This study reports the findings on the effect of Microcystin-LR (MCLR) on the gastrointestinal tract cells of mice and on two different cell lines, Caco2 and MCF-7. The cyanobacterium Microcystis aeruginosa produces the potent toxin, MCLR. This toxin has been implicated in a number of cases of ill-health. It was decided to investigate whether microcystin-LR induced apoptosis in the gastrointestinal tract of mice and also which possible mechanisms were involved in the induction in vitro. Balb/c mice were given a 75% LD50 intraperitoneal dose of pure microcystin -LR and sacrificed at 8, 16, 24 and 32 hours post-exposure. The small intestinal sections were stained with haematoxylin and eosin and examined for apoptotic cells. There was a time-dependent increase in the number of apoptotic cells with most in the duodenum and the jejunum. No change in glycogen content was evident at 24 hours post exposure when PAS-stained sections were examined. To determine that microcystin was the agent responsible for the changes, fluoroscein isothiocyanate (FITC) immunostaining for the toxin was done on the sections. Apoptosis in vitro was investigated in Caco2, a cell line that behaves like normal enterocytes when the cells are differentiated at confluency, and MCF-7, a breast cancer cell line deficient in pro-caspase-3, cells by 3-[dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and by staining with DAPI and Rhodamine 123. MCLR exposure induced apoptosis, as seen in decreased cell viability and increased leakage of LDH, as well as mitochondrial damage shown by Rhodamine staining. The MCF-7 cells, deficient in pro-caspase-3, and Caco2 cells did not show cleavage of poly(ADP)ribose polymerase (PARP) after exposure to 50μM MCLR after 72 hours exposure. Both micro- and milli-calpain activity was however significantly increased in both cell lines exposed to the toxin. There was a significant increase in H2O2, one of the key reactive oxygen species, production during the first 30 minutes that the cells were exposed to 50 mM MCLR.

A module and an inoculation technique were developed that would allow for the efficient immobilization of Microcystis aeruginosa PCC7806 on a synthetic membrane. A variety of module types, membranes (ceramic, tubular polyethersulfone and externally skinless polyethersulfone capillary membrane), and methods of immobilization (adsorption, pressure filtration and a developed technique that involved drying a cell slurry on a membrane) were assessed. The morphological properties that affected the immobilization of Microcystis aeruginosa PCC7806, as well as the effects of immobilization upon cell morphology were assessed. Cells in the stationary growth phase, which had a well-developed extra-cellular polysaccharide layer and no gas vesicles, were optimal for immobilization. Microcystin production under immobilized conditions was assessed under different nitrate concentrations, light intensities, biofilm thickness and immobilization times. Additional work included assaying for Microcystin production of two airlift-grown cultures under a high light intensity and complete nutrient deprivation and the inoculation of a ceramic membrane. An immunological technique was used to elucidate where toxin production was greatest within a biofilm immobilized upon an externally skinless polyethersulfone capillary membrane. The externally skinless polyethersulfone capillary membrane was evaluated to assess homogeneity and the physical differences between membrane batches that led to the erratic, incomplete biofilm formation, as a biofilm of a constant thickness could not be immobilized. Microcystis aeruginosa PCC7806 was exposed to a variety of solvents in order to permeabilize the cyanobacteria, as that would have enabled a truly continuous extraction process for the metabolite. FDA hydrolysis had to be optimized in order to use it as an indicator of cell viability. In addition a single-step extraction of Microcystin was attempted using live bacteria. A capillary membrane module, containing the externally skinless polyethersulfone capillary membrane, inoculated using pressure filtration, was the most efficient combination to establish a biofilm. Cells that were no longer actively dividing and that lacked buoyancy displayed superior immobilization to cells that were actively dividing and buoyant. The immobilized cells did produce Microcystin but in much lower concentrations to cells grown in an airlift culture. Biofilms grown with a higher nitrate concentration, a lower biofilm thickness and a lower light intensity had a higher specific microcystin content, while biofilms with a higher nitrate concentration a lower light intensity and a longer growth period displayed the a greater toxin production per mm2 of membrane. Microcystin occurred at its highest concentration in cells just above the pore opening. The diffusion of nutrients occurred relatively quickly to the outside layers of the biofilm, with a true gradient being established laterally from these nutrient veins that were above the pores. Permeabilization of the cells proved unsuccessful, as cells that remained viable did not release the intracellular compound into the surrounding medium.

