Uma característica associada à capacidade de Pseudomonas aeruginosa sobreviver e disseminar-se frente a situações adversas encontradas no ambiente e no organismo de hospedeiros é a regulação de genes de resposta a condições de estresse celular, que incluem o evento de choque térmico. A exposição do microrganismo a um ambiente de alta temperatura resulta na indução da síntese de proteínas específicas, representadas por chaperonas e proteases, que conferem um aumento da viabilidade celular microbiana em condições consideradas letais. Presume- se que o gene PA 0657 de P. aeruginosa O1 possa estar associado à indução ou supressão do processo transcricional de genes envolvidos na resposta ao choque térmico. Neste contexto, o objetivo deste trabalho foi caracterizar a função do gene PA0657 de P. aeruginosa O1. Para isso, análises bioinformáticas foram realizadas e o gene foi clonado e expresso em sistema bacteriano, produzindo uma proteína recombinante que foi purificada e caracterizada enzimaticamente. Além disso, uma linhagem de P. aeruginosa O1 com o gene PA0657 rompido por recombinação homóloga foi construída, sendo submetida, juntamente com a linhagem selvagem, a uma condição de estresse térmico, permitindo que a expressão de diversos genes associados ao evento de choque térmico pudessem ser avaliadas por qRT-PCR. Os resultados mostraram que a proteína recombinante PA0657 foi capaz de degradar adenosina trifosfato (ATP), dependente do íon Zn2+. Foi possível verificar também que a linhagem rompida perdeu a capacidade de produzir o pigmento piocianina quando crescida em meio cetrimida e não apresentou seu fenótipo mucóide. Em análise in silico observou-se que a região promotora do gene PA0657 é dependente de σ32 e a análise de expressão gênica evidenciou uma diminuição de expressão do gene rpoH na linhagem selvagem de PAO1 após choque térmico, sendo este gene significativamente expresso na linhagem rompida. Observou-se também um acentuado aumento na expressão do gene algU, nestas mesmas condições, na linhagem selvagem PAO1, com ausência de expressão na linhagem rompida. É possível concluir, dessa forma, que o gene PA0657 exerce participação importante no processo de regulação de resposta ao choque térmico e está relacionado com a conversão do fenótipo mucóide. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES / A feature associated with the ability of Pseudomonas aeruginosa survive and dissemination in adverse conditions found in the environment and in hosts‘ organisms is a specific genes regulation of stress response, including heat shock event. The exposure of microorganisms to a high temperature environment results in the induction of synthesis os specific proteins, represent by chaperones and proteases, conferring an increase og the microbial cell viability under conditions considered lethal. . It is presumed that the PA0657 gene of P. aeruginosa O1 can be related to the induction or suppression of transcriptional process of genes that are involved in a response to heat shock. In this context, the aim of this work was to characterize the function of the PA0657 gene of P. aeruginosa O1. In this way, bioinformatic analyzes were performed and the gene was cloned and expressed in bacterial system, producing recombinant protein which was purified and enzymatically characterized. Moreover, the PA0657 gene of P. aeruginosa O1 strain was disrupted by homologous recombination. The wild type strain and the disrupted strain were submitted to a thermal stress and the expression of various genes associated with heat shock event could be evaluated by qRT -PCR. The results showed that the PA0657 recombinant protein was capable of degrading adenosine triphosphate (ATP) of Zn2+ dependent manner. It was also verified that the ruptured strain lost its ability to produce pyocyanin pigment when grown on cetrimide means and its mucoid phenotype. It was possible to find in computational analysis that the promoter region of the PA0657 gene is dependent on σ32. The gene expression analysis showed a decrease in expression of the rpoH gene in the wild type strain after heat shock, this gene is significantly expressed in the disrupted strain. A significant increase in expression of the algU gene was observed, on the wild type strain with absence of expression in the disrupted strain. Therefore, it was possible to conclude that the PA0657 gene cts in the regulatory process of thermal shock response and is related to the conversion of the mucoid phenotype.

