No description available.

In this study, high-performance liquid chromatography (HPLC) was employed to detect and quantify pyrimidine salvage enzymes by monitoring the disappearance of substrates or formation of products.

Purification of lipase from Pseudomonas aeruginosa (from both a washed cell suspension and crude culture supernatant as the enzyme source) was performed utilizing affinity chromatography. Affinity chromatography was carried out using n-dodecylamine bound to Sepharose 4B. Chromatography of the concentrated crude culture supernatant resulted in a 65 to 95 fold purification with 5.8% recovery. Washed cells collected from a ten hour culture suspended in water also produced enzyme. Activity of the washed cell suspension supernatant was found to be 4.5 fold higher than the activity of the culture supernatant. A thirty percent recovery was obtained using the washed cell suspension supernatant. The washed cell suspension provides a cleaner preparation for use with the dodecylamine-agarose chromatography in purifying the enzyme.

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No. of bitstreams: 1 Dissertação- Mary Joyce Targino Lopes Magalhães.pdf: 1466870 bytes, checksum: 3202ba13f65ece33fb3c071651d2a444 (MD5) Previous issue date: 2013-07-26 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Pseudomonas aeruginosa is a leading bacterial cause of nosocomial infections. Possessing aquatic habitats, it presents a good indicator of water contamination. To verify that this bacterial species represents a potential source of contamination to the Mindu stream, we must carry out and analyze the following objectives: first, identify isolates of P. aeruginosa in samples of hospital effluent surface water obtained from the Mindu stream and second, to check whether the strains posses virulence factors as related mobility scourge, "twitching motility", biofilm formation and antimicrobial resistance, and finnaly, to assess the genetic similarity between isolates. Method: To identify the microbiological and biochemical composition, the 16S rRNA gene sequencing was used. For phenotype characterization we performed the following tests: first, we tested for mobility, using the scourge test "twithing motility", second we tested the biofilm formation and profile of antimicrobial resistance using the disk diffusion technique, the genetic similarity among isolates found was determined by PFGE. Results: We identified 17 isolates of P. aeruginosa in effluent water from the hospital. 8 isolates of the same species were in the surface water of the Mindu stream. The strains tested with mobility scourge; 100 % of the strains were found in water Mindu and 88 % of the strains were found in the hospital´s effluent samples were positive for " twitching motility ". All of the 25 isolates studied showed biofilm formation and more than 70 % of the strains found in hospital´s effluent water (raw and treated) had the phenotype of multidrug resistance.100 % of P. aeruginosa isolates found showed resistance to Ampicillin and 50 % of the strains were intermediately resistant to ceftriaxone. Among all the samples, there was genetic similarity; in the hospital´s effluent water and the hospital´s treated sewage, samples found in different seasonal periods, and among isolates found in hospital effluent and surface water of Mindu. Conclusion: The presence of P. aeruginosa containing virulence factors in surface water samples is indicative of the spread of nosocomial origin of microorganisms in the aquatic environment studied. There is strong evidence that the system of sewage treatment in the study is not efficient. Contaminants from P. aeruginosa containing multidrug resistance were in the samples of treated effluent water, and showed high genetic similarity among isolates of P. aeruginosa derived from raw wastewater. Therefore, it is necessary for action and more oversight on the part of health surveillance agencies with health services so that they meet the requirements of the laws in force in order to preserve the environment and people's health. / Pseudomonas aeruginosa é uma das principais bactérias causadora de infecções hospitalares, e por possuir habitat comumente aquático apresenta-se como boa indicadora de contaminação de águas, para verificar se essa espécie bacteriana representa uma fonte de contaminação em potencial para o igarapé do Mindu, o estudo se propôs a analisar os seguintes objetivos: identificar isolados de P. aeruginosa em amostras de efluentes hospitalares e água superficial do igarapé do Mindu; verificar se as cepas encontradas apresentavam fatores de virulência como, mobilidade ligada ao flagelo, “twitching motility”, formação de biofilme e resistência aos antimicrobianos, e avaliar a similaridade genética entre os isolados. Metodologia: Para a identificação microbiológica foram realizados testes bioquímicos e o sequenciamento do gene 16S rRNA dos isolados encontrados; Para a caracterização fenotípica foram realizados os seguintes testes: teste de mobilidade ligada ao flagelo, teste “twithing motility”, teste de formação de biofilme e perfil de resistência aos antimicrobianos pela técnica de disco-difusão; A similaridade genética entre os isolados de encontrados no estudo foi determinada por PFGE. Resultados: Foram identificados 17 isolados de P. aeruginosa em efluentes hospitalares e 8 isolados da mesma espécie na água superficial do igarapé do Mindu; Todas as cepas estudadas apresentaram mobilidade ligada ao flagelo; 100% das cepas encontradas na água do Mindu e 88% das cepas encontradas em amostras de efluentes hospitalares apresentaram “twitching motility” positivo; todos os 25 isolados estudados apresentaram formação de biofilme; mais de 70% das cepas encontradas nos efluentes hospitalares (brutos e tratados) apresentaram o fenótipo da multirresistência e 100% dos isolados de P aeruginosa encontrados na água do Mindu apresentaram resistência à Ampicilina e 50% dessas cepas apresentaram resistência intermediária a Ceftriaxona; houve similaridade genética entre isolados encontrados em efluente hospitalar bruto e efluente hospitalar tratado, entre isolados encontrados em diferentes períodos sazonais e entre isolados encontrados em efluentes hospitalares e água superficial do Mindu. Conclusão: A presença de P. aeruginosa contendo fatores de virulência, em amostras de água superficial, é indicativo de disseminação de microrganismos de origem nosocomial no ambiente aquático estudado. Há fortes indícios de que o sistema de tratamento de efluentes do estudo não está sendo eficiente, já que foram encontradas cepas de P. aeruginosa contendo fatores de virulência, inclusive a multirresistência em amostras do efluente tratado; e foi observada alta similaridade genética entre isolados de P. aeruginosa oriundos de efluente bruto e efluente tratado. Por isso, se faz necessário maior fiscalização por parte dos órgãos de vigilância sanitária junto aos serviços de saúde para que sejam cumpridas as exigências das legislações vigentes a fim de preservar o meio ambiente e a saúde da população.

