Dissertationen zum Thema „Bacterial proteome“

The explosive progress over the past decade in the fields of genomics, bioinformatics and mass spectrometry has resulted in an increased capability to investigate and compare the global protein expression of cells, tissues and organisms. The main focus of this study was on characterising the protein expression profiles of different serovars of Salmonella enterica and different serotypeso f Streptococcusp neumoniae in an attempt to identify protein factors associatedw ith host specificity and virulence. A novel approach for typing of bacterial isolates using SELDI TOF MS was developed. A thorough investigation on the effect of different factors on the quality of the SELDI profile was carried out, and the potential of several software programmes to perform cluster analysis of the SELDI data was assessedB. oth cytosolic and membrane-associatepdr oteins were separatedb y 2D GE, and detailed referencem aps of the proteins expressedu nder standardisedc. onditions were created.I n the caseo f Salmonella, it containedm ore than 300 proteins. The comparativea nalysisa t the subspeciesle vel revealedt hat, in many cases,t he variation in the expression patterns was greater between strains with the same serospecificity than betweens erotypes/serovarss, uggestingt hat the serological properties of bacteria do not correlate with differential protein synthesis. However, in the case of Salmonella, where the serovars have different host specificity, the high resolution 2D gel maps revealed several serovar-specific proteins, including enzymes involved in the catabolismo f various substrates,o r in the processo f cell detoxification. Changesi n the expression patterns of the serovars in different growth conditions, such as pH or oxygen availability, were mostly universal amongst the serovars, although a few serovar-specific proteins were also present. The findings revealed parts of the proteome that alter their expression when the microorganism are subjected to unfavourable conditions such as while colonising the host, amongst other parts that remain stable. Overall, the results demonstrated the importance of analysing many different isolates when performing protein expression studies in highly variable microbial populations.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Physics, May, 2020
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 315-348).
The quantitative composition of proteomes results from biophysical and biochemical selective pressures acting under system-level resource allocation constraints. The nature and strength of these evolutionary driving forces remain obscure. Through the development of analytical tools and precision measurement platforms spanning biological scales, we found evidence of optimization in bacterial gene expression programs. We compared protein synthesis rates across distant lineages and found tight conservation of in-pathway enzyme expression stoichiometry, suggesting generic selective pressures on expression setpoints. Beyond conservation, we used high-resolution transcriptomics to identify numerous examples of stoichiometry preserving cis-elements compensation in pathway operons. Genome-wide mapping of transcription termination sites also led to the discovery of a phylogenetically widespread mode of bacterial gene expression, 'runaway transcription', whereby RNA polymerases are functionally uncoupled from pioneering ribosomes on mRNAs. To delineate biophysical rationales underlying these pressures, we formulated a parsimonious ribosome allocation model capturing the trade-off between reaction flux and protein production cost. The model correctly predicts the expression hierarchy of key translation factors. We then directly measured the quantitative relationship between expression and fitness for specific translation factors in the Gram-positive species Bacillus subtilis. These precision measurements confirmed that endogenous expression maximizes growth rate. Idiosyncratic transcriptional changes in regulons were however observed away from endogenous expression. The resulting physiological burdens sharpened the fitness landscapes. Spurious system-level responses to targeted expression perturbations, called 'regulatory entrenchment', thus exacerbate the requirement for precisely set expression stoichiometry.
by Jean-Benoît Lalanne.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Physics

El género Brachyspira incluye varias especies patogénicas que afectan a cerdos, perros, pájaros y humanos. En cerdos, Brachyspira (anteriormente Serpulina y Treponema) hyodysenteriae y Brachyspira pilosicoli son patógenos intestinales bien conocidos. Estas especies son espiroquetas gram-negativas, flageladas y anaeróbicas, las cuales viven en el intestino grueso y que tienen una asociación estrecha con la mucosa del colon. Brachyspira hyodysenteriae es el agente causante de la disentería porcina, mientras que Brachyspira pilosicoli está relacionada con la espiroquetosis intestinal, una colitis más leve, no hemorrágica. La disentería porcina es una enfermedad con un impacto importante en la producción porcina debido a los costes vinculados a la mortalidad, morbididad, la producción ineficiente y la medicación de los animales. Aunque la enfermedad puede afectar animales de todas las edades, afecta raramente a lechones menores de tres semanas, produciéndose más frecuentemente durante los periodos de crecimiento y finalización, lo cual agrava las pérdidas económicas. Las estrategias para tratar estas enfermedades se basan principalmente en el uso de antibióticos como Tiamulin, Valnemulin, Tylosin, Tylvalosin o Lincomycin. Desafortunadamente, cepas resistentes a antibióticos han sido detectadas para ambas especies en diferentes países alrededor del mundo. A pesar de que hace mucho tiempo que se conoce el hecho de que los cerdos que se recuperan de una infección desarrollan Resistencia contra Brachyspira hyodysenteriae, no hay una vacuna disponible todavía. Los genomas de estas especies están disponibles desde los años 2009-2010, pero la información a nivel proteómico todavía es escasa. En este trabajo se presenta la caracterización de los proteomas de estos patógenos. Se proporciona evidencia experimental del perfil de expresión proteica en estas especies, incluyendo PTMs y SAS, y se ha llevado a cabo en el contexto de la búsqueda de potenciales candidatos para elaborar una vacuna. Esta caracterización se ha llevado a cabo a través del estudio del proteoma total, el proteoma expuesto y el inmunoproteoma de cepas comerciales y aislados de estas especies. - El proteoma total fue estudiado a través de una aproximación proteómica shotgun, usando una serie de herramientas informáticas dirigidas a optimizar la cantidad de información sobre la secuencia obtenida de los datos espectrométricos. En una primera etapa, los espectros fueron analizados con una combinación de seis motores de búsquedas diferentes usando la aplicación PeptideShaker. Aquellos espectros que no se identificaron en esta primera etapa fueron analizados con una combinación de novo y búsqueda en base de datos con motor de búsqueda usando la aplicación PEAKS. Las secuencias que no se identificaron después de esta etapa se buscaron en BLAST frente a base de datos de Brachyspira total y de mamíferos. En resumen, más de 1500 proteínas fueron identificadas para cada especie. La cobertura del proteoma estimada fue 67-70%. Además, se ha descrito el perfil de PTM, siendo la metilación la modificación más frecuente. A través del enriquecimiento especifico se identificaron 79 y 91 sitios de fosforilación, y 3221 y 5579 sitios de acetilación para B. hyodysenteriae y B. pilosicoli, respectivamente. - El proteoma expuesto fue estudiado usando sobrenadantes celulares y muestras obtenidas después de un tratamiento enzimático controlado de las células intactas. Entre las proteínas más abundantes identificadas en el proteoma expuesto hay proteínas relacionadas con movimiento/chemotaxis, proteínas ribosomales, enolase, NADH oxidasa, y Heat Shock Proteins. - El inmunoproteoma fue caracterizado por immunoblot de las proteínas de bacteria con suero de cerdos experimentalmente infectados. Se identificaron once proteínas inmunoreactivas para B. hyodysenteriae y 8 para B. pilosicoli. Dos de estas proteínas, enolase y PEPCK fueron identificadas como inmunoreactivas en las dos especies.
The genus Brachyspira includes several pathogenic species affecting pigs, dogs, birds and human. In pigs, Brachyspira (formerly Serpulina and Treponema) hyodysenteriae and Brachyspira pilosicoli are well known intestinal pathogens. These species are flagellated, anaerobic, gram negative spirochetes which inhabit the large intestine and have an intimate association with the colonic mucosa. Brachyspira hyodysenteriae is the causative agent of Swine dysentery, while Brachyspira pilosicoli is associated with Intestinal spirochetosis, a milder, non–haemorrhagic colitis. Swine dysentery is a disease with an important impact on pig production due to the costs associated with mortality, morbidity, inefficient production and medication of the animals. Although the disease can affect animals of all ages, it is rarely detected in piglets younger than three weeks old; occurring more frequently during growing/finishing periods, which aggravates the economic losses. Strategies to treat these diseases rely mainly in the use of antibiotics such as Tiamulin, Valnemulin, Tylosin, Tylvalosin or Lincomycin. Unfortunately, antibiotic resistant strains have been detected for both species in many countries around the world. Despite it is long been known that pigs generate resistance against B. hyodysenteriae after recovering from infection, no vaccine is available yet. The genomes of these species are available since 2009-2010 but proteome information is still scarce. In this work, a large-scale characterization of the proteomes of these pathogens is presented. The work provides experimental evidence of the protein expression profile in these pathogens, including PTMs and SAS and is carried out in the context of the search for potential vaccine candidates. This characterization has been performed through the study of the total proteome, the exposed proteome and the immunoproteome of commercial and environmental strains of these species. - The total proteome was studied through a shotgun proteomics strategy, using a range sofware tools directed to optimize the amount of sequence information extractable from the spectrometric data. In a first stage, spectra were analysed with a combination of six different search engines using the PeptideShaker application. Unmatched spectra were analysed by a combination of de novo and database search engines using the PEAKS application. Unmatched sequence tags after this stage were further BLASTed against Brachyspira and mammals databases. Overall, more than 1500 proteins were identified for each species. The estimated proteome coverage was 67-70%. In addition their PTM profile was described, being methylation the more frequent modification. Specific enrichment allowed identification of 79 and 91 phosphorylation sites and 3221 and 5579 acetylation sites for B. hyodysenteriae and B. pilosicoli, respectively. - The exposed proteome was studied using cell culture supernatants and samples obtained after a controlled enzymatic treatment of intact cells. Among the most abundant exposed proteins are proteins related to movement/chemotaxis, ribosomal proteins, enolase, NADH oxidase and Heat Shock Proteins. - The immunoproteome was characterized immunoblotting the bacterial proteins with sera from challenged pigs. Eleven immunoreactive proteins for B. hyodysenteriae and 8 for B. pilosicoli were identified. Two of these proteins, enolase and PEPCK, were found immunoreactive in the two species.