Orientadores: Angelica Zaninelli Schreiber, Plinio Trabasso / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T13:17:15Z (GMT). No. of bitstreams: 1 Nowakonski_AngelaVon_M.pdf: 1399484 bytes, checksum: ee465090318f9325ffdc02d5497ae055 (MD5) Previous issue date: 2007 / Resumo: Bacteriemias são ocorrências relacionadas a diferentes processos patológicos e em grande parte associadas a infecções hospitalares. Podem ser causadas por diferentes tipos de microrganismos, sendo os Gram negativos, em especial, os bacilos, responsáveis por grande número destas. O diagnóstico laboratorial envolve coleta de hemoculturas, isolamento e identificação do agente e avaliação de sua sensibilidade aos antimicrobianos. Com o aumento da sobrevida de pacientes com doenças de base muito graves e o uso indiscriminado de antimicrobianos de amplo espectro, são crescentes os relatos de desenvolvimento de resistência dos microrganismos aos antimicrobianos em uso. Trata-se este de estudo retrospectivo que buscou avaliar a prevalência e o perfil de resistência dos principais bacilos Gram negativos isolados a partir de hemoculturas de pacientes adultos, internados no HC-UNICAMP frente a antimicrobianos selecionados e correlação da tendência da resistência aos antimicrobianos com a estimativa de utilização destes antibióticos durante o período de 2000 a 2004. Neste período, os bacilos Gram negativos mais prevalentes foram Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa e Acinetobacter baumanii. Foi possível observar ao longo do período estudado uma alteração no perfil de freqüência dos bacilos Gram negativos encontrados nas hemoculturas, havendo permuta de Enterobacter cloacae por cepas de Klebsiella pneumoniae ao longo do tempo. O consumo aumentado de cefalosporinas de terceira e quarta geração no HC-UNICAMP pôde ser correlacionado de forma significativa a uma maior taxa de resistência de Klebsiella pneumoniae resistente. O aumento no consumo de quinolonas pôde ser correlacionado de forma significativa com aumento de resistência dos bacilos Gram negativos até o ano de 2002, excetuando a Klebsiella pneumoniae que manteve os níveis de resistência até 2004 / Abstract: Bacteriemias are events related to different diseases, mostly hospital infections. They can be caused by many different microorganisms, but the Gram negative bacilli are often responsible for them. The laboratory diagnosis includes blood sampling, isolation and identification of the microorganism, and evaluation of its susceptibility to the available antibiotics. Increases in prevalence of these resistant pathogens in hospitals are frequently related to high selective pressure commonly used in hospitalized patients, almost always represented by older and debilitated individuals. This is a retrospective study of the prevalence and resistance of pathogens isolated from the blood of patients in the Hospital das Clínicas UNICAMP, during the years 2000-2004, and the correlation of the intensive use of some proposed antibiotics. During this period the most commonly isolated microorganisms were: Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa and Acinetobacter baumanii. We observed an alteration of this profile during the study, having Klebsiella pneumoniae replaced the Enterobacter cloacae 's score. The increasing utilization of broad expect rum cephalosporin's in HC UNICAMP was related to the highest resistance of Klebsiella pneumoniae to these drugs. The ever increasing use of quinolones could be related in a very considerable way to the increasing resistance of all Gram negative bacilli until the year of 2002, when it declined but not for Klebsiella pneumoniae which was prolonged during all the time of the study / Mestrado / Patologia Clinica / Mestre em Ciências Médicas

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics. In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin. In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro. To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Este trabalho utiliza a Análise de Fluxos Metabólicos para estudar o aumento da eficiência da linhagem Burkholderia sacchari (LFM101) na produção de PHB. Foram avaliadas as eficiências de conversão de açúcares em PHA de LFM101. Esta também foi cultivada em batelada alimentada em reator, apresentando o máximo teórico durante um estado pseudo-estacionário sob oferta de glicose. Estes dados, submetidos ao software Metatool, resultaram em mapa metabólico contendo os fluxos das reações centrais ede PHA ocorrido no experimento. Através do cultivo de LFM101 e C. necator sob oferta de glicose marcada com 13C, determinou-se que estas utilizam as mesmas vias para produção de PHA, não justificando a baixa eficiência de LFM101. Em um projeto paralelo, estudou-se a eficiência da produção de PHB utilizando melaço de cana, glicerol cru e acetato, em produtores de hidrogênio e PHA, onde verificou-se não apenas o aumento de PHA em mutantes nifh- de R. capsulatus mas a interação dos parâmetros luz e nitrogênio a partir das metodologias DOE e RSM. / This work applies Metabolic Flux Analysis to discuss the eficiency of Burkholderia sacchari (LFM101) in PHA production from sugars . Conversion yields of LFM101 and Cupriavidus necator from carbohydrates to PHA were assessed. LFM101 was also grown in reactor fed-batch cultivations, and presented the theoretical maximum in a pseudo-steady stage while grown on glucose. These data were submitted to Metatool and resulted in a metabolic network containing the experimental fluxes of central and PHA metabolism. Cultivation of LFM101 and C. necator under 13C- labeled glucose showed that both species use the same metabolic pathways for the biodegradable polymer synthesis. On a parallel project, the efficiency of biopolymer production from molasses, raw glycerol and acetate in strains producing hydrogen and PHA was tested. Results showed that there was not only an increase in PHA production by R. capsulatus nifH- mutants but also the interaction of light and nitrogen effects were studied by DOE and RSM.