La fibrose kystique (FK) est une maladie génétique autosomale récessive conduisant à une défaillance pulmonaire mortelle. Celle-ci est causée par l’expression dysfonctionnelle de l’allèle CFTR codant pour une protéine transmembranaire impliquée dans le transport Cl- des cellules de l’épithélium pulmonaire vers la lumière des voies respiratoires. Cette mutation induit la production d’un mucus visqueux qui entrave les voies respiratoires et favorise l’accumulation de bactéries pathogènes. L’éradication de cette présence bactérienne est maintenant l’enjeu primordial chez les patients FK afin de procurer un bien-être et de prolonger l’espérance de vie de ceux-ci. Le cheval de bataille de cette lutte au mieux-être du patient FK s’appuie en partie sur l’efficacité des antibiotiques. Cependant, nous nous heurtons à la capacité d’adaptation des bactéries face à l’antibiothérapie, ce qui nous conduit à développer sans cesse de nouvelles molécules thérapeutiques. Ainsi, l’essence de ce projet de recherche a été de caractériser l’efficacité de la tomatidine (TO); l’aglycone de la tomatine (un glycoalcaloïde), produit par les plants de tomate et de son analogue : la molécule FC04-100. Par le passé, notre laboratoire a établi que la TO possède une action antibactérienne contre Staphylococcus aureus prototype via l’inhibition de l’expression des facteurs de virulences associés au système de régulation ARG ainsi qu’en agissant comme potentialisateur d’action des aminoglycosides (AMI). De plus, la TO est également un inhibiteur de la réplication intracellulaire de S. aureus small-colony variant (SCV) infectant des cellules épithéliales pulmonaires différenciées (Calu-3). Le premier volet de mon projet a été consacré à l’évaluation du potentiel d’action de la TO contre S. aureus et Pseudomonas aeruginosa; deux bactéries fréquemment co-isolées des poumons des patients. Ainsi, lors de cette étude, j’ai démontré pour la première fois que la TO peut être employée seule contre S. aureus en tant qu’agent bactéricide lorsque celle-ci est utilisée en présence de P. aeruginosa. Ce phénomène dépend de la production par P. aeruginosa, du 2-heptyl-4-quinolone-N-oxide (HQNO) de l’endopeptidase LasA. De plus, j’ai évalué la possibilité d’utiliser un AMI et TO lorsque S. aureus et P. aeruginosa sont en co-culture afin de réduire la population bactérienne de ces deux pathogènes. Les résultats obtenus ont permis de démontrer qu’une combinaison, de tobramycine (TOB) et TO, permet d’inhiber significativement la croissance bactérienne d’un S. aureus résistant à la méthiciline (MRSA) résistant à la TOB et P. aeruginosa, co-cultivés en condition planctonique. Le deuxième volet de ma maîtrise visait à mesurer l’efficacité antibactérienne de FC04-100 en collaboration avec ma collègue Isabelle Guay. Pour ce projet, j’ai démontré l’efficacité de la combinaison FC04-100 et la gentamicine (GEN) contre un biofilm de S. aureus. Pour ce faire, j’ai utilisé une méthode de culture en microplaque 96 puits, permettant ainsi de former plusieurs bioflims et de tester plusieurs concentrations d’antibiotiques. Les résultats suite à l’exposition des biofilms à la combinaison TO-GEN ont démontré qu’il était possible de réduire significativement la viabilité bactérienne de S. aureus en biofilm. De plus, j’ai comparé l’efficacité de FC04-100 à TO dans un essai d’infection de cellules Calu-3 différenciées et infectées par une souche clinique de S. aureus SCV. Les résultats révèlent que ces deux composés diminuent significativement la viabilité bactérienne, et ce, en proportion similaire par rapport aux cellules infectées non traitées. L’ensemble des résultats obtenus dans ces deux projets a permis de démontrer clairement le potentiel antimicrobien de la TO et de son analogue FC04-100. Cette découverte apporte donc un nouveau squelette de molécule qui pourrait s’avérer utilisable comme antibiotique.