Die hier vorgestellte Studie untersucht systematisch die angeborene Immunabwehr gegen L. pneumophila auf Ebene des gesamten Wirtsorganismus, sowie auf molekularer Ebene in Alveolar- und Knochenmarksmakrophagen. Mittels in vivo Transkriptomanalysen werden Typ I und II Interferone (IFN) als Hauptregulatoren der frühen pulmonalen Genexpression in der L. pneumophila-Infektion identifiziert. Infektionsexperimente in Wildtyp- und IFN-Rezeptor-defizienten Tieren offenbaren, dass Typ I und II IFNe maßgeblich die antibakterielle Abwehr gegen L. pneumophila vermitteln. Für die Bekämpfung der Infektion in der Lunge werden CD11c+ Zellen als wichtigste Empfänger der IFN-Signale identifiziert. Des Weiteren wird durch Behandlung von CD11c+ Alveolarmakrophagen mit IFNen ex vivo das intrazelluläre bakterielle Wachstum inhibiert. Mittels subzellulärer quantitativer Massenspektrometrie wird gezeigt, dass die Proteinkomposition der Legionellen-enthaltenden Vakuole substanziell durch beide IFNe modifiziert wird. In einer vergleichenden Netzwerkanalyse werden diese Proteomdaten mit eigenen und öffentlich zugänglichen Transkriptomdaten verglichen. Hierdurch können klar abgegrenzte Untergruppen von einerseits transkriptionell durch IFN-regulierten Proteinen sowie andererseits ausschließlich räumlich IFN-regulierten Proteinen unterschieden werden. Unter den durch IFN an der Vakuole angereicherten Proteinen wird Immunoresponsive gene 1 (IRG1) als zentraler Effektor identifiziert, welcher das Wachstum von L. pneumophila durch die Produktion des antibakteriellen Metaboliten Itaconsäure inhibiert. Zusammenfassend stellt diese Studie eine umfassende Ressource von IFN-vermittelten Effekten auf die Genexpression sowie auf das Proteom der bakteriellen Vakuole dar und deckt einen zellautonomen Abwehrmechanismus gegen L. pneumophila auf, welcher durch die IRG1-abhängige Produktion von Itaconsäure vermittelt wird.
The study presented here systemically examines the innate immune response against L. pneumophila on whole organism level as well as on a molecular level within macrophages, L. pneumophilas’ host cell. In vivo transcriptome analyses identify type I and II interferons (IFNs) as master regulators of the early pulmonary gene expression during L. pneumophila infection. Infection experiments in wild-type mice and mice lacking type I and/or II IFN signaling reveal a severe defect of antibacterial defense when IFN signaling is absent. CD11c+ cells were found to be the main targets of IFNs to restrict infection in the lung, and IFNs inhibited bacterial growth in CD11c+ alveolar macrophages ex vivo. Subcellular quantitative mass spectrometry shows that both IFNs substantially modify the protein composition of Legionella-containing vacuoles. Comparative network analysis, combining these proteome data with transcriptome data as well as public database data reveals distinct subsets of transcriptionally regulated IFN-stimulated genes (ISGs) on the one hand, but interestingly also exclusively spatially IFN-regulated vacuolar proteins. Among IFN-regulated vacuolar proteins, Immunoresponsive gene 1 (IRG1) was identified as a central effector that restricts growth of L. pneumophila through production of the antibacterial metabolite itaconic acid in macrophages. Collectively, this study provides a comprehensive resource of IFN-mediated effects on gene expression and the bacterial vacuolar proteome, and uncovers a cell-autonomous defense pathway against L. pneumophila, which is mediated by IFNs, IRG1 and itaconic acid.

The Gram-negative bacterium Helicobacter pylori is found in human gastric mucosa. H. pylori, one of the most common chronic bacterial infections of humans, is present in almost half of the world population. It is associated with chronic gastritis, non-ulcer dyspepsia, gastric and duodenal ulcers, and malignant neoplasms. The aim of this study was to detect microbial candidate protein markers whose presence might be correlated with the development of four different clinical consequences of H. pylori infection, gastric ulceration [GU], duodenal ulceration [DU], non-ulcer dyspepsia [NUD] and gastritis [GI]. Eleven H. pylori isolates associated with these outcomes were analysed. The total complement of protein from these H. pylori isolates were resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and compared using PDQUEST pattern analysis software. Relationships between the isolates associated with specific disease outcomes were determined by cluster analysis.Fifty six disease specific proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Up to 1165 protein species were resolved from each H. pylori strain. Proteome analysis revealed that only 470 (40%) of the proteins detected were common to all eleven isolates. Twenty six of the 56 disease specific proteins that were selected for identification consisted of spots whose expression is altered in response to stress conditions or those that can affect H. pylori cell division and the cell membrane. The remaining 30 proteins had no known function. This study has provided further confirmation of the extensive variation that the bacterium H. pylori exhibits at the proteome level. Most significantly this study has found, through the application of cluster analysis and protein matching, that isolates do form disease groups. Comparative proteome analysis is a useful method for highlighting the extensive strain variation that H. pylori exhibits and to determine if any disease specific proteins exist.

Orientadores: Francisco Carlos Groppo, Reginaldo Bruno Gonçalves
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O uso do cigarro tem sido associado com a progressão da periodontite bem como com a redução da resposta à terapia aplicada a essa doença. Porphyromonas gingivalis é um importante colonizador do biofilme subgengival além de ser um dos principais patógenos envolvidos no estabelecimento e progressão da doença periodontal. No entanto, os possíveis efeitos dos principais derivados do cigarro sobre P. gingivalis ainda não foram totalmente investigados. Dessa forma, os objetivos deste estudo foram avaliar os efeitos da nicotina e cotinina sobre a expressão de proteínas e sobre a capacidade de adesão e invasão celular de P. gingivalis. A fim de avaliar a expressão de proteínas, culturas de P. gingivalis W83 foram expostas à nicotina e cotinina nas concentrações de 6 e 600µg/mL, as proteínas foram extraídas, separadas por eletroforese bidimensional em gel de poliacrilamida (12.5% SDS-PAGE) e identificadas por LC-MS/MS. Os géis e suas corridas eletroforéticas foram feitas em triplicatas e a detecção de proteínas nos mesmos foi feita através de coloração com corante Coomassie. Proteínas diferentemente expressas foram digeridas com tripsina e as amostras de peptídeos sequenciadas utilizando um sistema Q-TOF API LC-MS/MS. A busca MS/MS foi realizada utilizando os bancos de dados MSDB e NCBI através do programa Mascot. Para examinar a capacidade de adesão e invasão de P. gingivalis, monocamadas de células KB e culturas de P. gingivalis ATCC 33277 foram expostas às concentrações de 0.1, 10 e 100 µg/mL de nicotina e cotinina. As células epiteliais foram incubadas por 24 h enquanto P. gingivalis foi exposta a essas substâncias até atingir a fase logarítmica. Após o período de incubação, P. gingivalis foi submetida aos ensaios de adesão e invasão às células KB. O número de bactérias associadas às células foi obtido através de contagem de unidades formadoras de colônia. Os resultados obtidos da análise expressão de proteínas mostraram que a adição de nicotina e cotinina promoveram alterações no proteoma de P. gingivalis. Entre os ± 430 spots de proteínas reproduzíveis detectados em cada gel, 20 proteínas foram menos expressas e 42 foram mais expressas em pelo menos um dos tratamentos (p<0.05; ANOVA - Tukey). Entre as proteínas identificadas, muitas estavam envolvidas em processos como produção de energia celular, síntese de proteínas, estresse oxidativo, virulência, transporte, etc. Em relação aos resultados obtidos nos ensaios de adesão e invasão, foi evidenciado que, quando as células epiteliais foram inoculadas com nicotina e cotinina, nenhuma diferença significativa na colonização de P. gingivalis foi encontrada. Quando P. gingivalis foi exposta à maior concentração de cotinina, sua capacidade de adesão e invasão às células epiteliais aumentou de forma expressiva (p<0.05; ANOVA - Tukey). No entanto, a nicotina e as outras concentrações de cotinina testadas não alteraram a capacidade de colonização. Esses achados indicam que a nicotina e a cotinina podem afetar a expressão de proteínas de P. gingivalis. Ainda, a cotinina pode alterar positivamente a eficiência de adesão e invasão de P. gingivalis.
Abstract: Cigarette smoking is associated with the development of periodontitis and the decreased response to periodontal therapy. P. gingivalis is an important colonizer of the subgingival biofilm and is one of the major pathogens involved in the initiation and progression of periodontal disease. However, the possible effects of major cigarette's derivatives on P. gingivalis were not fully investigated. Thus, the purpose of the present study was to evaluate the effects of nicotine and cotinine on the protein expression and cellular adhesion and invasion abilities of P. gingivalis. To evaluate protein expression, P. gingivalis W83 cultures were exposed to nicotine and cotinine 6 and 600µg/mL concentrations, the proteins were extracted, separated by two-dimensional polyacrylamide gel electrophoresis (12.5% PAGE) and identified with LC-MS/MS. The gels were run in triplicates and detection of proteins was obtained by staining the gels with Coomassie blue. Proteins differentially expressed were digested with trypsin, and the peptide samples sequenced using a Q-TOF API LC-MS/MS system. The MS/MS was searched against the MSDB and NCBI databank using Mascot program. In order to assess P. gingivalis adhesion and invasion abilities, KB cells monolayers and P. gingivalis ATCC 33277 cultures were exposed to 0.1, 10 and 100 µg/mL nicotine and cotinine concentrations. The epithelial cells were incubated for 24 h while P. gingivalis was exposed to these substances until early logarithmic phase. After incubation period, P. gingivalis were submitted to assays to evaluate adhesion to and invasion of KB cells. The number of bacteria associated with these cells was assessed by counting the colony-forming unities. The results from protein expression analyses showed that addition of nicotine and cotinine promoted alterations in proteome profile of P. gingivalis. Among ± 430 protein spots reproducibly detected on each gel, 20 protein spots were downregulated, and 42 were upregulated at least in one treatment (p<0.05; ANOVA - Tukey test). The identified proteins are involved in several processes, i.e. energy production, protein synthesis, oxidative stress, virulence, transport and binding activities. Data obtained from adhesion and invasion assays evidenced that epithelial cells inoculated with nicotine and cotinine did not show any significant differences in P. gingivalis colonization. When P. gingivalis was exposed to the higher concentration of cotinine, adherence and invasion of this bacterium to the epithelial cells markedly increased (p<0.05; ANOVA - Tukey test). However, nicotine and the other concentrations of cotinine did not alter the colonization ability. These findings indicate that nicotine and cotinine may affect P. gingivalis protein expression. In addition, cotinine may alter positively P. gingivalis adhesion and invasion efficiencies.
Doutorado
Farmacologia, Anestesiologia e Terapeutica
Doutor em Odontologia

