Assignment of novel functions to Helicobacter pylori 26695’s genome and reconstruction of a genome-scale metabolic model

Helicobacter pylori is a pathogenic organism associated with human gastric diseases. The development of mathematical models of metabolism is now considered a fundamental part of the study of the cell. For the particular case of microorganisms associated with human diseases, information on metabolic and regulatory networks can be used to understand the molecular factors of the microorganism that are likely to interact with the host and cause diseases. The availability of the genome sequence of H. pylori 26695 and its annotation has allowed in the past the construction of a metabolic model for this organism. The first genome-scale metabolic model for H. pylori 26695 was published in 2002 (iCS291) and a corrected reconstruction was published in 2005 (iIT341 GSM/GPR). The main goal of the present work was to update H. pylori’s genome-scale metabolic model based on the new information made available. For that purposes, using new annotation methodologies and data available in databases, an assignment of novel functions to H. pylori 26695’s genome was performed. For a total of 510 “hypothetical proteins” (almost 1/3 of the genes) identified in the last re-annotation, 137 new functions were attributed. A total of 581 E.C. numbers were assigned to CDS, being 528 complete E.C. numbers. This new information was used as the basis of the model reconstruction. In addition, transport reactions in the model were updated. The biomass equation was reviewed and H. pylori biomass coefficients and composition were adjusted. The obtained model successfully predicted the nutritional requirements and amino acids essentialities, which were experimentally validated. As a result, the present work presents a new H. pylori 26695 genome-scale metabolic model with more accurate and reliable predictions and can be used to identify potential targets for designing more effective drugs for H. pylori inactivation

Assignment of novel functions to Helicobacter pylori 26695’s genome

Helicobacter pylori is a pathogenic bacterium that colonizes the human epithelia, causing duodenal and gastric ulcers as well as gastric cancer. The genome of H. pylori 26695 has been sequenced and annotated. In addition, two genome-scale metabolic models have been developed. In order to maintain accurate and relevant information on coding sequences (CDS) and to retrieve new information, the assignment of new functions to Helicobacter pylori 26695’s genes was performed. The use of software tools, on-line databases and an annotation pipeline for inspecting each gene allowed the attribution of validated E.C. numbers to metabolic genes, and the assignment of 177 new functions to the CDS of this bacterium. This information provides relevant biological information for the scientific community dealing with this organism and can be used as the basis for a new metabolic model reconstruction.(undefined

Evaluation of antibiotics residues levels in Portuguese honey: a concerted study with the Portuguese Beekeepers Associations

The antibiotics residual presence in honey is a current problem with negative implications, mainly commercial since according to European legislation antibiotic occurrence in honey samples is forbidden

Análise de sulfonamidas no mel: validação e optimização de um método de HPLC-fluorescência

