Pseudomonas aeruginosa est un pathogène opportuniste pouvant causer des infections pulmonaires chroniques chez les gens atteints de la Fibrose Kystique (FK). Pour réussir à s’implanter dans les voies respiratoires des patients FK, P. aeruginosa dispose d’un important arsenal de facteurs de virulence, dont la sécrétion de protéases, de lipases, de phospholipases ainsi que la production de toxines spécifiques. Le séquençage complet du chromosome de P. aeruginosa souche PAO1 (6.3 Mb) a révélé une organisation génomique hautement régulée. Dans le but de mieux comprendre l’interaction hôte-pathogène, nous avons utilisé la technique de mutagenèse à étiquette (STM). La STM a permis d’isoler 214 mutants incapables de maintenir l’infection pulmonaire chronique chez le rat. Parmi ceux-ci, le mutant STM2895, contenant un transposon dans le gène PA2895, a été retenu pour des analyses plus approfondies. L'étude phénotypique de STM2895 a permis de constater un défaut dans la production d’exoprotéases lorsque comparé à la souche sauvage PAO1. La caractérisation biochimique de ce défaut, utilisant des tests de dégradation spécifiques et l’immunobuvardage, a démontré qu’au moins deux des quatre protéases majeures sécrétées par P. aeruginosa sont impliquées. En effet, les élastases LasA et LasB ont été démontrées non fonctionnelles probablement dues à un problème de repliement. PA2895, une protéine possédant un domaine transmembranaire prédit, ne code pour aucune fonction connue, mais il est co-transcrit avec le gène PA2896, un facteur sigma putatif de type ECF (extracytoplasmic function). Des analyses en transcriptome sur le mutant STM2895, ainsi que sur un mutant de délétion de PA2896, ont permis de lier l’opéron au métabolisme du fer. De plus, des études in vivo dans le modèle d’infection pulmonaire chronique chez le rat ont clairement démontré que le mutant STM2895 est incapable de se maintenir dans l’hôte au même niveau que la souche sauvage PAO1. Le gène PA2895 est donc essentiel au maintien in vivo de P. aeruginosa dans le poumon de rat. / Pseudomonas aeruginosa is an opportunistic pathogen that can cause pulmonary infections in cystic fibrosis patients (CF). To overcome innate self defense, P. aeruginosa possesses a wide arsenal of virulence factors. These include degradation enzymes such as proteases, lipases and phospholipases and the production of three specific toxins: exotoxin A and exoenzymes S and T. Sequencing of the complete P. aeruginosa chromosome (strain PAO1) of 6.3 Mb revealed a highly regulated and complex genomic organization. In order to better understand host-pathogen molecular interactions, we developped a new signature-tagged mutagenesis (STM) approach based on PCR screening. The PCR-based STM technology lead to the identification of 214 mutants deficient in their ability to maintain a chronic pulmonary infection in the rat lung. In that pool of STM mutants, STM2895, which contains a transposon insertion in functional PA2895, was the most frequently drafted during the whole mutant library screening. Phenotypic analyses of the STM2895 strain allowed us to identify an exoprotease production defect as compared with wild type strain PAO1. The biochemical characterization of that proteolytic default using specific degradation assays combined with western blotting revealed that at least two (LasA and LasB) of the four major exoproteases from P. aeruginosa STM2895 strain are inactive. In fact, LasA and LasB elastases were shown to be present in the STM2895 culture supernatant, correctly processed but inactive due to a probable misfolding of proteins. The PA2895 gene (unknown function) encodes a protein with a predicted transmembrane domain. Basic genomic context analyses strongly suggest a cotranscription unit with the downstream gene PA2896, a putative sigma 70 factor from ECF (extracytoplasmic function) type. Microarray experiments on the STM2895 strain and an insertional mutant of the PA2896 gene were performed to establish a link between the putative PA2895-PA2896 operon and the metabolism of iron. Transcriptome analysis also demonstrated a repressive action of PA2895 on the transcription of PA2896 putative sigma factor. Finally, in vivo studies in the rat lung chronic infection model clearly showed a ten-fold decrease in survival capacity of the mutant strain when compared to the PAO1 wild-type strain.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 203-212; 260-261). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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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.

In der vorliegenden Arbeit wurden Untersuchungen zur mikrobiellen Aktivität von Pseudomonas putida DSM12735 durchgeführt. Als Messgröße diente die mikrobielle Wärmeleistung, basierend auf dem Stoffumsatz durch die Mikroorganismen. Ziel war es, die Vor- und Nachteile der verwendeten Kalorimeter herauszuarbeiten. Dafür wurden klassische Batch-Wachstumskurven aufgenommen. Ein weiteres Ziel bestand darin, eine Methode zur schnellen kalorimetrischen Detektion der mikrobiellen Aktivität insbesondere für die stationäre Phase zu entwickeln. In dieser Phase findet kein signifikanter Stoffumsatz statt. Durch das gezielte Auslösen einer zweiten Wachstumsphase und damit einem Stoffumsatz wird die mikrobielle Aktivität kalorimetrisch wieder messbar. Eingesetzt wurden folgende Kalorimeter: der Thermal Activity Monitor 2277 (TAM) mit den Kalorimetern Micro Reaction System 2250-4 ml und 2250-20 ml (kurz: TAM-4ml, TAM-20ml), das IC-Chip-Kalorimeter FCC22 (Institut für Physikalische Chemie, TU Freiberg) und das Kalorimeter Micro-DSC II (MDSC).

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.

Chemotherapeutic treatment of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is substantially challenged by several membrane-spanning, multidrug-efflux pumps of the three-component RND family. Of these pumps, MexXY-OprM contributes to the intrinsic resistance of this organism by exporting clinically relevant antibiotics, most notably the ribosome-targeting aminoglycosides. Overproduction of MexXY-OprM is the most common mechanism providing pan-aminoglycoside resistance to P. aeruginosa cystic fibrosis clinical isolates. The mexXY genes are located in an operon, the expression of which is induced by ribosome-targeting antimicrobials. The mexXY operon is negatively regulated by MexZ, a repressor protein encoded by the divergently-transcribed gene mexZ. A second gene, PA5471, is also induced by ribosome-targeting antibiotics and is required for antibiotic induction of mexXY expression. One possibility is that PA5471 interacts with MexZ to alleviate repression of mexXY, thereby providing a mechanism for PA5471-dependent drug inducibility of mexXY. PA5471 interaction with MexZ was confirmed using a bacterial two-hybrid assay. To identify residues/regions of PA5471 important for interaction with MexZ, random chemical mutagenesis of the mexZ and PA5471 genes was carried out and the effects of these mutations on interaction of their protein products was assessed using the bacterial two-hybrid assay. Mutations of PA5471 that compromised interaction with MexZ included P68S, G76C, R216C, R221W, R221Q, G231D, and G252S, which occur within or in close proximity to a predicted surface-exposed α-helix of a PA5471 structural model that may contribute to the MexZ-interaction domain. Representative mutations P68S, G76C, R216C and R221W were placed into the chromosome of P. aeruginosa to assess their impact on drug-inducible mexXY expression. All of these mutations significantly reduced mexX upregulation in the presence of spectinomycin, where mutations R216C and R221W resulted in the near complete ablation of this antibiotic induction. These data suggest that PA5471 acts as a direct antirepressor of MexZ and that this interaction is key to mexXY upregulation in response to ribosome-targeting induction signals. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-26 13:32:39.307

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.