Made available in DSpace on 2015-05-14T12:59:32Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 730230 bytes, checksum: c9ea66b757014e0b2d6d2b07b2de3af1 (MD5) Previous issue date: 2012-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Acinetobacter species have gained importance in recent years due to their increased involvement in serious infections and antimicrobial resistance. Since multidrug resistant bacteria pose a challenge to the treatment of infections, it is necessary to find new substances with antimicrobial properties in combating these microorganisms. Thus, medicinal plants are studied, since they are used for treatment of various illnesses. Therefore, many studies on biological activity have been performed with essential oils obtained from medicinal plants, such as the essential oil from Citrus limon (EOCL), attempting to help overcome this problem. Thus the present study aimed to evaluate the effect of the essential oil of EOCL against multidrug resistant strains of Acinetobacter spp. Its chemical composition was determined by GC/MS and its antibacterial activity was assessed by determining their MIC and MBC by microplate bioassay, its Time kill too was determinated. Also was analyzed the effect of modulating action of conventional antibiotics by microplate bioassay. Among phytochemicals, Neral (29.4%) presented as the major essential component of EOCL. The oil caused growth inhibition in 16 (67%) of 24 strains tested, showing a MIC of 625 μg/ml and MBC of 1250 μg/mL. In a time kill study, the oil displayed a concentration-dependent antibacterial activity. Results of combining antibiotics and EOCL had shown us a synergistic and additive effect with EOCL /antibiotics combinations. The results of this study suggest that essential oil of C. limon may suppress the growth of Acinetobacter species and could be a source of metabolites with modified antibacterial activity. / Espécies do gênero Acinetobacter ganharam importância nos últimos anos devido ao seu envolvimento crescente em infecções graves e resistência antimicrobiana. Uma vez que bactérias resistentes a múltiplas drogas representam um desafio para o tratamento de infecções, é necessário encontrar novas substâncias com propriedades antimicrobianas no combate a estes microrganismos. Para tentar superar este panorama, várias pesquisas vêm sendo desenvolvidas em busca de novos produtos antibacterianos, dentre elas estão os estudos com os metabólitos de plantas, como o óleo essencial de Citrus limon (OECL). Assim, o presente estudo teve como objetivo avaliar o efeito do óleo essencial de Citrus limon contra cepas de Acinetobacter multidroga resistentes. Sua composição química foi determinada por CG/EM e sua atividade antibacteriana avaliada pela determinação de sua CIM e CBM pela técnica de microdiluição, sua cinética de morte microbiana também foi analisada. Verificou-se também seu efeito modulador da ação dos antibióticos convencionais por técnicas de microdiluição. Entre os fitoconstituintes, neral (29,4%) apresenta-se como o principal componente de OECL. O referido óleo inibiu o crescimento de 67% das 24 cepas ensaiadas, tendo sua CIM estabelecida em 625 μg/mL e a CBM em 1250 μg/mL Este óleo possui atividade antibacteriana dependente de sua concentração e do tempo de exposição no microrganismo e é um modulador da ação dos antibióticos convencionais. Os resultados deste estudo sugerem que o referido óleo pode suprimir o crescimento de espécies de Acinetobacter e poderia ser uma fonte de metabólitos com atividade modificadora antibacteriana.