Orientadores: Adriana Zerlotti Mercadante, Fabio Marcio Squina
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Espécies reativas de oxigênio (ERO) e nitrogênio (ERN) são geradas dentro das células pela exposição a agentes endógenos e exógenos, estas espécies, quando em níveis normais, encontram-se envolvidas na produção de energia, regulação do crescimento celular, sinalização intercelular e síntese de substâncias biológicas importantes. Por outro lado, se produzidas em excesso, podem provocar oxidação lípidica, de proteínas ou do DNA causando o que conhecemos por estresse oxidativo. Para combater o excesso de espécies reativas, os organimos produzem moléculas antioxidantes tais como os carotenoides e enzimas como superóxido dismutase e catalase. No entanto, é difícil apontar as estratégias de adaptação dos micro-organismos em resposta a diferentes condições de estresse através do estudo individual de moléculas produzidas. Diante do exposto, esta pesquisa teve por objetivo elucidar o genoma, proteoma e transcriptoma bem como a produção de carotenoides da bactéria termófila Thermus filiformis quando submetida à algumas condições de cultivo: presença e ausência de H2O2 e temperatura de crescimento abaixo (63 ?C) e acima (77 ?C) do seu ótimo (70 ?C). Para tanto, o genoma e transcriptoma foram analisados com o emprego de tecnologias de sequenciamento de última geração e ferramentas computacionais, e a proteômica e os carotenoides foram caracterizados por cromatografia líquida e espectrometria de massas. Além disso, devido à conhecida capacidade antioxidante e alto potencial de aplicabilidade na indústria farmacêutica, cosmética e de formulação de alimentos, foi feita a clonagem, expressão e caracterização da enzima superóxido dismutase de Thermus filiformis (TfSOD). A TfSOD apresentou atividade enzimática utilizando como cofator tanto manganês quanto ferro e termoestabilidade a até 80 ?C. O sequenciamento de DNA produziu um total de 9.680.471 reads pareados e uma montagem com n50 = 85,2Kb, n90 = 17,1kb, contig de maior tamanho com 275,5kb e um tamanho total de 2,46MB. A predição genética resultou em 2.403 genes codificadores de proteínas. Na análise de transcriptoma, 97,1% dos genes codificadores de proteínas preditos apresentaram expressão com valores detectáveis de RSEM (RNA-Seq by Expectation-Maximization). Através da análise do transcriptoma foram identificados 37% e 5,86% dos genes diferencialmente expressos (p-valor<0,05) nos ensaios com diferentes temperaturas e com e sem adição de H2O2, respectivamente. Através da análise do proteoma, no ensaio com diferentes temperaturas, foi encontrado um total de 27,7% proteínas diferencialmente expressas com um FDR (False Discovery Rate) < 0,05%, sendo 20% significativamente diferentes (p-valor<0,05, teste T) e, no ensaio com e sem adição de H2O2, um total de 28,3% com um FDR < 0,05%, sendo 6% significativamente diferentes (p-valor<0,05, teste T). Algumas diferenças foram observadas na produção de carotenoides de acordo com cada condição de cultivo. Quanto ao perfil de carotenoides, nas condições a 70 ?C e a 77 ?C os carotenoides majoritários foram termozeaxantina-15 e termozeaxantina-13, enquanto que para condição a 63 ?C foram termozeaxantina-15 e zeaxantina livre. A amostra cultivada a 70 ?C sem adição de H2O2 apresentou a maior quantidade de carotenoides totais (1.516 ?g/g), por outro lado o extrato rico em carotenoides que apresentou maior capacidade de desativação do radical peroxila (50,5) foi o da amostra com adição de H2O2. Os resultados do presente estudo mostram que os principais processos afetados pela mudança de temperatura e adição de peróxido de hidrogênio foram: catabolismo, transcrição e tradução de proteínas. Observou-se também que a alteração na temperatura teve uma maior influencia na expressão diferencial de genes e proteinas do que a adição de peróxido. Através das análises do trancriptoma e do proteoma de T. filiformis foram identificadas enzimas termo-estáveis com potencial de aplicação industrial, como por exemplo alfa-amilases, alfa-galactosidases e esterases. Além disso, o extrato rico em carotenoides dessa bactéria apresentou capacidade de desativar o radical peroxila superior à capacidade de extratos de frutas e até mesmo de padrões de carotenoides
Abstract: Reactive oxygen (ROS) and nitrogen (RNS) species are produced in the cells by exposure to endogenous and exogenous agents, these species, when at normal levels, are involved in energy production, cell growth regulation, intercellular signaling and synthesis of important biological substances. On the other hand, if overproduced, can cause lipid, protein and DNA oxidation, leading to what is known as oxidative stress. To combat excessive reactive species, organisms produce antioxidant molecules such as carotenoids and enzymes such as superoxide dismutase and catalase. However, it is difficult to point out the adaptation strategies of microorganisms in response to different stress conditions through the study of individual molecules. Therefore the aim of this research was to elucidate the genome, proteome and transcriptome, as well as the carotenoid production of Thermus filiformis when submitted to the some cultivation conditions under stress: without and with hydrogen peroxide and temperature below (63 ?C) and above (77 ?C) the optimum (70 ?C). In order to achieve this aim, the genome and transcriptome were analyzed using next generation technology and computational tools, and proteome and carotenoids were characterized by liquid chromatography and mass spectrometry. Moreover, due to its known antioxidant capacity and potential application on pharmaceutical, cosmetics and food formulations, a superoxide dismutase from Thermus filiformis (TfSOD) was cloned, expressed and characterized. The TfSOD showed cambialistic characteristics, once it had enzymatic activity with either manganese or iron as cofactor and thermostability until 80 ?C. The DNA sequencing produced a total of 9,680,471 paired reads and the produced assembly had an n50 = 85.2Kb, n90 = 17.1kb, the largest contig size = 275.5kb and total size of 2.46MB. Gene prediction resulted in 2,403 protein coding genes. In the transcriptome analysis, 97.1% of predicted protein coding genes showed detectable expression with RSEM values (RNA-Seq by Expectation-Maximization). Through the computational analysis of T. filiformis transcriptome 37% and 5.86% of the genes significantly different (p-value < 0.05) in the assays with different temperatures and with and without H2O2 were identified, respectivelly. In the total proteome analysis a total of 27.7% proteins were differentially expressed with a FDR (False Discovery Rate) < 0.05%, being 20% significantly different (p-value < 0.05, T-test) in the temperature assay and 28.3% proteins with a FDR (False Discovery Rate) < 0.05%, being 6% significantly different (p-value < 0.05, T-test) in the H2O2 assay. Some changes were observed in the carotenoid production according to the cultivation condition. Regarding to the carotenoid profile, the major carotenoids under conditions at 70 ?C (without and with H2O2) and at 77 ?C were thermozeaxanthin-15 and thermozeaxanthin-13 while at 63 ?C were thermozeaxanthin-15 and free-zeaxanthin. The sample cultivated at 70 ?C without H2O2 showed the highest amount of total carotenoid (1516 ?g/g of dry mass), on the other hand the sample with the highest antioxidant capacity was the one cultivated at 70 ?C with H2O2. The carotenoid rich extract of all conditions studied showed a peroxyl scavenging capacity higher than those carotenoid rich extracts from some fruits and from some carotenoid standards, demonstrating the potential applicability of T. filiformis extracts in industry. The results of this study show that the main processes affected by temperature change and addition of H2O2 were: catabolism, transcription and protein translation. It was also observed that the change in temperature has greater influence on the differential expression of genes and proteins than the H2O2 addition. Through trancriptome and proteome analysis of T. filiformis thermostable enzymes have been identified with potential industrial applications, such as alpha-amylases, alpha-galactosidases and esterases. Moreover, the extract rich in carotenoids of this bacterium had a greater peroxyl radical scavenging capacity than the capacity of fruit extracts and even carotenoids standards
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Orientador: Hélia Harumi Sato
