aurea são portadoras de toxinas paralisantes, semelhantes às PSP, em pequenas concentrações e que um programa de monitoração é necessário em nosso país para verificação da presença dessas toxinas em organismos que são usados como alimento pela população. / The Paralytic Shellfish Poisoning (PSP) toxins are well-known natural bioactive compounds due to their accidental consumption in contaminated seafood. These molecules, of which the most potent representative is saxitoxin (STX), are a class of neurotoxic alkaloids, having different isoforms and varied toxicities, that are produced by some cyanobacteria and some species of dinoflagellates from the genus Alexandrium, Gymnodinium and Pyrodinium. PSP toxins are water-soluble neurotoxins that act on nerve and muscle cells by blocking sodium channels voltage-dependent, thus preventing the conductance of neuron signal leading to muscular paralysis. In severe cases, death may result due to respiratory failure. Diarrhetic Shellfish Poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP is usually a consequence of consuming contaminated shellfish that have ingested dinoflagellates of the genera Dinophysis and Prorocentrun through their filter feeding activities. Contamination of seafood by PSP and DSP toxins has posed serious problems to the fisheries industry as well to public health. In this study, was stabilized a monitoring program to shellfish (Perna perna) and finfish (Sardinella brasiliensis, Anchoviella lepidentostole and Brevoortia aurea) collected in fish markets in Caraguatatuba and São Sebastião cities, São Paulo state. The extracts for PSP were prepared by two ways: according to AOAC (Association of Official Analytical Chemists), through the heating for 5 min of blend of 100 g of well mixed sample with 0.1 N HCl; or through of the concentration of ethanolic extracts from finfish’s muscle + skin. The PSP mouse bioassay for PSP toxins involves intraperitonial injection (i.p.) of 1 mL of the acid extract into each of three mice (~ 20 g). The mice were observed for classical PSP symptoms and the time to mouse death was recorded and the toxicity was determinate (in mouse units, MU) from the Sommer’s table. To DSP toxins, the extracts was prepared trough the extraction of digestive glands with acetone, and i.p injection in mice was used to determine the presence of theses toxins. In the mouse bioassay for the extracts prepared by AOAC method no positive results was obtained. For the mouse bioassay with ehtanolic extracts was obtained positive results to 77.8 % of the tested extracts. The media of MU of all samples, in this case, was 0,147 MU/g. To the mouse bioassay for the DSP toxins, three samples gives evidence of presence of the diarrhetic toxins, because the mice showed signal like diarrhea. The ethanolic extracts, that was positive to the PSP toxins, was fractionated by a Sep-Pak C18 cartridge. The first elution, with 0.1 M acetic acid, was analyzed by using prechromatographic oxidation and liquid chromatography with fluorescence detection. The HPLC analysis indicated the presence of the PSP toxins, confirming the bioassays. Therefore, in the first time in Brazil was demonstrated that the species S. brasiliensis, A. lepidentostole and B. aurea are carriers of toxins like PSP in little concentrations and that a monitoring program is necessary in our country to verify the presence of these toxins in organisms that are used as food by the population.

Major constituents of the venom of various animals are peptidogenic in nature. Marine snails belonging to the species Conus are venomous predators that use small, structurally constrained peptides present in their venom for prey capture and defense. It is known that ~500 Conus species are present in nature and the venom of each of these Conus species is a complex mixture of nearly 100 peptides accounting for > 50,000 peptides with little overlap among the different species. The peptides isolated from the venom of Conus species are commonly known as conotoxins or conopeptides. Some of the common targets of these peptides include the different ion channels like Na+, K+, and Ca2+, and receptor subtypes such as nicotinic acetylcholine and NMDA receptors. The ion channels and receptor subtypes were targeted by conopeptides with high degree of specificity and selectivity. The structural information on the peptides from cone snails can prove to be a valuable starting tool for the understanding of the function of different ion channels and hence in the design of neuropharmacologically active drugs. Conotoxins are disulfide-rich peptides and the number of disulfide generally ranges from two to five. Based on the arrangement of cysteines in their primary sequence, they are classified into different superfamilies. The signal sequences of the precursors belonging to a particular superfamily are highly conserved and hence the members within the same family have, in common, the unique disulfide arrangement and pharmacological activity. Conotoxins are classified into eleven superfamilies till date. In order to understand the underlying the principles involved in the action of these peptides on different ion channels, one needs to know the three-dimensional structures which, in potential, will help in the identification of the pharmacophores responsible for the observed pharmacological activity. With the aim of studying the structure-activity relationships found among the conotoxins, we have initiated a study on the peptides isolated from the marine snails found in the Indian coastal waters. This thesis is focused in the structural studies of the peptide toxins from marine cone snails and a terrestrial scorpion. The tool used for the structural studies of these peptide toxins is Nuclear Magnetic Resonance Spectroscopy. Chapter 1 provides an overview of the peptide toxins found among various animal species with more emphasis on conotoxins and scorpion toxins. In addition, the rationale behind the present study has also been explained. Chapter 2 describes the structure determination of two conopeptides isolated from Conus amadis, δ-Am2766 and Am2735, which are active on mammalian sodium channels. The structural aspects and comparison with other known conopeptides belonging to the same superfamily as that of these two peptides have also been described. Solution NMR studies of Ar1446 and Ar1248, two conopeptides isolated from the species Conus araneosus have also been studied using Homonuclear NMR methods. Ar1446 is a three disulfide-bonded peptide. Our studies have revealed that this peptide has a novel disulfide connectivity not previously observed in the M superfamily or any other superfamily of conotoxins. The structural features of Ar1446 will be described along with the NMR studies on two-disulfide bonded peptide, Ar1248, belonging to the A-superfamily of conotoxins. The main problem faced in the kind of study of peptides isolated from natural sources is the amount that can be isolated and purified to homogeneity. In order to obtain large quantities of peptides, we have successfully used Cytochrome b5 as fusion host to clone, over express and purify these peptides using recombinant methods. The use of recombinant methods has aided in the preparation of isotopically enriched peptides. The use of cyt b5 as fusion host for the large scale production of some of the peptides from Indian marine snails is described in Chapter 4. A novel pharmacologically active linear peptide, Mo1659 isolated from Conus monile, have been studied using Heteronuclear NMR methods. This peptide was cloned, over expressed and purified using Cytochrome b5 as a fusion host. Another linear peptide, Mo1692 (also from Conus monile), has been prepared using the same method and was studied using Homonuclear NMR methods. Both these peptides were liberated from the fusion host using cyanogen bromide cleavage and were subsequently purified using RP-HPLC. The results of the biosynthetic preparation and NMR studies of these two peptides have been described in Chapter 5. Chapter 6 describes the solution structure determination of a novel scorpion toxin characterized in the venom of the Indian red scorpion Buthus tamulus. The cloning, over expression, folding and purification of BTK-2 is described here. The structure and the function of this recombinantly produced BTK-2 will also be described.

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: [70]-74)