De acordo com a Organização Mundial de Saúde (World Health Organization - WHO), aproximadamente 40 milhões de pessoas ao redor do mundo estão infectadas com HIV/AIDS. Atualmente, a epidemia tem sido controlada em grande parte do mundo ocidental, porém, projeções sugerem que, até o fim desta década, o número de incidência da doença poderá duplicar. Apesar das significantes melhoras na morbidade e mortalidade de pacientes infectados pelo HIV, o rápido surgimento de cepas resistentes aos agentes anti-HIV, além dos efeitos adversos e o alto custo de fármacos de última geração, torna-se necessário o continuo desenvolvimento de novas classes de agentes anti-HIV. A transcrição e multiplicação do RNA viral são dependentes das interações seqüência-específica entre duas proteínas reguladoras virais essenciais, Tat e Rev, com seus respectivos sítios no RNA, TAR e RRE. Durante a última década, os aminoglicosídeos foram introduzidos como ligantes universais do RNA, sendo capazes de se ligar ao TAR e ao RRE. A literatura apresenta diversos aminoglicosídeos que são capazes de se ligar ao TAR e inibir a interação Tat-TAR bem como, inibir competitivamente a ligação da proteína Rev ao RRE, como, por exemplo, a neomicina e tobramicina. Considerando a importância dos aminoglicosídeos e análogos nucleosídicos, conhecidamente eficazes na terapia antirretroviral, o trabalho foi direcionado para a síntese de conjugados de aminociclitol, 2- desoxi-estreptramina, e adenosina, bem como, dímeros de adenosina via estratégia de click chemistry por reação de cicloadição azido-alcino catalisada por Cu(I) (CuAAC). Para a síntese destes produtos, o precursor adenosina foi convertido no derivado 5\'-azido-5\'- desoxi-adenosina, o qual foi condensado com diversos diinos terminais comerciais, contendo diferentes grupos espaçantes, com a finalidade de explorar suas influências nas propriedades eletrônicas e estéricas nos conjugados de interesse frente à atividade anti-HIV. Os derivados alcinos presentes na posição C-5\' de adenosina, via grupo triazol, foram empregados para a síntese dos monômeros nucleosídeo-aminociclitóis, assim como, na síntese de dímeros nucleosíde0-aminociclitóis, via reação de cicloadição 1,3-dipolar, na presença de CuSO4, quantidade catalítica, e ascorbato de sódio, para geração in situ de Cu(I). Adicionalmente, alguns dos compostos, na concentração de 1mM, foram testados empregando o ensaio de ELISA para detecção da presença de proteína viral p24 em linhagem células H9 e avaliação de sua atividade antirretroviral. De acordo com o ensaio biológico, um dos compostos preparados apresentou, proporcionalmente ao crescimento da linhagem H9 (HIV) controle, atividade de inibição de formação da proteína viral p24 similar ao composto padrão zidovudina (AZT). Além disso, outros dois compostos também apresentaram um resultado relevante uma vez que suas atividades foram similares ao composto padrão lamivudina (3TC). Estes resultados sugerem que a presença de uma cadeia metilênica mais extensa, como cadeia lateral ou grupo espaçante, pode influenciar positivamente a atividade biológica por efeito hidrofóbico ou estérico. Por fim, os ensaios de viabilidade celular demonstraram que os compostos testados não foram citotóxicos nas condições testadas. / According to World Health Organization - WHO about 40 million of people are infected with HIV/AIDS. Currently, the epidemic has been controlled largely in the western world, since, projections suggest that, until this decade end, the disease incidence could increase. Despite significant improvements in morbidity and mortality of HIV-patients, quick emergence of resistant strains to anti-HIV agents, in addition to adverse effects and high cost of recent drugs, becomes necessary the ongoing development of new classes of HIV agents. Transcription and translation of the viral RNA are dependent of sequence-specific interactions among two essential viral regulatory proteins, Tat and Rev, and their corresponding TAR and RRE sites in HIV-1 RNA. Over the past decade, aminoglycosides were established as universal RNA linkers, being able to link to TAR and RRE. The literature reports several aminoglycosides that bind to TAR and inhibit Tat-TAR interaction, as well as, competitively inhibit the bind between Rev protein and RRE, such as: neomycin and tobramycin. Considering the importance of nucleosides analogs, effective in antiretroviral therapy, and aminoglycosides, the work was driven to the synthesis of aminocyclitol, 2- deoxy-streptamine, conjugated to the adenosine, as well as, conjugated dimers of adenosine by molecular duplication via click chemistry strategy involving copper-catalysed azide-alkyne cycloaddition reaction (CuAAC). For the synthesis of these products, the starting material adenosine was converted to 5\'-azide-5\'-deoxy-adenosine, which was conjugated with several commercials terminal dialkynes containing different intercalating groups in order to explore the influences of the steric and electronic properties of conjugates towards anti-HIV activity. Alkynes derivated at C-5\' position of adenosine, via triazole group, were used in the synthesis of nucleoside-linked aminocyclitols, as well as, nucleoside conjugated dimers by 1,3-dipolar cycloaddition reaction under microwave-assisted conditions (MW), using the catalytic system CuSO4/sodium ascorbate for the in situ generation of Cu(I). Additionally, several compounds, at concentration of 1mM, were tested in vitro by ELISA for detection of p24 protein in H9 cells to antiretroviral evaluation. According with the biologic assay, one of the compounds showed inhibition of p24 protein production similar to zidovudine (AZT), when compared to H9 cells line growth control, Furthermore, two compounds also showed important activities similar to lamivudine (3TC). These results suggest that the presence of a longer methylenic chain, as side chain or intercalating groups, could influence positively in the biologic activity due to hydrophobic or steric effects. Ultimately, the cell viability assays showed that compounds were not cytotoxic in the tested conditions.