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Proteases bacterianas são enzimas de elevada importância comercial, amplamente aplicadas em diversas áreas como nas indústrias de detergentes, de alimentos, farmacêutica e têxtil. Este trabalho teve como principais objetivos selecionar entre 59 linhagens de Bacillus sp., da coleção de culturas do Laboratório de Bioquímica de Alimentos da FEA, aquelas que apresentam potencial de maior produção de proteases com características tais como estabilidade em diferentes condições de temperatura, pH, detergentes e solventes orgânicos, atividade em ampla faixa de pH e capacidade de lisar células de Xanthomonas campestris. Em seguida, visou-se otimizar a produção de proteases pela linhagem selecionada, determinar as características bioquímicas da protease parcialmente purificada e estudar a aplicação do extrato enzimático bruto e preparação parcialmente purificada. Entre as cinquenta e nove linhagens de Bacillus sp. testadas foram selecionadas nove linhagens que produziram maior atividade de proteases. A produção de protease pelas nove linhagens foi testada em frascos agitados contendo o meio de cultura nº 1 (10g/L de caseína, 1g/L de extrato de levedura e sais), meio nº 2 (35 g/L de melaço de cana de açúcar, 20g/L de água de maceração de milho, 3g/L de extrato de levedura Prodex-Lac SD® e 20g/L de soro de queijo), e por fermentação em meio sólido nº 3 (farelo de trigo e água, na proporção 1:1, m:m). As linhagens de Bacillus sp. LBA 07, Bacillus sp. LBA 46 e Bacillus sp. LBA 08 fermentadas nos meios de cultura nº 1, nº 2 e nº 3 produziram 222 U/mL, 548 U/mL e 13480 U/grama de substrato seco (gss) respectivamente. As proteases dos extratos enzimáticos brutos obtidos das nove linhagens fermentadas nos três meios de cultura apresentaram atividade ótima na faixa de pH 7 a 9 e 60° C, estabilidade na faixa de pH 5 a 9 por 24h a 4º C , e após 1 h de tratamento a 50° C. Entre os extratos enzimáticos brutos de proteases testados, aqueles obtidas da fermentação de Bacillus sp. LBA 46 nos três meios de cultura foram as mais estáveis em detergente Ariel®. Quando incubadas em solventes orgânicos alguns extratos enzimáticos brutos proteases mantiveram mais de 60% de atividade residual após 24h em acetona (Bacillus sp. LBA 8 e 44), hexano (Bacillus sp. LBA 19, 29, 44, 46 e 60), clorofórmio (Bacillus sp. LBA 44 and 60) e etanol (Bacillus sp. LBA 60). Os extratos enzimáticos brutos de proteases obtidos do cultivo da linhagem de Bacillus sp. LBA 46 nos meios n° 2 e n° 3 foram as mais eficientes na lise de células de Xanthomonas campestris, aumentando cerca de 30% a transmitância a 620 nm (Trans 620nm) do meio fermentado de goma xantana. A linhagem de Bacillus sp. LBA 46 foi selecionada como melhor produtora de protease e estudos preliminares de identificação biomolecular indicam que se trata de uma linhagem de Bacillus licheniformis. Utilizando-se a linhagem de Bacillus sp LBA 46 e o meio de cultura otimizado (meio n° 4) por metodologia de superfície de resposta (MSR), composto de 40g/L de melaço de cana de açúcar, 6g/L de água de maceração de milho, 2g/L de extrato de levedura Prodex-Lac SD® e 20g/L de soro de queijo, foi obtido 3000 U/mL de protease após 96h de fermentação a 30° C e 200 rpm. No estudo da aplicação da enzima para a remoção de manchas de tecidos de algodão foram obtidos melhores resultados de remoção de manchas de sangue e molho de tomate com carne moída, utilizando-se a combinação de extrato bruto de protease (100 ou 1000U) com o detergente Omo®. O extrato enzimático bruto da linhagem de Bacillus sp. LBA 46 foi parcialmente purificado por fracionamento com sulfato de amônio (80% de saturação), diálise e cromatografia de filtração em gel (Sephadex G100), resultando em fator de purificação de 3,69. Após caracterização com MSR observou-se que a protease da preparação parcialmente purificada apresentou atividade ótima a 55° C e pH 7,5 e considerável estabilidade (95% de atividade residual) na faixa de pH 5,7 ¿ 9,3 após 1h de incubação a 30 ¿ 36° C, e acima de 78,9% quando incubadas por 1h em pH 7,5 e 50° C. A condição ótima de lise das células de X. campestris do meio fermentado de goma xantana utilizando-se o extrato enzimático bruto de protease e a preparação parcialmente purificada de proteases, foi observada utilizando 42 U de protease /mL de suspensão celular de X. campestris a 60° C, resultando em aumento de mais de 20% da Trans 620nm do meio fermentado de goma xantana. Um aumento de quase 40% de Trans 600nm foi observado após 2h de reação utilizando extrato enzimático bruto de protease (42 U de protease/mL de suspensão celular de X. campestris) a 65° C. A produção de proteases de Bacillus sp. LBA 46 por fermentação em estado sólido foi otimizada utilizando MSR, sendo obtido 5000 U/grama de substrato seco utilizando-se meio de cultura composto de farelo de trigo e água (60%:40%) após 96h de fermentação a 30° C
Abstract: Proteases are commercially relevant enzymes widely applied in several industrial areas, such as in detergent, food, pharmaceutical and textile industries. Proteases from Bacillus sp. can present advantages compared to the proteases from other sources, including better thermostability, stability in pH range from slightly acid to alkaline pH values and stability in organic solvents. The aims of this work were selecting Bacillus sp. strains with capability of producing proteases with better biochemical properties, such as stability in different conditions of temperature, pH, detergents and organic solvents, activity in a wide range of pH and capability of lysing cells of Xanthomonas campestris. Afterwards, it was aimed the optimization of the production of proteases by the selected Bacillus sp. strain and the determination of the biochemical characteristics of the partially purified protease and the application of the crude and partially purified protease. Nine Bacillus sp. strains were selected as the best protease producers among fifty nine Bacillus sp. strains tested. The protease production by the nine strains was carried out in Erlenmeyer flasks containing medium no. 1 (10g /L of casein, 1g/L of yeast extract and salts), medium no. 2 (35 g/L of sugar cane molasses, 20g/L corn steep liquor, 3g/L of yeast extract Prodex-Lac SD® and 20g/L of dried whey), e by fermentation using solid substrate medium no. 3 (wheat bran and water, 1:1, m:m). The strains Bacillus sp. LBA 07, Bacillus sp. LBA 46 and Bacillus sp. LBA 08 when fermented in medium no. 1, no. 2 e no. 3 produced 222 U/mL, 545 U/mL and 13480 U/gram of dried substrate (gds) respectively. Proteases from the crude enzymatic extracts obtained from the fermentation of the nine Bacillus sp. strains in the three media showed optimal activity in pH range 7-9 and 60° C, stability in pH range 5-9 for 24 hours at 4° C and after 1h at 50° C. The protease preparations from the fermentation of Bacillus sp. LBA 46 in the three media were the most stable when incubated in detergent Ariel®, among the proteases tested from the Bacillus sp. strains. In addition, some proteases presented more than 60% residual activity after 24h in the organic solvents acetone (Bacillus sp. LBA 8 and 44), hexane (Bacillus sp. LBA 19, 29, 44, 46 and 60), chloroform (Bacillus sp. LBA 44 and 60) and ethanol (Bacillus sp. LBA 60). The protease preparations obtained from the cultivation of Bacillus sp. LBA 46 in medium no. 2 and no. 3 presented the best results on the lysis of Xanthomonas campestris cells, resulting in an increase of approximately 30% in transmittance at 620 nm (Trans 620nm) of the fermented broth of xanthan. Bacillus sp. LBA 46 strain was selected as the best protease producer and after preliminary biomolecular analysis of identification, the results indicate that this microorganism correspond to a Bacillus licheniformis strain. Protease preparation containing 3000 U/mL was obtained from Bacillus sp. LBA 46 cultivated in Erlenmeyer flasks containing medium no. 4 composed of 40g/L of sugar cane molasses, 6g/L of corn steep liquor, 2g/L of yeast extract Prodex-Lac SD® and 20 g/L of dried whey after 96h of fermentation at 30° C and 200 rpm, optimized with response surface methodology (RSM). In the the washing tests, the best results of the removal of blood and tomato sauce with ground beef stains from cotton fabrics were observed using the combination of crude extract of protease (100 or 1000U) with detergent Omo®. Crude protease extract of the Bacillus sp. LBA strain was partially purified by ammonium sulfate fractionation (80% saturation), dialysis and gel filtration chromatography (Sephadex G100), resulting in the purification fold of 3.69. After characterization with RSM it was observed that the crude protease extract and partially purified proteases presented optimal activity at 55° C and pH 7.5 and considerable stability (95% of residual activity) in pH range 5.7 ¿ 9.3 after 1h incubation at 30-36° C and more than 78.9% when incubated at pH 7.5 and 50 °C for 1h. The optimal conditions of the lysis of X. campestris cells contained in the fermentation broth using crude and partially purified protease preparations were observed using 42 U of protease/mL of cell suspension of X. campestris at 60° C, resulting in a increase of more than 20% in Trans 620 nm of the fermented broth of xanthan. It was observed an increase of almost 40% in Trans 620 nm after 2h reaction using crude protease (42 U de protease/mL of cell suspension of X. campestris) at 65° C. The production of proteases by Bacillus sp. LBA 46 under solid state fermentation was optimized using RSM, resulting in 5000 U/gram of dry substrate utilizing wheat bran and water (6g:4g) after 96h of fermentation at 30° C
Doutorado
Ciência de Alimentos
Doutor em Ciência de Alimentos