Os antibióticos são usados no tratamento de abelhas com o intuito de combater infecções associadas às doenças da Loque Americana e Loque Europeia que atacam as larvas das abelhas levando à destruição da colmeia. As sulfonamidas são um dos grupos de antibióticos mais usados pelos apicultores. Como a Legislação Europeia, proíbe a comercialização do mel na presença de quaisquer resíduos de antibióticos, a implementação de métodos analíticos que permitam a detecção e quantificação destes resíduos afigura-se uma tarefa fundamental. O presente trabalho teve como objectivo a optimização, validação e aplicação de um método de HPLC, com detector de fluorescência e derivatização pré-coluna, para a análise de 11 sulfonamidas em amostras de mel: sulfatiazol, sulfamerazina, sulfametazina, sulfametizol, sulfametoxipiridazina, sulfisoxazol, sulfadoxina, sulfametoxazol, sulfadimetoxina, sulfaquinoxalina e sulfameter (padrão interno). O método de calibração usado foi o de sobreposição de matriz de mel com padrão interno. A separação foi efectuada numa coluna de fase reversa à temperatura de 32 ºC em modo de gradiente usando 2 eluentes preparados com diferentes proporções de tampão acetato (pH 4,0) e acetonitrilo. O tempo da corrida foi de 40 minutos. Os comprimentos de onda de excitação e de emissão usados foram 405 nm e 495 nm, respectivamente. A extracção de sulfonamidas das amostras de mel ou de soluções padrão de sulfonamidas com sobreposição de matriz de mel foi efectuada em SPE-C18, após um passo de hidrólise para quebra das ligações entre sulfonamidas e açúcares. Foram efectuadas calibrações usando soluções padrão de calibração de matriz de mel “claro” e de mel “escuro”, atendendo aos efeitos de matriz de mel obtidos nas sensibilidades das medições analíticas de algumas sulfonamidas. A linearidade do método para cada sulfonamida foi testada no intervalo de concentrações de 1-100 μg/L. Foi compensado o efeito de matriz pelo uso do método de sobreposição de matriz e da sulfonamida sulfameter como padrão interno. Os limites de detecção e quantificação para cada uma das sulfonamidas analisadas foram da ordem dos 0,4-3 μg/kg e 1-9 μg/kg de mel, respectivamente. O método de HPLC apresentou repetibilidade e precisão intermédia aceitáveis (CV em geral, inferiores a 5,0% e 15%, respectivamente). A exactidão do método foi aferida recorrendo a ensaios de recuperação, tendo-se obtido percentagens de recuperação entre 70-120%. Este método foi aplicado na detecção e quantificação de resíduos de antibióticos em amostras de mel após classificação da cor da amostra em mel “claro” ou mel “escuro”, e os resultados foram comparados com os obtidos pelo método qualitativo CHARM II (método aprovado pelo US-FDA para a análise de resíduos em mel).The antibiotics are used in the treatment of bees to treat infections associated with American Foulbrood and European Foulbrood diseases, that attack the bees larvae taking to the destruction of the beehive. Sulfonamides are one of the groups of antibiotics widely used by the beekeepers. As the European Legislation, prohibit the commercialization of honey in the presence of any residues of antibiotics, the implementation of analytical methods that allow the detection and quantification of these residues is a fundamental task. The objective of this work was the optimization, validation and application of a HPLC method, with fluorescence detection and pre-column derivatisation, for the analysis of 11 sulfonamides in honey samples: sulfathiazole, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxipyridazine, sulfisoxazole, sulfadoxine, sulfamethoxazole, sulfadimethoxine, sulfaquinoxalina and sulfameter (internal standard). The calibration method used was the sobreposition matrix method with internal standard. The separation was carried out in a reverse phase column at temperature of 32 ºC, with gradient using 2 eluents prepared with different proportions of buffer acetate solution (pH 4,0) and acetonitrile. The time of analysis was 40 minutes. The excitation and emission wavelengths were 405 nm and 495 nm, respectively. The sulfonamides extraction from honey samples or standard solution of sulfonamides prepared with honey sobreposition matrix, was performed with SPE-C18, after an hydrolysis step to liberate the sugar-sulfonamides bounds. Calibration were made using sulfonamide standard solutions either with “light” honey matrix and “dark” honey matrix, taking into account the honey matrix effects obtained in analytical measurement sensitivities for some sulfonamides. The linearity of the method for each sulfonamide was tested in the interval of concentrations from 1 to 100 μg/L. The matrix effect was compensated using of the sobreposition matrix method and sulfameter as internal standard. The detection and quantification limits for each analyzed sulfonamides were in the order of 0,4-3 μg/kg and 1-9 μg/kg of honey, respectively. The HPLC method presented acceptable repeatibility and intermediate precision (RSD%, in general, less than 5,0% and 15%, respectively). The method accuracy was checked with recovery assays and giving recovery percentages of 70-120%. This method was applied in the detection and quantification of antibiotics residues in honey samples after their colour classification, and the results were compared with the ones obtained by the qualitative method CHARM II (approved method for US-FDA for the analysis of residues in honey).Projecto “Rastreio Nacional de Antibióticos no mel: Avaliação das vias de contaminação dos resíduos no mel”, financiado pelo Programa Apícola Nacional, e com o apoio da Federação Nacional de Apicultores de Portugal