[pt] Recentemente, os nanocristais semicondutores, também conhecidos como pontos quânticos, tornaram-se muito atrativos em abordagens de detecção por fotoluminescência devido as suas propriedades ópticas peculiares, tais como fluorescência intensa e com perfil estreito, comprimento de onda máximo ajustável através do controle do tamanho das partículas e maior fotoestabilidade em comparação com os corantes orgânicos convencionais. As nanopartículas sintetizadas foram avaliadas como sondas fotoluminescentes (na forma de dispersão aquosa) para a determinação de captopril, histamina, canamicina e tiroxina (analitos não fotoluminescentes na temperatura ambiente) evitando o uso de procedimentos complexos de derivatização química e permitindo quantificações de forma simples e com sensibilidade. Nanopartículas de CdTe modificadas com o ácido tioglicólico (TGA) e com o ácido 2-mercaptopropiônico (2MPA) e também nanopartículas de ZnS modificadas com L-cisteína foram sintetizadas pela abordagem em fase aquosa coloidal. Estas foram caracterizadas usando métodos microscópicos e espectroscópicos adequados. A fotoluminescência da nanopartícula 2MPA-CdTe foi consideravelmente mais intensa quando na presença de captropil. Sob condições ótimas, o modelo de calibração (isoterma de ligação de Langmuir) foi linear até 4,8 x 10-4 mol L-1 com constante de equilíbrio de ligação de 3,2 x 104 L mol-1 e limite de detecção (LOD) de 6,2 x 10-6 mol L-1 (1,3 (micro)g mL-1). Aplicações em soro sanguíneo humano fortificado com captropil e em formulações farmacêuticas foram demonstradas. A fotoluminescência das nanopartículas de TGA-CdTe foi reduzida (supressão) após adição de diferentes concentrações de histamina seguindo o modelo de Stern- Volmer. A resposta linear cobriu uma faixa de concentração até 5,7 x 10-4 mol L-1, com LOD de 9,6 x 10-6 mol L-1 (1,1 (micro)g mL-1). A abordagem proposta foi utilizada para determinação de histamina em carne de atum. Já a presença de aminoglicosídeos aumentou a fluorescência das nanopartículas de TGA-CdTe (seguindo o modelo da isoterma da adsorção de Langmuir). A kanamicina foi o aminoglicosídeo escolhido para estudar o efeito do aumento da intensidade da fotoluminescência das nanopartículas de TGA-CdTe disperso em solução aquosa. A faixa linear estendeu-se até 8,2 x 10-7 mol L-1 com LOD de 2,5 x 10-8 mol L-1 (14,2 ng mL-1). As constantes de ligação entre diversos aminoglicosídeos e TGACdTe foram calculadas e indicou que existe uma relação entre o número de grupos amino primários disponíveis e o aumento da luminescência. Essa abordagem foi aplicada com sucesso para a análise de amostras de leite e água de riacho, ambos fortificados com kanamicina, usando procedimento de extração em fase sólida com um polímero impresso molecularmente (MIP). A intensidade da fotoluminescência da nanopartícula cisteína-ZnS em solução contendo brometo de cetiltrimetilamônio (CTAB) foi reduzida (quenched) após adição de tiroxina. A redução total do sinal (quenching) seguiu o modelo de Stern-Volmer com resposta linear até 4,0 x 10-6 mol L-1 de concentração do analito, o LOD foi 6,2 x 10-8 mol L-1 (48,3 ng mL-1). A dispersão aquosa da