RIASSUNTO I batteri psicrotrofi sono i principali responsabili del deterioramento del latte crudo o termotrattato poiché sono in grado di sintetizzare proteasi e lipasi extracellulari termostabili, causa di formazione di odori e sapori sgradevoli, fenomeni di gelificazione, riduzione delle proprietà schiumogene del latte, perdita di qualità sensoriale e riduzione della shelf-life. Ad oggi, non esiste ancora una sufficiente conoscenza degli specifici pathways proteolitici e lipolitici di questi enzimi termostabili. Inizialmente questo lavoro ha riguardato la caratterizzazione dell’attività enzimatica di 80 ceppi di psicrotrofi isolati da latte crudo. I batteri appartenenti al genere Pseudomonas sono stati i più isolati (78.75%) e Pseudomonas fluorescens è risultata la specie predominante (30.16%); tra le Enterobacteriaceae (21.25%), Serratia marcescens è stata la specie più frequentemente isolata (52.94%). Quarantuno ceppi di psicrotrofi mostravano tutte le attività enzimatiche. Il più alto numero di ceppi positivi a tutte le temperature di incubazione è stato osservato per l’attività lipolitica (59) e, a seguire, proteolitica (31) e lecitinasica; i tratti enzimatici variavano tra i ceppi di Pseudomonas e Enterobacteriaceae ed erano marcatamente influenzati dalla temperature di incubazione, essendo 30 °C quella ottimale. Il gene aprX è stato ritrovato in 19 ceppi di Pseudomonas ed è risultato esser diffuso tra i ceppi di P. fluorescens (15 su18). La seconda parte della ricerca è stata focalizzata sulla valutazione della produzione di composti organici volatili (VOCs) e del rilascio di acidi grassi liberi (FFAs) da ceppi di batteri psicrotrofi. Diverse specie del genere Pseudomonas e il ceppo Serratia marcescens hanno mostrato profili di VOCs complessi e dipendenti dal ceppo batterico inoculato in campioni di latte UHT, durante le differenti condizioni di incubazione. In particolare, sono stati identificati 56 VOCs appartenenti a aldeidi, chetoni, acidi grassi, alcoli, composti solforati e idrocarburi. Generalmente, il numero di VOCs tendeva ad incrementare con il progredire del tempo di incubazione, sia nel latte controllo non inoculato sia nei campioni di latte contaminati. Tra i VOCs rilevati, alcune molecole sono state individuate solo quando il latte era contaminato da uno specifico ceppo microbico. Nel dettaglio, 3-metilbutan-2-olo, 3-metilesan-2-olo, pentan-1-olo e 3,3-dimetilesano sono stati rilevati solo a seguito dello sviluppo di P. fragi. P. rhodesiae è stata l’unica specie a produrre pentano-2,3-dione, eptano e 3-metilesano, mentre l’esano è stato rilasciato solo in campioni di latte contaminati con P. fluorescens. La maggior produzione di composti solforati e alcoli è stata individuata nello spazio di testa del latte contaminato con P. mosselii e P. fragi, rispettivamente. Lo sviluppo e l’attività di P. rhodesiae e S. marcescens sono risultati associati ad un più alto numero di acidi grassi e chetoni mentre P. fluorescens ha mostrato la più scarsa produzione di composti volatili. Alcuni VOCs come 3-metilbutan-1-olo, 2-metilpropan-1-olo, 3-idrossibutan-2-one, butano-2,3-dione, acido butanoico ed acido esanoico potrebbero perciò rappresentare potenziali marker per il riconoscimento dell’attività enzimatica di batteri psicrotrofi e per la precoce individuazione del deterioramento del prodotto. Per quanto riguarda il rilascio di FFAs, diverse quantità di questi composti sono state rilasciate dai batteri psicrotrofi appartenenti a specie diverse e alla stessa specie conseguentemente alla diversa capacità di produrre lipasi. Gli acidi palmitico (16:0), oleico (18:1) e linoleico (18:2) sono risultati i più presenti tra gli acidi grassi saturi, monoinsaturi e polinsaturi. P. fluorescens PS73 e P. fluorescens PS81 sono state le specie che hanno prodotto la maggior quantità di FFAs, a 24 h e 4 giorni di incubazione, rispettivamente. Al contrario, H. alvei PS57 e P. fragi PS55 hanno rilasciato la minor quantità di FFAs ad entrambi i tempi di incubazione. Le lipasi dei ceppi di psicrotrofi hanno mostrato una specificità variabile nei riguardi degli esteri degli acidi grassi con diversa lunghezza della catena carboniosa. P. fragi PS55, P. putida PS17, P. fluorescens PS14 and P. fulva PS10 sono risultate le specie più attive nell’idrolisi dei trigliceridi. La lipasi del ceppo di P. rhodesiae PS62 ha mostrato la più scarsa capacità idrolitica verso tutti i trigliceridi testati. Infine, è stata effettuata la caratterizzazione proteomica di proteasi extracellulari di ceppi di P. fluorescens. Una proteasi termostabile di circa 45 kDa è stata individuata su casein zymogram gel in ciascun surnatante dei ceppi batterici selezionati. L’estratto enzimatico del ceppo P. fluorescens PS19, concentrato per ultrafiltrazione (10 kDa), ha mostrato un’elevata attività proteolitica e due ulteriori bande proteolitiche di circa 15 e 25 kDa. I risultati delle analisi nLC/MS/MS dopo digestione sia in gel che in solution hanno evidenziato che la proteasi di 45 kDa corrisponde ad una AprX metalloproteasi di P. fluorescens (acc. no. C9WKP6, UniProt). La proteasi di 15 kDa è stata riconosciuta come un frammento della stessa AprX metalloproteasi, mentre la proteasi di 25 kDa non ha mostrato nessuna omologia con alcuna delle proteine note di Pseudomonas. La caratterizzazione tramite LC/MS del profilo peptidico generato dall’azione delle proteasi termostabili dello stesso ceppo sulle frazioni caseiniche del latte è in corso di studio. In generale, questo studio fornisce ulteriori conoscenze per la lo studio delle attività enzimatiche di batteri psicrotrofi nel latte.
ABSTRACT Psychrotrophic bacteria are responsible for the highest spoilage of unprocessed or heated milk during storage because of their capacity to synthesize thermostable extracellular proteases and lipases. The activities of these enzymes lead to formation of off-odours/flavours, gelation of milk, lowering of milk foaming properties, loss of sensory quality and shortening of the shelf life. To date, still little is known about the specific proteolytic and lipolytic pathways of these thermostable enzymes. Initially we evaluated the enzymatic traits of 80 raw milk-associated psychrotrophic strains. Among psychrotrophic isolates, Pseudomonas were the most commonly occurring contaminants (78.75%) being Pseudomonas fluorescens the predominant isolated species (30.16 %), along with Enterobacteriaceae (21.25%), primarily Serratia marcescens (52.94 %). Forty-one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all incubation temperatures was found for the lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities. The enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains and were markedly influenced by incubation temperature being 30 °C the optimal one. The aprX gene was detected in 19 out of 80 psychrotrohic strains and it resulted widespread among P. fluorescens strains (15 out of 18). The second part of the research was focused on the evaluation of spoilage potential of psychrotrophic strains by analyzing the production of volatile organic compounds (VOCs) and the release of free fatty acids (FFAs). From results of SPME-GC/MS analysis, different species of the genus Pseudomonas and Serratia marcescens produced a complex and strain-dependent VOCs profiles in UHT milk samples at different storage and time conditions. Fifty-six VOCs belonging to 7 chemical groups (aldehydes, ketones, fatty acids, esters, alcohols, sulphur compounds and hydrocarbons) were identified. Generally, the VOCs went to increase during the storage time both in the control and contaminated milk samples, some compounds being detected only in the latter samples. Compounds such as 3-methylbutan-2-ol, 3-methylhexan-2-ol, pentan-1-ol and 3,3-dimethylhexane were detectable only for P. fragi. P. rhodesiae was the only species producing pentane-2,3-dione, heptane and 3-methylhexane while hexane was released only by P. fluorescens. P. mosselii and P. fragi produced the highest number of sulphur compounds and alcohols, respectively. The highest number of FFAs and ketons was detected in the headspace of milk samples contaminated by P. rhodesiae and S. marcescens. P. fluorescens provided the lowest development of VOCs. 3-methylbutan-1-ol, 2 methylpropan-1-ol, 3-hydroxybutan-2-one, butane-2,3-dione and butanoic and hexanoic acids could be regarded as potential markers of psycrotrophic contamination useful for the early detection of milk bacterial spoilage. Regarding the release of FFAs, different quantities of these compounds have been released from milk fat by tested bacteria, between and within species, in relation to diverse capacity for production of lipolytic enzymes. Palmitic (16:0), oleic (18:1) and linoleic (18:2) acids levels were found to be the highest among the SFAs, MUFAs and PUFAs, respectively. P. fluorescens PS73 and P. fluorescens PS81 were the major FFAs producers, at 24 h and 4 days of incubation, respectively while H. alvei PS57 and P. fragi PS55 were the less active in lipid breakdown at both the incubation conditions. Lipases from psychrotrophic strains showed a variable range of specificity toward fatty acid esters with different fatty acid chain lengths, being P. fragi PS55, P. putida PS17, P. fluorescens PS14 and P. fulva PS10 the more active to hydrolyse triglycerides. Lipase from P. rhodesiae PS62 showed the highest hydrolytic resistance toward all tested fatty acid triglycerides. Finally, proteomic characterization of extracellular proteases of P. fluorescens strains has been performed. One thermostable protease of approximately 45 kDa was detected in each of the cell-free supernatant of the selected strains on a casein zymogram gel. After concentration by ultrafiltration (10 kDa), the protease extract of P. fluorescens PS19 showed a high proteolytic activity and two additional proteolytic bands with molecular masses of approximately 15 and 25 kDa on casein zymography. This extract was subjected to proteomic characterization by nLC/MS/MS analysis of both in gel and in solution digestion. Results showed the protease of 45 kDa to correspond to P. fluorescens AprX metalloprotease (acc. no. C9WKP6, UniProt). In addition, the same results leaded to recognize the 15 kDa protease as a fragment of this AprX metalloprotease. On the contrary, the 25 kDa protease showed no homology to any known protein of Pseudomonas spp. The characterization by LC/MS of the peptides profile generated by the action of thermostable proteases of the same strain on milk caseins is still under investigation. Overall, this study provides a better understanding of the enzymatic activities of psychrotrophic bacteria in milk.