Background: Multidrug-resistant Gram-negative bacteria (MDRGN) are found with rising prevalence in non-hemodialysis risk populations as well as hemodialysis (HD) cohorts in Asia, Europe and North America. At the same time, colonization and consecutive infections with such pathogens may increase mortality and morbidity of affected individuals. We aimed to monitor intestinal MDRGN colonization in a yet not investigated German HD population. Methods: We performed cross-sectional point-prevalence testing with 12 months follow-up and selected testing of relatives in an out-patient HD cohort of n = 77 patients by using microbiological cultures from fresh stool samples, combined with Matrix Assisted Laser Desorption Ionization—Time of Flight Mass Spectrometry (MALDI-TOF-MS) and antimicrobial susceptibility testing. Results: We detected MDRGN in 8 out of 77 patients (10.4%) and 1 out of 22 relatives (4.5%), indicating only colonization and no infections. At follow-up, 2 patients showed phenotypic persistence of MDRGN colonization, and in 6 other patients de-novo MDRGN colonization could be demonstrated. Pathogens included Escherichia coli and Klebsiella pneumoniae (with extended-spectrum beta-lactamase [ESBL]-production as well as fluoroquinolone resistance), Stenotrophomonas maltophilia and Enterobacter cloacae. Conclusions: In a single-center study, MDRGN colonization rates were below those found in non-HD high-risk populations and HD units in the US, respectively. Reasons for this could be high hygiene standards and a strict antibiotic stewardship policy with evidence of low consumption of fluoroquinolones and carbapenems in our HD unit and the affiliated hospital.

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Several Gram-negative bacteria modify their outer most surface structure, lipopolysaccharide (LPS), to evade immune surveillance and survive within the host. Many of these changes occur within the lipid A domain, a region that is recognized by the innate immune system via Toll-like receptor-4 (TLR4). One such pathogen, Porphyromonas gingivalis, orchestrates chronic inflammatory disease by disrupting immune homeostasis. P. gingivalis initially synthesizes a penta-acylated lipid A that functions as a weak TLR4 agonist but displays tetra-acylated forms that are either immunologically silent or TLR4 antagonists. The impact of lipid A modifications on downstream signaling and antigen-specific immunity are unclear. TLR4 signals from the plasma membrane through a MyD88-dependent pathway and intracellularly through a TRIF-dependent pathway. Here we show that expression of immunological silent or antagonistic lipid A enables P. gingivalis to evade TRIF-dependent signaling in dendritic cells (DCs). Evasion of TRIF signaling accelerated antigen degradation and impaired priming of pathogen-specific T cells. In contrast, a P. gingivalis strain expressing agonist lipid A potently activated TRIF signaling and delayed antigen degradation, thereby preserving peptides for optimal T cell activation. We propose that lipid A modifications control the endocytic activity of DCs and the efficiency at which microbe-specific T cells are primed. We next investigated the impact of purified P. gingivalis LPS on innate signaling and antigen presentation. All P. gingivalis LPS species induced a program of DC maturation that allowed for constitutive antigen uptake and cross-presentation. This was independent of TLR4 agonist activity and required CD14, a protein that transports TLR4 to endosomes where TRIF signaling can occur. Agonist LPS induced signaling through both MyD88 and TRIF and elicited T cell priming. Antagonistic LPS potently accelerated CD14 endocytosis and induced TRIF-biased signaling leading to comparable degree of cross-priming. Immunologically silent LPS promoted CD14 endocytosis but failed to activate signaling and induced T cell tolerance. Collectively, our results demonstrate that modification of lipid A structure enables Gram-negative bacteria to direct the host immune system towards tolerance or immunity. We propose that these findings can be harnessed for therapeutic modulation of the immune system to treat a variety of immune-mediated diseases. / 2021-06-14T00:00:00Z

No description available.