cisteína-ZnS foi usada como sonda óptica para a determinação de tiroxina em formulações farmacêuticas e em saliva humana fortificada com analito. / [en] Recently, semiconductor nanocrystals, also known as quantum dots, have become very attractive for photoluminescence based sensing approaches due to their unique optical properties like intense photoluminescence with narrow profile, maximum wavelength adjustable by the control of particle size and higher photostability in comparison of conventional organic dyes. Different synthesized nanoparticles were evaluated as photoluminescent probes (as aqueous dispersions) for the determination of captopril, histamine, kanamycin and thyroxine (nonphotoluminescent analytes at room-temperature) avoiding the use of complex chemical derivatization procedures and enabling simple and sensitive quantifications. Thioglycolic acid (TGA) and 2-mercapoprionic acid (2MPA) modified CdTe nanoparticles and L-cysteine modified ZnS nanoparticles were synthesized via the colloid aqueous phase route. Their characterization was made using proper microscopic and spectroscopic methods. The emission intensity of 2MPA-Cdte is greatly enhanced in the presence of captopril. Under optimum conditions, the calibration model (Langmuir binding isotherm) was linear up to 4.8 x 10-4 mol L-1 with equilibrium binding constant of 3.2 x 104 L mol-1 and limit of detection (LOD) of 6.2 x 10-6 mol L-1 (1.3 (micro)g mL-1). Applications in captopril fortified human serum and in pharmaceutical formulations were demonstrated. The photoluminescence of TGA-CdTe nanoparticles was quenched by histamine in a concentration dependent manner (Stern-Volmer model). The linear response covered the concentration range up to 5.7 x 10-4 mol L-1 with LOD of 9.6 x 10-6 mol L-1 (1.1 (micro)g mL-1). The proposed method was used for the analysis of tuna fish. The presence of aminoglycosides enhanced the photoluminescence of the TGA-CdTe nanoparticles (following a Langmuir binding isotherm model). Kanamycin was used as a model aminoglycoside in order to study its effect on the photoluminescence enhancement of TGA-CdTe quantum dots dispersed in aqueous solution. The linear range extended up to 8.2 x 10-7 mol L-1 with LOD of 2.5 x 10-8 mol L-1 (14.2 ng mL-1). Binding constants were calculated for several aminoglycosides indicating that there is a relationship between the number of available primary amino groups and the increasing in photoluminescence. This approach was successfully applied for determination of kanamycin fortified milk and in stream water samples after solid phase extraction using a molecular imprinted polymer produced using a kanamycin template. The photoluminescence intensity of cysteine-ZnS in solution containing cetyltrimethyl ammonium bromide (CTAB) was quenched by thyroxine. The overall quenching followed a Stern-Volmer model with linear response coveing an analyte concentration range up to 4.0 x 10-6 mol L-1. LOD was 6.2 x 10-8 mol L-1 (48.3 ng mL-1). The aqueous dispersion of cysteine-ZnS was used as optical probe for the determination of thyroxine in pharmaceutical formulations and in analyte fortified human saliva.