Identifica los tipos de RL contenidos en las bandas de un halo revelado en agar CTAB/MB. Desarrolla una técnica para extraer RL a partir del agar CTAB/MB implementando, además, una metodología de superficie de respuesta Box-Behnken que maximizó las separaciones entre las 4 bandas de un patrón de RL producidos por P. aeruginosa 6K-11. Identifica, mediante UPLC-MS/MS, 9 congéneres de RL en la banda 1 (RC8C10, RC10C8, RC10C10, RRC8C10, RRC10C8, RRC10C10, RRC10C12:1, RRC12C10 y RRC10C12), 7 en la banda 2 (RC8C10, RC10C8, RC10C10, RRC8C10, RRC10C8, RRC10C10 y RRC10C12:1), 4 en la banda 3 (RC10C10, RRC8C10, RRC10C8 y RRC10C10) y 3 en la banda 4 (RRC8C10, RRC10C8, RRC10C10). La estructura molecular es probablemente el factor más importante en la migración de los RL puesto que se observa que la relación de la presencia de isómeros se mantiene en las cuatro bandas. Además de existir predominancia de diRL. Concluye que la cantidad y la diversidad de tipos de RL disminuye en la migración de cada banda según su complejidad estructural (cantidad de ramnosas, isomería y masa) lo que comprueba las diferencias en cuanto a composición química y abundancia de los RL que se encuentran dentro de cada banda. / Tesis

Tesis presentada a la Universidad de Chile para optar al grado académico de Magíster en Bioquímica, área de especialización Toxicología y Diagnóstico Molecular y Memoria para optar al Título de Bioquímico / Las bacteriocinas son péptidos antimicrobianos de síntesis ribosomal producidos por microorganismos, principalmente del dominio bacteria. Las bacteriocinas producidas por bacterias Gram positivo o Gram negativo, presentan una actividad tóxica sobre especies bacterianas estrechamente relacionadas con la bacteria productora. Para ejercer su acción antimicrobiana las bacteriocinas utilizan receptores específicos localizados exclusivamente en la bacteria blanco, por lo tanto raramente se observa actividad citotóxica sobre células eucariontes. No obstante, algunas bacteriocinas inhiben específicamente el crecimiento de líneas celulares neoplásicas. Debido a estas propiedades, las bacteriocinas tienen una potencial aplicación biotecnológica por su posible uso como alternativa a los tratamientos con antibióticos tradicionales, o como nuevos antibióticos contra patógenos multirresistentes, como preservantes o desinfectantes en la industria alimenticia, y en el área biomédica como antineoplásicos para combatir algunos tipos de cáncer. En nuestro laboratorio encontramos un nuevo compuesto antimicrobiano del tipo bacteriocina producido por la cepa de Pseudomonas aeruginosa O400, al que hemos denominado gicina A. Este compuesto es capaz de inhibir, selectivamente, el crecimiento de bacterias Gram positivo. Debido a que no existen informes en la literatura de bacteriocinas producidas por bacterias Gram negativo que presenten actividad antimicrobiana solamente sobre bacterias Gram positivo, el estudio del mecanismo de acción y las propiedades bioquímicas de gicina A constituye un valioso aporte en la búsqueda de nuevas sustancias antimicrobianas. El objetivo general de esta tesis fue “Purificar, caracterizar bioquímicamente, y evaluar tanto el mecanismo de acción antibacteriano de gicina A, como su toxicidad in vitro sobre líneas celulares eucariontes”. Para lograr este objetivo se realizó la purificación de gicina A mediante cromatografías en columnas hidrofóbicas y HPLC. Mediante electroforesis de proteínas y espectrometría de masas se determinó que esta proteína tiene una masa molecular de 7925 Da. La evaluación de sus propiedades bioquímicas demostró que la actividad de gicina A es estable a un amplio rango de temperaturas, conserva su actividad al ser solubilizada en una amplia gama de solventes y presenta una mayor actividad al ser solubilizada a un pH neutro. Por otra parte empleado gicina A purificada se confirmó que esta posee una actividad antibacteriana selectiva por bacterias Gram positivo. Estudios de alteración de la cinética de crecimiento de la bacteria blanco y ensayos de microscopía de fluorescencia indicaron que gicina A posee un efecto bactericida sobre bacterias Gram positivo y que este efecto es producido por una permeabilización en la membrana celular. La evaluación de la citotoxicidad in vitro de gicina A sobre células eucariontes mostró que esta bacteriocina ejerce una leve toxicidad en las líneas de origen tumoral, sin embargo no se observó efecto citotóxico sobre las líneas celulares no tumorales. / Bacteriocins are antimicrobial peptides ribosomally synthesized produced by microorganisms, mainly from Bacteria domain. Bacteriocins produced by Gram positive or Gram negative bacteria, present a toxic activity on bacterial species closely related to the bacteriocin producing bacteria. To exert its antimicrobial action, bacteriocins use specific receptors exclusively located in the target bacteria, therefore rarely cytotoxic activity on eukaryotic cells can be observed. However, some bacteriocins specifically inhibit growth of neoplastic cell lines. Because of these properties, bacteriocins have a potential for biotechnological application for its possible use as an alternative to the traditional antibiotic treatments, or as new antibiotics against multi-resistant pathogens, also as preservatives or disinfectants in food industry, and in biomedical field as antineoplastics. In our laboratory we have found a new antibacterial compound with bacteriocin properties produced by the Pseudomonas aeruginosa O400 strain, which we called gicin A. This compound is able to selectively inhibit the growth of Gram positive bacteria. Because there are no reports in the literature of bacteriocins produced by Gram negative bacteria that have antimicrobial activity only on Gram-positive bacteria, the study of the mechanism of action and biochemical properties of gicin A is a very valuable contribution to the search of new antimicrobial substances. The overall aim of this thesis was to “Purify, characterize biochemically, and assess both the antibacterial action mechanism of gicin A, and its in vitro toxicity on eukaryotic cell lines”. To achieve this objective the purification of gicin A was performed by a hydrophobic column chromatography and HPLC. Protein electrophoresis and mass spectrometry determined that this protein has a molecular mass of 7925 Da. The evaluation of its biochemical properties showed that the activity of gicin A is stable at a wide temperature range, it retains its activity when is solubilized in a wide range of solvents and has a higher activity when is solubilized at a neutral pH. Moreover, employing purified gicin A was confirmed that it has a selective antibacterial activity against Gram positive bacteria. Alteration studies of the growth kinetics of the target bacteria and fluorescence microscopy indicated that gicin A has a bactericidal effect on Gram positive bacteria and that this effect is produced by cell membrane permeabilization. The evaluation of cytotoxicity in vitro of gicin A on eukaryotic cells shows that this bacteriocin has a mild toxicity on tumoral origin cell lines, however we noticed no cytotoxic effect on non tumoral origin cell lines.