The sialidases are a group of glycosyl hydrolases that specifically remove terminal sialic acid (Neu5Ac) residues from various glycans. In the two common human pathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these enzymes have been shown to be key virulence factors directly involved in bacterial colonization and infection. However, little is known about their detailed structural and mechanistic features and lack of this information significantly slows down the progress of new drug discovery targeting these enzymes. Therefore, we embarked structural and kinetic studies towards the three distinct sialidases (designated as NanA, NanB and NanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA) from P. aeruginosa. Full-length NanA failed to crystallize due to the presence of some natively disordered regions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was crystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with close contacts between the two monomers. The second pneumococcal sialidase NanB only shares 24% sequence identity with NanA. Crystal structure of NanB was also determined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In general, the core catalytic domain of both CNanA and NanB adopts the classic six- bladed β-propeller fold (or called sialidase fold), with a set of highly conserved residues stacking around the proposed active sites. NanC is a close homologue of NanB, sharing over 50% sequence identity. However, NanC crystallization is not successful so far. To compare the three sialidases in more detail, a computational NanC model was made based on the structure of NanB. Mapping of the active sites of CNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the probe. Although sharing many common features, NanA, NanB and NanC present different topologies around the catalytic centre, give these enzymes a high level of diversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR studies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to be an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles multiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3- sialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S. pneumoniae thus expresses NanA, NanB and NanC for disparate but cooperative roles. Such a working pattern of three sialidases in one microbe is unusual in nature, which might be essential for pneumococcal pathogenesis at various stages. Based on the crystal structures of CNanA and NanB, preliminary work towards S. pneumoniae sialidases inhibitor design is under way, in which, a variety of techniques, such as the fluorescence-based thermal shift assay, NMR spectroscopy, computational docking and X-ray crystallography, are incorporated in. The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a unique trimer in crystal that is associated, in part, by the immunoglobulin-like trimerization domain around a three-fold crystallographic axis. The core catalytic domain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase activity was detected with this enzyme. In addition, two key catalytic residues including one of the arginine in the arginine triad and the acid/base catalyst aspartic acid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate selectivity towards pseudaminic acid, which is a specific carbohydrate superficially similar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed mutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA into a poor sialidases. Moreover, the crystal structure of PaNA also indicates that His45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic acid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315 to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA could be a pseudaminidase with structural and mechanistic variations. This enzyme, together some other uncharacterized fellow proteins, might form a novel subclass in the sialidase superfamily. The various findings in the current projects provide meaningful insights towards several sialidases that have been linked to bacterial virulence, which may contribute to a more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression control (Crc) protein was indirectly involved in pyr gene regulation, and shown to negatively regulate expression of PyrR at the posttranscriptional level.

El presente trabajo investigó la adición de un inóculo de Pseudomonas aeruginosa en ciertos efluentes grasos recolectados de locales de expendio de comida rápida. Se consideró evaluar el comportamiento del inóculo en un medio apropiado que estimule o facilite la biodegradación de lípidos como parte de un futuro proceso de tratamiento de desechos. Para esto se utilizó el método de titulación con NaOH 0.05 N mediante el cual se cuantificó la cantidad de ácidos grasos liberados debido a la actividad de las lipasas de Pseudomonas. Los parámetros a evaluar fueron: temperatura, tiempo de incubación, pH inicial, concentración de sales y cantidad de inóculo (% V/V). Se comprobó que los mejores resultados (0,823, 0,747 y 0,781 U) se dieron con una temperatura de 37 ºC, un tiempo de 24 h y un pH inicial de 7. Los resultados mostraron que con tiempos mayores a 24 h (48 y 72 h) la actividad de enzima decrece y esto puede ser debido a la acción de otras enzimas por medio de los cuales Pseudomonas consume también los ácidos grasos liberados y por consiguiente se reduce la cantidad de NaOH usada para el punto final. Palabras clave: Efluentes grasos, Tratamiento de desechos grasos, Pseudomonas aeruginosa, lipasas.