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Metronidazole is an essential component of the triple therapy regimen against Helicobacter pylori infection. The development of resistance towards metronidazole results in failure to eradicate H. pylori completely. The main aim of the investigation was to understand further the mechanism(s) of resistance in H. pylori. The investigation focussed upon studying the role and function of NADH oxidase in metronidazole resistance. The NADH oxidase levels were shown to be significantly higher in metronidazole susceptible strains than in resistant strains. The purification and characterisation of the enzyme responsible for the oxidation of NADH resulted in isolation of a protein shown to be catalase. The results suggest that NADH oxidase activity in susceptible strains is a function of a bifunctional catalase rather than that of a distinct enzyme. This was confirmed by isolation of catalase from E. coli cells containing cloned H. pylori catalase and demonstration that catalase and NADH activities co-purified. The catalase activity of the purified protein from the bacterial strains used was retained but the oxidation of NADH was not significant in the resistant strain. The base sequence of the catalase gene from the susceptible strain was determined and shown to be 99% identical to that from the cloned gene. The comparison of the derived amino acid sequence of catalase from H. pylori and Proteus mirabilis showed that the heme-binding site is highly conserved. The amino acids in the NAD(P)H binding site are conserved in both strain NCTC 11639 (Mtz s ) and the genomic strain ATCC 26695 (Mtz s) of H. pylori but show significant differences compared with P. mirabilis. A three-dimensional model of catalase from a metronidazole-susceptible H. pylori strain showed stearic hindrance around the NAD(P)H binding site and a substitution of an acidic for a basic residue within the phosphate binding site. Both effects could result in NAD(P)H being less tightly bound and, hence able to leave the catalase in exchange for NADH. Other substitutions may account for the ability of the modified binding site to oxidise NADH. The oxidation of NADH aids in the activation of metronidazole, which damages DNA. The absence of NADH oxidase activity in resistant strains results in the inability of the enzyme to activate metronidazole leading to resistance. The finding that this NADH oxidase activity is a function of a modified catalase in susceptible strains suggests a novel mechanism of metronidazole resistance in H. pylori.

Ulm, Univ., Diss., 2008.

Stuttgart, Univ., Diss., 2007.

Universiẗat, Diss., 2004--Kiel.

A criptococose é uma doença invasiva capaz de apresentar-se de forma fatal podendo acometer pacientes imunocompetentes e imunocomprometidos. Os agentes etiológicos Cryptococcus neoformans var. grubii e C. neoformans var. neoformans apresentam distribuição cosmopolita, sendo as excretas de pombos o seu principal reservatório. Com o advento de terapias imunossupressoras e a pandemia de HIV, observou-se o aumento significativo de casos de pacientes com criptococose. Atualmente, o diagnóstico é baseado na apresentação clínica, na observação microscópica de líquor corado com tinta da Índia e/ou no isolamento em cultura. Neste trabalho desenvolveu-se um ELISA para detecção de anticorpos contra C. neoformans var. grubii em soro de pacientes utilizando como antígeno um extrato protéico total de uma linhagem clínica isolada de um paciente com criptococose (HC6). Foram testados através de ELISA 40 amostras de soros de pacientes com criptococose, sendo 67,5% positivos e 32,5% falsos negativos. Como controles negativos foram testados 82 amostras de soros de indivíduos hígidos, dos quais 26,82% apresentaram resultados positivos para os testes realizados. Para testar a reatividade cruzada, foram utilizadas 10 amostras de pacientes com histoplasmose (20% de reatividade cruzada), 9 amostras de pacientes com paracoccidioidomicose (66,6% de reatividade cruzada), 9 amostras de pacientes com candidose (13,3% de reatividade cruzada) e 7 amostras de pacientes com aspergilose (14,28% de reatividade cruzada). Visando solucionar o problema da reatividade cruzada, identificamos proteínas antigênicas de C. neoformans var. grubii por eletroforese bidimensional seguida por western blot e espectrometria de massa (MALDI-TOF MS). Das 75 amostras analisadas, quatro foram identificadas: uma proteína hipotética, 2 isoformas de HSPs 70 e uma catalase-2. As proteínas identificadas apresentaram baixa similaridade com ortólogas de outros fungos patogênicos, sendo, dessa forma, possíveis alvos para a padronização do ELISA e diagnóstico da criptococose. / Cryptococosis is an invasive and potentially fatal disease. Cryptococcus neoformans is the etiological agent, which can affect both immunocompromised and immunocompetent individuals. C. neoformans var. grubii and C. neoformans var. neoformans are cosmopolitan and their major natural reservoir is the excrement from pigeons. With the advent of immunosupressor therapies and the pandemic HIV infection, a significant augmentation of cryptococosis cases in humans was observed. Nowadays cryptococcosis diagnosis is based on the clinical presentation, India ink sample preparation methods and/or in vitro culture isolation. In this work we had developed an ELISA to detect antibodies against C. neoformans var. grubii in serum from patients with cryptococcosis using as antigens a whole cell protein extract from a clinical cell line isolate (HC6). Sera from 40 patients with cryptococcosis were tested by ELISA. From these, 67.5% were positives and 32.5% were false-negatives. As a negative control 82 samples from health subjects were also tested, from these 26.82% were positives. To test cross-reactivity, samples from 10 patients with histoplasmosis (20% cross-reactivity), 9 from patients with paracoccidioidomicosis (66.6% cross-reactivity), 9 from patients with candidosis (13.3% cross-reactivity) and 7 from patients with aspergilosis (14.28% cross-reactivity) were tested. To solve the cross-reactivity problem, we searched immunogenic proteins which were specific to C. neoformans var. grubii applying two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) followed by western blot and mass spectrometry (MALDI-TOF MS). From the 75 sample analyzed, four were identified: one as a hypothetic protein, two HSPs 70 isoforms and the protein catalase 2. These proteins showed low similarity with orthologues from other pathogenic fungi, and are potential targets to further of the standardizing cryptococosis diagnosis by ELISA.