No description available.

Please read the abstract in the section 00front of this document / Dissertation (MSc(Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

The nature of the reserves of Pseudomonas aeruginosa that are oxidized during endogenous respiration was studied by following the changes in the chemical constituents and in the distribution of radioactivity of starving cells that had been grown on C¹⁴-labeled substrates. The total protein and nucleic acid of cell suspensions decreased during starvation. Deoxyribonucleic acid increased slightly, whereas ribonucleic acid decreased. C¹⁴O₂ was evolved from endogenously respiring cells specifically labeled in the nucleic acid fraction and from cells specifically labeled in the protein fraction. Chemical fractionation of C¹⁴-labeled cells showed a decrease in hot trichloroacetic acid-soluble and insoluble compounds, indicating that the C¹⁴O₂ arose from the degradation of RNA and protein and not from free pool compounds. A decrease in ribosomal RNA and protein was evident from physical fractionations of starved labeled cells. An enzyme responsible for the initiation of ribosomal degradation was found to be associated with the ribosome fraction and was identified as polynucleotide phosphorylase. The enzyme was inactive in high magnesium concentrations but was active under conditions which allowed the dissociation of the large ribosomal units into 50S and 30S components. Polynucleotide phosphorylase was not solubilized by the dissociation of the 70S ribosomes but remained firmly attached to the 50S subunit. The oxidation of exogenous substrates resulted in varying degrees of suppression of the oxidation of endogenous RNA and this suppression was attributed to the relative stabilizing effect that the exogenous substrates exerted on the ribosomes. The oxidation of endogenous protein was depressed during the oxidation of exogenous glucose, aspartic acid and adenosine and was increased during the oxidation of α-ketoglutaric acid. The response of endogenous respiration to the oxidation of exogenous substrates appeared to be related to a requirement for ammonium ions for assimilation of carbon. / Land and Food Systems, Faculty of / Graduate

The response of Pseudomonas aeruginosa to phosphate starvation and subsequent refeeding was studied by following changes in turbidity, cell count and chemical composition on incubation in phosphate deficient medium. In shaken, phosphate deficient cultures, the turbidity and viable cell count were shown to increase significantly, with the latter reaching a maximum level at 24 hours under the conditions employed. A linear response of phosphate starved cells to low levels of phosphate supplied exogenously was evident from turbidity measurements, and a threshold requirement for phosphate analogous to the "energy of maintenance" (McGrew and Mallette, 1962) was not detected. In still, phosphate deficient cultures, the turbidity and total cell count increased and the viable cell count decreased slightly at 24 hours. The levels of protein and deoxyribonucleic acid (DNA) per ml of culture increased during this period, and the amount of ribonucleic acid (RNA) decreased. Extensive ribosomal degradation was apparent from sucrose density gradient centrifugation patterns. An enzyme having an alkaline pH optimum and displaying activity against a wide variety of phosphomonoesters was demonstrated in phosphate-starved cells. The enzyme was inhibited by inorganic phosphate, and was considered to be the counterpart of the repressible phosphomono-esterase reported in other microorganisms and studied in detail in Escherichia coli (Torriani, i960; Garen and Levinthal, I960; Heppel, Harkness and Hilmoe, 1962). The enzyme activity of cell free extracts of P. aeruginosa was shown to be associated mainly with the ribosomal fraction. / Land and Food Systems, Faculty of / Graduate