Das Ziel dieser Arbeit war die Synthese niedermolekularer Verbindungen, die an die prä-miRNA binden und dadurch die Reifung zur miRNA inhibieren. Daher sollten die RNA-Binder 2-Desoxystreptamin sowie Neamin mit Alkinen funktionalisiert und durch Kupfer-katalysierte Azid-Alkin 1,3-dipolare Cycloaddition (CuAAC) mit verschiedenen bivalenten Aziden verknüpft werden. Im Rahmen dieses Projekts wurde der synthetische Zugang zu den benötigten Alkin- sowie Azid-funktionalisierten Grundbausteinen optimiert. Ferner wurde ein effektives und zuverlässiges Protokoll für die CuAAC erarbeitet, welches es ermöglichte, 88 Testsubstanzen in guter Ausbeute und hoher Reinheit zu isolieren. Anschließend wurde die Substanzbibliothek in einem BRCA-Reifungsassay unter kompetitiven Bedingungen auf die Inhibition der miRNA-Reifung getestet. Dabei wurden mehrere potente Inhibitoren der miRNA-Reifung mit IC50-Werten von bis zu 0.5 µM identifiziert. Der zweite Schwerpunkt dieser Arbeit lag auf der Etablierung einer chemo-enzymatische RNA-Funktionalisierungsstrategie um prä-miRNA-Sonden herzustellen: In-vitro-Transkriptionen mit der T7-RNA-Polymerase sowie Ligationen mit der T4 RNA Ligase 1 waren das Fundament der enzymatischen RNA-Synthese, während die Funktionalisierung der RNA durch CuAAC erzielt wurde. Dafür wurden die neuen Verbindungen O-(5‘-Guanosin)-O-propargylmonophosphat sowie 3‘,5‘-O,O-Bisphosphat-5-ethinyluridin synthetisiert, durch in-vitro-Tran¬skription an das 5‘-Ende von prä-miRNAs eingeführt und anschließend durch CuAAC mit einem Fluoreszenzlöscher modifiziert. Das Uridinbisphosphat wurde durch CuAAC mit einem Fluorophor markiert und anschließend effizient mit der T4 RNA Ligase 1 an das 3‘-Ende verschiedener prä-miRNAs ligiert. Darüber hinaus war es auch möglich, das Alkin-modifizierte Uridinbisphosphat an das 3‘-Ende von prä-miRNAs zu ligieren, dieses durch eine zweite Ligation an eine definierte interne Position zu verschieben und abschließend durch CuAAC zu funktionalisieren. / The objective of this work was the synthesis of small molecules, which bind to pre-miRNAs to prevent their maturation to fully active miRNAs. To create a substance library of bivalent inhibitors, the RNA binding motifs 2-deoxystreptamine as well as neamine were alkyne modified and linked with several bisazides via a copper catalyzed alkyne-azide cycloaddition (CuAAC). Hence, optimized syntheses of the basic building blocks along with an effective and reliable CuAAC-protocol were established. 88 test substances were isolated in good yield and high purity. Finally they were analyzed with regard to their potential to selectively inhibit the miRNA maturation. For this purpose, the assay was performed under competitive conditions with a set of two pre-miRNA-pairs. The initial screening revealed several inhibitors with IC50-values in the lower µM range. The second focus of this work was on the development of a synthetic access to alkyne modified ribonucleotides to establish a chemo-enzymatic functionalization strategy for RNAs using in-vitro-transcription, ligation and CuAAC. In this context, the syntheses of 3‘,5‘-O,O-bisphosphate-5-ethinyl uridine and O-(5‘-guanosine)-O-propargyl monophos-phate are described for the first time. The guanosine monophosphate was used as transcription starter to address the 5’-end of RNA and was consecutively labeled with an azido-tagged quencher. The uridine bisphosphate was conjugated with a fluorophor and introduced to the 3’-end of RNAs by T4 RNA ligase 1. Moreover, the uridine bisphosphate can be ligated to the 3’-end without a fluorophor attached, to serve as a connecting point for a further ligation with an oligonucleotide of any length. Thereby, the former terminal alkynylated uridine was shifted to a defined internal position by successive enzymatic reactions and was successfully derivatized with a fluorophor by CuAAC.