Systems analysis of metabolism in Helicobacter pylori

Tese de doutoramento em Engenharia Química e BiológicaHelicobacter pylori is associated with gastric diseases, such as gastritis, peptic and duodenal ulcers, mucosa associated lymphoid tissue lymphoma and gastric adenocarcinomas. Despite more than half of the global population being infected with this bacterium, not all individuals will develop clinical symptoms. Nevertheless, its association with gastric cancer, the high infection rate, as well as the failures on eradication efforts and vaccine development lead to an important health concern. In order to better understand the mechanisms leading to disease and also to develop effective ways to fight H. pylori infection, it is important to understand its physiological and metabolic behavior. H. pylori is a microaerophilic, gram negative, fastidious organism, for which no effective defined medium has been developed. Furthermore, current cultivation procedures use media that, even when semi-defined, have a variety of nutrients that can be used as carbon and energy sources. Under those conditions, the metabolic and physiological characterization of H. pylori is very difficult. The aim of this work was thus to study the physiology and metabolism of H. pylori to allow a better understanding of the behavior and pathogenicity of this organism. For that aim, a systems biology approach was used where Gas Chromatography-Mass Spectrometry (GC-MS) based metabolomics and genomics data were used to characterize the nutritional requirements and the preferred carbon sources and to build a reliable metabolic model. Data on H. pylori’s exometabolome was for the first time collected from experiments performed with semi-defined media and allowed to establish the essentiality of several amino acids: L-histidine, L-leucine, L-methionine, L-valine, L-isoleucine and Lphenylalanine. Moreover, this analysis indicated that L-aspartate, L-glutamate, L-proline and L-alanine could be potential carbon sources for H. pylori. Thus, a comparative analysis of different amino and organic acids as carbon sources was performed and glutamine / glutamate emerged as the most effective compounds. However, since glutamine is unstable under the conditions used, glutamate was selected the most adequate carbon source for growing H. pylori. Then, a simplified medium containing only the essential amino acids and Lglutamate, besides vitamins and salts, as well as 5% of fetal bovine serum (FBS) was used to perform physiological studies. Under those conditions, the specific growth rate was determined to be 0.126 h-1, while the use of uniformly labeled 13C L-glutamate together with GC-MS measurements of proteinogenic amino acids allowed to confirm the essential amino acids and raise some clues about L-proline and L-alanine which, given their presence in FBS, had still unclear roles. L-proline, although not labeled, is probably not essential and could be obtained from arginine, while L-alanine showed very low labeling patterns and is hypothesized to originate from pyruvate. Finally, labeling experiments allowed to confirm the presence of a complete tricarboxylic acid cycle. These collected data, together with genomics and other information available, were used to build and validate the first genome-scale metabolic model of H. pylori that is able to make quantitative predictions, covering roughly 23% of the genome. This model can be used, in the future, to investigate novel drug targets and elucidate the metabolic basis of infection. Furthermore, a comparative study was performed that analyzed different methods for assessing growth, viability, culturability and morphology, as these are important physiological parameters.A caracterização fenotípica é de extrema importância para entender o metabolismo celular, especialmente em organismos patogénicos, para os quais, na sua maioria, a informação disponível é limitada. Compreender o metabolismo de um organismo patogénico é fundamental para desvendar os mecanismos de interação patogénio-hospedeiro, com vista à identificação de alvos para desenvolvimento de fármacos. Neste trabalho estudou-se o metabolismo da bactéria Helicobacter pylori, um micro-organismo patogénico associado a doenças gástricas, usando uma estratégia de Biologia de Sistemas. As experiências realizadas usando um meio liquido semi-definido para crescer H. pylori provaram a existência de um fator de stress associado a uma paragem do seu crescimento, acompanhada de uma mudança de morfologia. Sendo conhecido o caracter pleiomórfico da H. pylori quando sujeita a determinadas condições de stress, e dado que os requisitos nutricionais não se encontravam completamente definidos, foi levantada a hipótese deste fator de stress estar associado à falta de um nutriente essencial. Assim, foi efetuada uma análise de exometaboloma com vista a determinar os seus requisitos nutricionais e preferências. Verificou-se a existência de aminoácidos essenciais ao crescimento, e comprovou-se a preferência dos aminoácidos como fonte de carbono (glutamato, prolina, alanina e aspartato). No entanto, algumas dúvidas persistiram relativamente à essencialidade de prolina e alanina. Para identificar a fonte de carbono preferencial foram testados diversos aminoácidos. Identificou-se que o aminoácido L-glutamato é o mais indicado ao crescimento de H. pylori, tendo-se efetuado a caracterização do seu crescimento com o referido aminoácido como fonte de carbono. Como forma de comprovar a efetiva utilização de L-glutamato como fonte de carbono, foram efetuadas experiências com 13C L-glutamato, tendo-se utilizado cromatografia gasosa com espectroscopia de massas para a análise das amostras. A análise dos aminoácidos do hidrolisado de biomassa de H. pylori permitiu comprovar a utilização de L-glutamato como fonte de carbono. Comprovou-se ainda a essencialidade dos aminoácidos isoleucina, histidina, leucina, metionina, fenilalanina e valina. Foi possível também identificar algumas vias metabólicas ativas para as quais residiam dúvidas, como o caso da existência do ciclo de Krebs completo. As experiências realizadas permitiram ainda levantar hipóteses acerca da biossíntese de L-prolina e L-alanina em H. pylori a partir de L-arginina e piruvato, respetivamente. Com a informação recolhida, associada à anotação do genoma recentemente efetuada foi possível reconstruir um modelo metabólico à escala genómica para H. pylori 26695. As previsões obtidas com o referido modelo estão de acordo com os dados experimentais. Este modelo contém informação biológica relevante para este organismo, com possível aplicação na identificação de potenciais alvos metabólicos, para a obtenção de fármacos mais efetivos na erradicação de H. pylori.Research Grant SFRH/BD/47596/2008 financed by FCT (Fundação para a Ciência e a Tecnologia). This work was supported in part by the ERDF - European Regional Development Fund thought the COMPETE Programme (operational programme for competitiveness), and National Funds through the FCT within the project FCOMP-01-0124-FEDER-009707 (HeliSysBio-molecular Systems Biology in Helicobacter pulori)