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms encoding the N-terminal extension of L27. Cleavage of L27 by Prp represents a new target for potential antibiotic therapy. In order to characterize this protease, Prp has been overexpressed and purified. Using an assay we have developed, based on cleavage of a fluorogenic peptide derived from the conserved L27 cleavage sequence, we have undertaken an analysis of the enzyme kinetics and substrate specificity for Prp cleavage and tested predictions made based on a structural model using active-site mutants.

O uso de enzimas como agentes de modificação das propriedades funcionais de proteínas tem se tornado bastante difundido na indústria de alimentos. As proteases, apresentam inúmeras vantagens, principalmente, devido a sua atividade em baixas concentrações e a sua ausência de toxicidade, que faz com que se elimine a necessidade da sua remoção do produto final. O objetivo deste trabalho foi determinar as condições ótimas de produção da protease de Microbacterium sp. kr10, caracterizar e purificar parcialmente a enzima, assim como verificar a sua utilização como agente de modificação das propriedades funcionais da proteína de soja. Através da metodologia de superfície de resposta foram determinadas as condições ótimas de produção da protease, pH de 7,0, temperatura de 25°C e 12,5 g L-1 de farinha de pena (p/v). O padrão proteolítico da enzima tanto no extrato cru quanto na parcialmente purificada indicam que esta é uma metaloprotease, com pH e temperaturas ótimos nas faixas de 6,5 a 7,5, e 45 a 55°C, respectivamente. A atividade enzimática foi totalmente inibida por EDTA, fenantrolina, HgCl2 e CuCl2 e parcialmente inibida por ZnCl2, MnCl2 e SnCl2. A enzima foi parcialmente purificada através de cromatografia de gel filtração e troca iônica resultando num fator de purificação de 250. Um aumento gradativo do grau de hidrólise da proteína de soja foi observado à medida que se aumentou a razão enzima/substrato utilizada, assim como a redução da formação de espuma e o aumento da capacidade emulsificante de uma solução composta pelo hidrolisado de soja e óleo de soja, mesmo sob condições de alta temperatura e alta concentração de sal. Desta forma, esta protease apresenta potencial para aplicação como agente de modificação protéica de proteína de soja isolada.

The Staphylococcus genus comprises a diverse group of Gram-positive bacteria that are opportunistic pathogens of humans and other mammals. S. epidermidis and S. aureus are the most common human pathogens of the staphylococci, causing a variety of infections including biofilm-based medical device infections, skin infections, and pneumonia. Both of these organisms produce proteases whose functions in virulence are not fully characterized. In S. epidermidis, protein-mediated biofilm formation requires a cell wall-anchored adhesin called Aap that must be proteolytically processed in order to allow intercellular adhesion. The S. epidermidis protease(s) responsible for cleaving Aap were unknown. Chapter II describes our findings that the secreted metalloprotease SepA is required for Aap-mediated biofilm formation and cleaves Aap at two different sites. Further, this protease is negatively regulated by the global regulator SarA. Chapter III discusses studies of the S. aureus Spl (serine protease-like) proteases. Although they are produced in vivo, their substrates and role in virulence are unknown. We found that in a rabbit model of pneumonia, a mutant lacking the spl protease operon caused more localized disease compared to wild type S. aureus. Proteomics studies of the secreted and surface proteins in wild type compared to spl mutant S. aureus revealed several changes. We also found that the SplA protease cleaves human Mucin-16, the first identification of a biological substrate of the Spls. Finally, we found that the animal-associated species S. caprae produces an autoinducing peptide (AIP) that is a potent inhibitor of S. aureus quorum sensing. We identified the S. caprae AIP structure as an 8-residue thiolactone ring. A synthetic version of the peptide inhibits S. aureus virulence and quorum sensing induction in a murine skin infection model. This is a novel example of quorum sensing cross talk between staphylococcal quorum sensing systems. These studies are described in Appendix A. On the whole, this work identified two substrates of S. aureus proteases and demonstrated their importance in biofilm formation and infection. We also characterized a novel inhibitor of S. aureus quorum sensing that attenuates virulence. These findings shed light on the importance of staphylococcal secreted proteases and quorum sensing cross talk in the modulation of virulence factor production and the ability to cause disease.

This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of Neisseria gonorrhoeae, N. meningitidis and Streptococcus pyogenes on host epithelial cells and in vivo.

N. gonorrhoeae cause the sexually transmitted disease gonorrhoea and N. meningitidis is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, N. meningitidis generally induced an earlier response compared to N. gonorrhoeae, probably as a result of the meningococcal capsule. The role of N. meningitidis serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure.

S. pyogenes may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. S. pyogenes ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of S. pyogenes is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of S. pyogenes.

Preterm birth, birth prior to 37 weeks gestation, is the leading cause of neonatal mortality and morbidity worldwide. While the uterine cavity and amniotic fluid largely remain sterile throughout gestation, bacterial infections can occur and are associated with preterm birth and/or preterm premature rupture of the fetal membranes (PPROM). Sneathia amnii can be detected as a component of the vaginal flora in healthy women; however, it’s also associated with bacterial vaginosis and preterm birth. Sn35, an isolate of S.amnii, was identified and sequenced through the Vaginal Human Microbiome Project at VCU. Our objective was to classify potential virulence determinants in Sn35 and we successfully identified a putative zinc endopeptidase. The zinc endopeptidase appeared to cleave itself in a site-specific manner under calcium-depleted conditions, resulting in a truncated protein. The truncated protein did have collagenase activity and bacteriolytic activity as well.

Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene, chlorinated solvents often used in plastics and dry cleaning solvent manufacturing. The treatment of VC-contaminated sites by bacteria that can biodegrade VC has been demonstrated to be a practical and potentially cost-effective alternative to traditional "pump and treat" site cleanup options. However, little is known about the biochemical pathways involved in VC-assimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. The results of these studies demonstrated the successful extraction and identification of proteins involved in VC-assimilation from ethene-enriched groundwater samples. The presence and diversity of VC-assimilating bacteria in several ethene-enriched groundwater samples were examined using tandem mass spectrometry analysis to identify the protein biomarkers EtnC and EtnE. VC-assimilating organisms can evolve in vitro from bacteria that grow on ethene but very little is known about the molecular changes involved. Proteomic investigations comparing three strains of Mycobacterium strain JS623, a wild type and two VC-adapted strains, validated previous studies indicating that protein expression changes are involved in VC-adaptation. Tandem mass spectrometry and spectral counting were used to identify proteins and semi-quantitatively estimate protein expression levels in the three ethene-grown JS623 variants. The results of this study suggest that multiple VC-adaptation mechanisms are involved in the two VC-adapted strains

The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. These metabolites are mostly small peptides and have unusual components and chemical bonds. These metabolites are synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. One of those small peptides, bacilysin, is a dipeptide antibiotic composed of L-alanine and L-anticapsin which is produced and excreted by certain strains of Bacillus subtilis. Proteins that are responsible to synthesize bacilysin are encoded by bac operon. It has been shown that the biosynthesis of bacilysin is under the control of quorum sensing global regulatory pathway through the action of ComQ/ComX, PhrC (CSF), ComP/ComA in a Spo0K (Opp)-dependent manner. The objective of the study is to identify the functional roles of bacilysin biosynthesis in the regulatory cascade and idiophase cell physiology operating in B. subtilis by using gel-based and gel-free proteomics techniques. For this, we employed comparative proteome-wide analysis of the bacilysin producer B. subtilis PY79 and its bacilysin nonproducer derivative bacA::lacz::erm OGU1 strain which was recently constructed by our group. Identification via GeLC analysis of 76 differentially expressed proteins from total soluble proteome of wild-type PY79 and bacilysin minus OGU1 strain indicated the direct or indirect multiple effects of bacilysin on metabolic pathways, global regulatory systems and sporulation.

Background: Increased numbers of intestinal E. coli are observed in inflammatory bowel disease, but the reasons for this proliferation and it exact role in intestinal inflammation are unknown. Aim of this PhD-project was to identify E. coli proteins involved in E. coli’s adaptation to the inflammatory conditions in the gut and to investigate whether these factors affect the host. Furthermore, the molecular basis for strain-specific differences between probiotic and harmful E. coli in their response to intestinal inflammation was investigated. Methods: Using mice monoassociated either with the adherent-invasive E. coli (AIEC) strain UNC or the probiotic E. coli Nissle, two different mouse models of intestinal inflammation were analysed: On the one hand, severe inflammation was induced by treating mice with 3.5% dextran sodium sulphate (DSS). On the other hand, a very mild intestinal inflammation was generated by associating interleukin 10-deficient (IL-10-/-) mice with E. coli. Differentially expressed proteins in the E. coli strains collected from caecal contents of these mice were identified by two-dimensional fluorescence difference gel electrophoresis. Results DSS-experiment: All DSS-treated mice revealed signs of a moderate caecal and a severe colonic inflammation. However, mice monoassociated with E. coli Nissle were less affected. In both E. coli strains, acute inflammation led to a downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower caecal concentrations of bacterial fermentation products than the control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterised protein YggE. NfuA was upregulated nearly 3-fold in both E. coli strains after DSS administration. Reactive oxygen species produced during intestinal inflammation damage Fe-S clusters and thereby lead to an inactivation of Fe-S proteins. In vitro data indicated that the repair of Fe-S proteins by NfuA is a central mechanism in E. coli to survive oxidative stress. Expression of YggE, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC under control and inflammatory conditions. In vitro growth experiments confirmed these results, indicating that E. coli Nissle is better equipped to cope with oxidative stress than E. coli UNC. Additionally, E. coli Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold higher than E. coli UNC. In turn, caecal indole concentrations resulting from cleavage of tryptophan by TnaA were higher in E. coli Nissle- associated control mice than in the respective mice associated with E. coli UNC. Because of its anti-inflammatory effect, indole is hypothesised to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle. Results IL-10-/--experiment: Only IL-10-/- mice monoassociated with E. coli UNC for 8 weeks exhibited signs of a very mild caecal inflammation. In agreement with this weak inflammation, the variations in the bacterial proteome were small. Similar to the DSS-experiment, proteins downregulated by inflammation belong mainly to the central energy metabolism. In contrast to the DSS-experiment, no upregulation of chaperone proteins and NfuA were observed, indicating that these are strategies to overcome adverse effects of strong intestinal inflammation. The inhibitor of vertebrate C-type lysozyme, Ivy, was 2- to 3-fold upregulated on mRNA and protein level in E. coli Nissle in comparison to E. coli UNC isolated from IL-10-/- mice. By overexpressing ivy, it was demonstrated in vitro that Ivy contributes to a higher lysozyme resistance observed for E. coli Nissle, supporting the role of Ivy as a potential fitness factor in this E. coli strain. Conclusions: The results of this PhD-study demonstrate that intestinal bacteria sense even minimal changes in the health status of the host. While some bacterial adaptations to the inflammatory conditions are equal in response to strong and mild intestinal inflammation, other reactions are unique to a specific disease state. In addition, probiotic and colitogenic E. coli differ in their response to the intestinal inflammation and thereby may influence the host in different ways.
Hintergrund: Chronisch entzündliche Darmerkrankungen zeichnen sich unter anderem durch eine starke Proliferation intestinaler E. coli aus. Unbekannt ist jedoch, ob diese Vermehrung eine Ursache oder eine Folge der Erkrankung darstellt. Ziel der vorliegenden Doktorarbeit war es daher, E. coli-Proteine zu identifizieren, welche der Anpassung an die entzündlichen Bedingungen im Darmtrakt dienen und unter Umständen einen Effekt auf den Gesundheitszustand des Wirtes haben. Weiterhin sollten die molekularen Ursachen für stammesspezifische Unterschiede zwischen probiotischen und gesundheitsschädlichen E. coli näher untersucht werden. Methoden: In den tierexperimentellen Analysen wurden keimfreie Mäuse entweder mit dem probiotischen E. coli Nissle oder dem adhärent-invasiven E. coli UNC monoassoziiert und in zwei verschiedenen Entzündungsmodellen näher untersucht. Einerseits wurde eine starke Darmentzündung durch die Gabe von 3,5% Natrium-Dextransulfat (DSS) ausgelöst. Andererseits wurde in Interleukin 10-defizienten (IL-10-/-) Mäusen eine sehr milde Form der Entzündung durch Besiedlung mit E. coli induziert. Die E. coli Bakterien wurden am Ende der Versuche aus den Caecuminhalten der Mäuse isoliert und die bakterielle Proteinexpression wurde mittels zwei-dimensionaler Gelelektrophorese analysiert. Ergebnisse des DSS-Versuchs: Alle Tiere des DSS-Versuchs entwickelten unabhängig vom E. coli Stamm, mit dem sie besiedelt waren, eine moderate Entzündung im Caecum und eine starke im Colon, wobei die Entzündungsreaktion durch die Monoassoziation mit E. coli Nissle leicht abgeschwächt wurde. In beiden E. coli Stämmen führte die Darmentzündung zu einer verringerten Expression von Enzymen des Kohlenhydratabbaus und der Energiegewinnung. In Folge dessen waren die intestinalen Konzentrationen bakterieller Fermentationsprodukte in den entzündeten Tieren geringer als in den gesunden Kontrolltieren. Weitere differentiell exprimierte Proteine umfassen das Fe-S- Cluster Reparaturprotein NfuA, die Tryptophanase TnaA und das uncharakterisierte Protein YggE. In beiden E. coli Stämmen, welche aus den DSS-Tieren isoliert wurden, war das NfuA Protein dreifach höher exprimiert. Eine Darmentzündung führt zu einer vermehrten Bildung reaktiver Sauerstoffspezies, welche die Fe-S-Cluster in Eisen-Schwefel-Proteinen zerstören und damit zu einer Inaktivierung dieser Proteine führen. In vitro Untersuchungen bestätigten, dass die Reparatur der Eisen-Schwefel-Proteine durch NfuA ein wichtiger Mechanismus ist um oxidativem Stress entgegenzuwirken. Das YggE Protein, welches laut Literaturangaben einen hemmenden Einfluss auf die Bildung reaktiver Sauerstoffspezies hat, war in E. coli Nissle 4- bis 8-fach erhöht (verglichen mit E. coli UNC unter Kontroll- und Entzündungsbedingungen). In vitro Versuche bestätigten diese Daten und zeigten, dass E. coli Nissle im Vergleich zu E. coli UNC eine erhöhte Resistenz gegenüber oxidativem Stress aufweist. Außerdem wurde im Vergleich E. coli Nissle vs. E. coli UNC (unter Entzündungs- und Kontrollbedingungen) ein 4- bis 7-fach erhöhter TnaA-Gehalt nachgewiesen. Indol, das Produkt der TnaA-katalysierten Tryptophanspaltung wurde in erhöhten Mengen im Intestinaltrakt E. coli Nissle-assoziierter Kontrolltiere detektiert. Seit längerem werden entzündungshemmende Eigenschaften für Indol postuliert, die aufgrund der Ergebnisse dieser Doktorarbeit nun auch mit den gesundheitsfördenden Eigenschaften von E. coli Nissle in Zusammenhang gebracht werden können. Ergebnisse des IL-10-/-- Versuchs: Nach einer 8-wöchigen Assoziationsdauer wurde nur in den mit E. coli UNC besiedelten IL-10-/- Tieren eine schwache Entzündungsreaktion nachgewiesen. Bedingt durch diese sehr schwach ausgeprägte Entzündungsantwort waren auch die Veränderungen im bakteriellen Proteom von E. coli UNC nur gering. Wie im DSS-Versuch waren Proteine des bakteriellen Energiestoffwechsels reprimiert, allerdings wurde keine Induktion von NfuA beobachtet. Daher scheint die Induktion von NfuA nur der Anpassung an eine starke Entzündung zu dienen. Weiterhin wurde nachgewiesen, dass E. coli Nissle aus IL-10-/- Tieren den Hemmer für das vertebrate C-Typ Lysozym (Ivy) sowohl auf mRNA- als auch auf Proteinebene stärker exprimiert als E. coli UNC. Überexpression von Ivy unter in vitro Bedingungen zeigte, dass es an der erhöhten Lysozymresistenz von E. coli Nissle beteiligt ist und somit eine Rolle als möglicher Fitnessfaktor von E. coli Nissle spielt. Schlussfolgerungen: In dieser Doktorarbeit wurde gezeigt, dass Darmentzündungen die Proteinexpression eines im Darm lebenden Bakteriums beeinflussen. Einige der aufgedeckten bakteriellen Anpassungsreaktionen werden sowohl bei einer starken als auch bei einer schwachen Entzündung ausgelöst; andere wiederum sind spezifisch für nur einen dieser Entzündungszustände. Weiterhin wurde deutlich, dass sich E. coli-Stämme hinsichtlich ihrer Reaktion auf eine Darmentzündung unterscheiden und damit möglicherweise den Wirt beeinflussen. 