Selenoproteine spielen eine wichtige Rolle in der antioxidativen Abwehr und bei Immunreaktionen. Der Selen(Se)metabolismus wird von Hepatozyten gesteuert, die das Se-Transportprotein Selenoprotein P (SEPP) synthetisieren und sezernieren. SEPP nimmt bei kritischen Erkrankungen, z. B. Sepsis ab und führt zu niedrigen Se-Spiegeln. Sepsis triggert die übermäßige Produktion von proinflammatorischen Zytokinen. Aminoglykosid-Antibiotika (AG), die oft bei schwerer Sepsis eingesetzt werden, induzieren Fehlinterpretationen der mRNA inklusive des Stoppcodons UGA welches für die Selenoprotein-Biosynthese notwendig ist. Es wurden daher die molekularen Wechselwirkungen zwischen den Zytokinen IL-6, IL-1b und TNFa, AG und dem Se-Status mit der Biosynthese in Leberzelllinien untersucht. IL-6 führte zu einer starken Reduktion der SEPP-mRNA und einer dosisabhängigen Reduktion von SEPP. Parallel dazu reduzierte IL-6 das Transkriptlevel, die Proteinexpression und die Enzymaktivität der Typ-I-Dejodase (DIO1). Auf die Expression der antioxidativ-wirkenden Glutathionperoxidasen (GPX) wirkte IL-6 isozymspezifisch; während die Transkriptkonzentrationen von GPX2 anstiegen und die von GPX4 abnahmen, blieb GPX1 unbeeinflusst. Die IL-6-abhängigen Effekte bestätigten sich auch in Reportergenassays von SEPP-, DIO1-, GPX2- und GPX4-Promotorkonstrukten. Um die Wirkungen von AG auf die Selenoprotein-Translation besser zu verstehen, wurden die SECIS-Elemente von GPX1-, GPX4- und SEPP-Transkripten in ein Reportersystem kloniert und auf eine Regulation durch AG und Se analysiert. Die Ergebnisse zeigen, dass der korrekte Se-Einbau vom Se-Status, von der AG-Konzentration und dem spezifischen SECIS-Element abhängig ist. Auf transkriptionaler und translationaler Ebene führten AG zu stark erhöhten SEPP-Spiegeln, während die Expression und Enzymaktivität von GPX und DIO1 nur in geringerem Ausmaß beeinflusst wurden. Eine Analyse der Se-Beladung zeigte, dass der Se-Gehalt von SEPP stark durch AG reduziert und vom Se-Status abhängig war. / Selenoproteins play important roles in antioxidant defence and immunoregulation. Selenium (Se) metabolism is controlled by hepatocytes synthesizing and secreting the Se-transporter selenoprotein P (SEPP) declining in critical illness, e.g., sepsis. Sepsis triggers excessive production of pro-inflammatory cytokines. Aminoglycoside (AG) antibiotics applied in sepsis in induce mRNA misinterpretation including the stop codon UGA required during selenoproteins biosynthesis. The molecular interplay between the cytokines IL-6, IL-1b and TNFa, AG and Se-status on selenoprotein expression was investigated in hepatic-derived cell lines. IL-6 strongly reduced the level of SEPP mRNA and secreted SEPP in a dose-dependent manner. Likewise, expression of selenoenzyme type 1 deiodinase (DIO1) declined at the transcript, protein and enzyme activity level. The effects of IL-6 on the expression of antioxidative-acting glutathione peroxidases (GPX) were isozyme-specific; while transcript level of GPX2 increased and those of GPX4 decreased, GPX1 remained unaffected. IL-6-dependent effects were reflected in reporter gene experiments of selenoprotein promoter constructs. Characterising the effects of AG on selenoprotein translation, the SECIS-elements of GPX1, GPX4 and SEPP transcripts were cloned into a reporter system and analysed for their response to AG and Se. The results indicate that the correct co-translational Se-insertion depends on the Se-status, AG concentration and the specific SECIS-element. At both transcriptional and translational levels, SEPP levels were strongly increased in response to AG, whereas the expression and enzyme activity of GPX and DIO1 were affected to a lower degree. Analysis Se-status indicate that the Se-content of SEPP was strongly reduced by AG and depends on Se-status.