The non-phosphorylated oxidative pathway of glucose dissimilation has been established in Pseudomonas aeruginosa and evidence for phosphorylated pathways, other than the Embden-Meyerhof scheme, has been obtained. In the present study the non-phosphorylated and phosphorylated pathways of glucose degradation have been investigated with cell-free extracts of this organism. Gluconolactone was shown to be an intermediate in the oxidation of glucose to gluconic acid. The enzymatic hydrolysis of the lactone ring has an absolute magnesium ion, or divalent cation requirement. In the presence of phosphate buffer magnesium was chelated and effectively removed from participation in the enzymatic reaction. As has been reported in the literature, the product of glucose and gluconic acid oxidation was identified as 2-ketogluconate. In the presence of adenosine triphosphate (ATP), glucose and gluconate are phosphorylated and the kinases involved, therefore, link the non-phosphorylated with the phosphorylated pathways. The demonstration of triphosphopyridine nucleotide (TPN) linked dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, as well as the production of glucose-6-phosphate and 3-phosphoglyceraldehyde from cell-free extracts with gluconate or ribose plus ATP, illustrated the presence of a functional pentose phosphate cycle in this organism. An active 6-phosphogluconate dehydrase and a 2-keto-3-deoxy-6-phosphogluconate aldolase were demonstrated by the production of pyruvic acid from 6-phosphogluconate and indicated the presence of the Entner-Doudoroff pathway. The oxidation of 3-phosphoglyceraldehyde to 3-phosphoglyceric acid initiated by a TPN specific 3-phosphoglyceraldehyde dehydrogenase, and the conversion of phospho-enol-pyruvate to pyruvic acid was shown. It is suggested that the trioses are immediately concerned in the observed CO₂ fixation by this organism. Fructose-1,6-diphosphate aldolase, fructose-1,6-diphosphate phosphatase and phosphohexoisomerase may be involved in the formation of glucose-6-phosphate from triose phosphates. A direct link between 2-ketogluconate and the phosphorylated pathways could not be shown but the reduction of the phosphate ester of the compound was demonstrated. The feasibility of 2-ketogluconate undergoing a 3:3 split is presented. No attempt has been made to estimate the relative importance of the various pathways of glucose dissimilation as it is felt that this is determined by the conditions and stages of growth of the organism. / Land and Food Systems, Faculty of / Graduate

An effort has been made to demonstrate that the major pathway for glucose oxidation in Pseudomonas aeruginosa (ATCC 9027) involves the sequence of reactions: glucose →gluconate → 2-ketogluconate → 2-keto-6- phosphogluconate → 6-phosphogluconate. It appears however, that extracts of this organism are capable of phosphorylating glucose directly, that is, to yield glucose-6-phosphate and subsequently 6-phosphogluconate. A study of this latter pathway was felt to be necessary in order to evaluate the likelihood of it being a major alternative to the established non-phosphorylated pathway. Since it is known that glucose-6-phosphate dehydrogenase from P. aeruginosa and other microorganisms as well as from certain animal tissues exhibits a marked sensitivity to various nucleotides particularly to adenosine triphosphate. A study of this inhibition was made in order to assess the possible role that this sensitivity may play in determining the importance of this pathway as the major route of glucose oxidation. Enzyme fractionation studies revealed that hexokinase and glucose-6-phosphate dehydrogenase could be separated either by an ethanol fractionation or by an alkaline ammonium sulfate fractionation. The best separation of dehydrogenase was obtained using ethanol although hexokinase could only be isolated using the alkaline ammonium sulfate method. Cell free extracts of P. aeruginosa oxidize glucose to 2-keto-gluconate but carry the reaction no further. This represents a consumption of l µM of oxygen per µM glucose. In the presence of ATP the amount of oxygen consumed was reduced to a maximum of 0.5 µM per µM glucose, indicating the accumulation of a compound less oxidized than 2-ketogluconic acid. 6-phosphogluconate appeared to conform to the requirements of such a compound. Chromatographic analysis of reaction mixtures containing ATP revealed the accumulation of a phosphorylated compound which could not be identified. Under in vitro conditions both pathways appear to be operable but the non-phosphorylated pathway accounts for most of the glucose in the metabalizing organism. / Land and Food Systems, Faculty of / Graduate