Re-annotation of the genome sequence of Helicobacter pylori 26695

Helicobacter pylori is a pathogenic bacterium that colonizes the human epithelia, causing duodenal and gastriculcers, and gastric cancer. The genome of H. pylori 26695 has been previously sequenced and annotated. In addition, two genome-scale metabolic models have been developed. In order to maintain accurate and relevant information on coding sequences (CDS) and to retrieve new information, the assignment of new functions to Helicobacter pylori 26695s genes was performed in this work. The use of software tools, on-line databases and an annotation pipeline for inspecting each gene allowed the attribution of validated EC numbers and TC numbers to metabolic genes encoding enzymes and transport proteins, respectively. 1212 genes encoding proteins were identified in this annotation, being 712 metabolic genes and 500 non-metabolic, while 191 new functions were assignment to the CDS of this bacterium. This information provides relevant biological information for the scientific community dealing with this organism and can be used as the basis for a new metabolic model reconstruction.This work was supported by the project FCOMP-01-0124-FEDER-009707, entitled HeliSysBio molecular Systems Biology in Helicobacter pylori (Ref.: FCT PTDC/EBB-EBI/104235/2008). Daniela Correia is grateful for financial support from the FCT (PhD grant: SFRH/BD/47596/2008)

System analysis of metabolism in Helicobacter pylori

Systems biology integrates different levels of information for understanding biological systems. The availability of the genome sequence of Helicobacter pylori has allowed the construction of a genome-scale metabolic model for this organism. In order to study the behaviour of H. pylori and understand the mechanisms associated with infection using systems biology tools and controlled cultivation conditions, fermentations in a chemically defined medium were performed and several conditions were tested. The experimental data obtained were compared with simulated data generated by the existing model. The simultaneous use of both approaches allows to correct the in silico model and, on the other hand, to rationally adjust the medium components. The improvement of the genome-scale metabolic model will allow the identification of potential targets in order to design more effective drugs for the inactivation of H. pylori

Uncovering the metabolic capacities of H. pylori 26695 using 13C labeling experiments

The determination of nutritional requirements of pathogenic organisms is of great significance for understanding host-pathogen interactions. Despite the knowledge obtained so far concerning amino acid requirements in H. pylori, it is still unclear which are the metabolic pathways used for biosynthesis and catabolism. Thus, information on the carbon flow in this organism is required. Glutamate is a very important metabolite in bacterial metabolism that can be used as a carbon and nitrogen source. 13C flux analysis has been largely applied to characterize phenotypes by quantifying in vivo the carbon fluxes. One of the most important applications of this approach is the identification of active pathways in less-studied organisms. Thus, in order to clarify the metabolic pathways used by H. pylori 26695, 13C labeling experiments with 13C-glutamate were conducted and labeled amino acids in biomass hydrolysates were analyzed by GC-MS. The obtained results confirmed L-glutamate as a potential sole and effective carbon source for H. pylori. Overall, all non-essential amino acids, except proline, presented a 13C labeling pattern. We hypothesized that L-proline is produced from L-arginine, while L-alanine is probably produced from pyruvate by alanine dehydrogenase. Additionally, the full usage of complete TCA cycle, under the conditions used, was also demonstrated

β-D-Glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H(2)O(2 )production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-β-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val(204), from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala(204). RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val(204), but not strains with the Bgl isoform Ala(204), do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., β-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val(204 )show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-β-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala(204). Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H(2)O(2 )production. Rather, the viability during addition of β-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val(204 )than for those with the isoform Ala(204). CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val(204 )with low hydrolysing activity are more prone to survive in environments that contain high levels of β-D-glucosides, thus contributing in some extent to mycoplasmaemia

Comparison of H. pylori in silico metabolic model predictions with experimental data

[Excerpt] The Systems Biology approach has been replacing the reductionist view that dominated biology research in the last decades. Present biochemical knowledge and genomic databases allowed the development of metabolic models for several organisms, which, however, are still incomplete. The availability of the genome sequence of H. pylori has allowed the construction of a genome-scale metabolic model for this organism. The purposes of this work were to study the growth of H. pylori in a chemically defined medium, to compare the experimental data obtained with the simulated data supplied by the model and analyse the composition of the in silico media used. [...