Les antibiotiques sont des molécules incontournables dans le traitement des infections bactériennes. L’émergence et la dissémination de la résistance aux antibiotiques chez la pathogène opportuniste Pseudomonas aeruginosa, ont amené l’Organisation Mondiale de la Santé à déclarer indispensable le développement de nouvelles approches thérapeutiques pour lutter contre cette bactérie. Bien que certaines alternatives aient été envisagées, la préservation de l’activité d’antibiotiques majeurs tels que les aminosides et la colistine est primordiale. La caractérisation des mécanismes de résistance à ces médicaments est nécessaire pour la mise au point de nouvelles molécules et mieux prendre en charge les patients. Dans ce contexte, nous montrons que des mutations dans le gène fusA1 (codant le facteur d’élongation EF-G1A) et dans l’opéron pmrAB (système à deux composants PmrAB) entrainent une augmentation de la résistance aux aminosides chez des mutants isolés au laboratoire et des souches issues de patients, atteints ou non, de mucoviscidose. Certaines substitutions d’acide aminé dans EF-G1A accroissent les niveaux de résistance de 2 à 16 fois aux quatre sous-classes d’aminosides. Par ailleurs, des changements d’acide aminé dans le système à deux composants PmrAB activent l’expression des gènes PA4773-PA4774-PA4775, et la production de norspermidine et de spermidine. La synthèse de ces polyamines va de pair avec une baisse de 4 à 16 fois de la sensibilité aux aminosides à noyan 2-désoxystreptamine bisubstitué en 4,6 (gentamicine, amikacine et tobramycine). De plus, il apparaît que la résistance des mutants pmrB à la colistine est en partie dépendante de la pompe d’efflux MexXY(OprM), un système impliqué dans la résistance naturelle, adaptative ou acquise aux aminosides. Enfin, nous montrons que les mutants pmrB surproduisent des alcaloïdes contenant un motif azétidine, par une voie de synthèse non-ribosomale et dépendante du quorum sensing. Ces alcaloïdes diminuent la virulence de P. aeruginosa dans le modèle Galleria mellonella. / Antibiotics are invaluable drugs to combat bacterial infections. Emergence and spread of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa have led the World Health Organization to consider as a crucial priority the development of new therapeutic approaches to fight this bacterium. In addition to other alternatives, preservation of activity of major antibiotics such as aminoglycosides and colistin is primordial. Consequently, characterization of the resistance mechanisms to these drugs is a prerequisite to design novel molecules, and improve patient care. In this context, we show that mutations in gene fusA1 (encoding elongation factor EF-G1A) and in operon pmrAB (two-component system PmrAB) lead to an increased resistance to aminoglycosides in in vitro-selected mutants and strains isolated from cystic fibrosis (CF) and non-CF patients. Certain amino acid substitutions in EF-G1A confer a 2- to 16-fold increased resistance to the four aminoglycoside subclasses. On the other hand, amino acid variations in two-component system PmrAB activate the expression of genes PA4773-PA4774-PA4775, and production of norspermidine and spermidine. This upregulated polyamine biosynthesis is associated with a 4- to 16-fold decreased susceptibility to 4,6-di-substituted deoxystreptamine aminoglycosides (gentamicin, amikacin and tobramycin). Moreover, our work reveals that the acquired resistance of pmrB mutants to colistin partially depends upon pump MexXY(OprM), a system that otherwise mediates intrinsic, adaptive and acquired resistance to aminoglycosides. Finally, we show that pmrB mutants overproduce azetidine-containing alkaloids by a quorum-sensing-regulated, nonribosomal peptide synthetase pathway. These alkaloids impair the virulence of P. aeruginosa in a Galleria mellonella infection model.

No / Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins (BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hypertension (PAH). However, the precise molecular consequences of PAH causing mutations on the function of the receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based techniques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD) and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD, providing further evidence that haplo-insufficiency is a major molecular consequence of disease-related BMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impaired type I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leading to perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intracellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally and to address the potential for resolution of stoichiometric balance, we investigated an agent that promotes translational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway. We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor-receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have identified an important target for early therapeutic intervention in familial PAH.