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d₂, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d2, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

The worldwide rise of antibiotic resistance stimulates the search of new strategies for killing bacteria, whose mechanisms of action are different from those of antibiotics. Two promising strategies in this respect are the use of cationic antimicrobial peptides (CAMPs) and photodynamic therapy (PDT). Both approaches have been considered and some aspects elucidated during my PhD. Therefore, studies along two different research lines, having the same final aim and merging in the last part of the project, have been carried out. The first research line focused on PDT, in particular I investigated how several experimental factors affect the phototoxic activity against bacteria of cationic porphyrins, and I identified some of their molecular targets in Staphylococcus aureus. The second research line focused on the mechanism of action of a CAMP, apidaecin 1b, that was then conjugated to a photoactive porphyrin to obtain a new and broad-spectrum antimicrobial agent. PDT utilizes a visible light absorbing molecule, called photosensitizer (PS), that in the presence of oxygen generates cytotoxic reactive species that kill bacterial cells previously loaded with the PS. I studied the effects of some experimental factors that could affect the binding of the PS to the bacterial cell, and, as a consequence, the efficiency of bacteria photoinactivation. In particular, the effects of the cell washing after incubation with the PS, and of the presence of cations in the incubation medium, were considered. For these studies a dicationic porphyrin was used, and its ability to photoinactivate S. aureus and E. coli was measured after irradiation of the bacteria with increasing doses of blue light. Illumination was carried out with the unbound PS left in the suspension, as well as after one or four washings of the cells. The washings produced a completely different effect in the two microorganisms: slightly decreased the photokilling of S. aureus and strongly increased the photokilling of E. coli. The increased photokilling of E. coli was explained with a re-localisation, occurring during the time needed for the washing procedure, of the porphyrin molecules in inner cellular sites critical for survival. This is very likely to occur, considering the short (5 min.) incubation time of the cells with the PS. The presence of a monovalent cation (Na+) in the cell medium during incubation and illumination did not affect negatively the photoinactivation of S. aureus, that was slightly increased by the presence of Ca2+ and Mg2+. On the contrary, the phoinactivation of E. coli was slightly decreased in the presence of both mono- and divalent cations, and the effect was more evident in cells illuminated without washings. In all cases, the bacteria photoinactivation with this dicationic porphyrin was little affected by the presence of cations, in comparison to other cationic porphyrins. A classical proteomic approach, that included two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis, was used to identify some proteins that are potential primary molecular targets of PDT. Towards this aim, the change of the proteomic profile of S. aureus after PDT treatments was investigated using two different cationic porphyrins. For each of these two porphyrins, that strongly differ in their photoinactivation activity, I have selected two PDT treatments on the base of the different survival of treated bacteria: a sublethal one, which allows a bacterial survival of 60 to 80%, and a stronger one, which allows only a bacterial survival of about 1%. 2-DE maps from PDT-treated and untreated samples were compared using the Proteomweaver software for 2-DE maps analysis. All proteins showing a significant difference in their intensity levels between untreated and PDT treated 2-DE maps were identified by mass spectrometry (MALDI-TOF/TOF). All identified proteins were then divided into functional classes on the basis of their cellular function, in order to identify the pathways that were more damaged by PDT. Among the 265 proteins identified by the Proteomweaver analysis, 70 changed their intensity levels after PDT treatments; most of these are involved in the response to oxidative stress, in the energy metabolism and in the uptake of sugar. The comparison of the effects of PDT treatments between the two porphyrins revealed that several proteins were modified by both of them, and generally showing the same trend. Interestingly, the modifications of the proteomic patterns induced by PDT treatments are not consistent with a response of S. aureus cells to oxidative stress induced by other oxidizing agents (hydrogen peroxide, superoxide or diamide), therefore suggesting a selective targeting of specific proteins. These findings provide new insight in the understanding of the mechanism of action of PDT, and may be useful to design new photosensitizing agents with improved antibacterial activity. In the second research line I studied the antibacterial activity, and the ability to translocate into the bacterial cell, of the insect antimicrobial peptide apidaecin 1b. The improved understanding of the translocation mechanism of apidaecin was then used to investigate the possibility to use apidaecin itself as a cargo delivering molecule, by conjugating it with a porphyrin, in an attempt to obtain a new broad-spectrum antimicrobial agent. Apidaecin 1b is a small (18 residues) peptide that is extensively studied because of its unique properties, which include the ability to inhibit some Gram (-) bacteria growth and a high capability to translocate into cells with a non pore-forming mechanism. Moreover, no toxicity against eukaryotic cells has been reported even at concentrations that totally inhibit bacterial growth. In order to study its mechanism of action, each of the three arginine residues (or all of them together) were replaced with the corresponding peptoid residue, N-(3-guanidinopropyl) glycine (NArg), and the minimal inhibitory concentrations (MICs) of all of these peptide-peptoid hybrids were evaluated against E. coli, P. aeruginosa and S. aureus. No antimicrobial activity was detected for apidaecin and its peptide-peptoid hybrids against S. aureus and P. aeruginosa. On the contrary, against E. coli apidaecin showed a MIC value of 8-16 µM, while its peptide-peptoid hybrids substituted in arginine 4 and 12 showed a little decrease of antimicrobial activity and those substituted in arginine 17 or in all three arginines completely lost their activity. These results suggested that the arginine in position 17 is particularly important in the translocation mechanism into the bacterial cell. Therefore, apidaecin and its peptide-peptoid hybrids were labelled with fluorescein, in order to monitor by means of fluorescence microscopy and flow cytometry their ability to tightly bind to bacterial cells. The fluorescein-labelling completely abolished the antimicrobial activity of all our compounds and inhibited to a great extent their binding and translocation into bacterial cells. This effect was mainly due to the additional mass brought by fluorescein to apidaecin and its hybrids. Anyway, it was observed that the peptide-peptoid hybrid substituted in arginine 17 did not show any binding to bacterial cells. This finding confirmed the importance of this residue for antimicrobial activity, and, in general, the importance of the C-terminal of apidaecin in the binding and translocation into the bacterial cells. The conjugate (called T-api), resulting from the coupling of an anionic porphyrin (monocarboxy-tetraphenylporfine, cTPP) with the N-terminal of apidaecin 1b, did not show any antimicrobial activity in the dark. On the contrary, following activation with blue light, T-api caused a significant reduction of bacteria survival with an efficacy strongly dependent on the bacteria species. The phototreatment was extremely successful against E. coli and S. aureus, while the photokilling efficiency against P. aeruginosa was much lower but sufficient to induce a significant reduction of survival. Irradiation experiments were also performed by treating bacteria with cTPP alone, apidaecin 1b alone or the two agents together but not conjugated. None of these treatments affected the survival of both E. coli and P. aeruginosa, while S. aureus survival was decreased by cTPP, but to a lesser extent in comparison to T-api. Thus, it was demonstrated that the conjugation of an antimicrobial peptide with a photoactive molecule can lead to the formation of antimicrobial agents very effective and displaying a broader activity in comparison to the single components.
cIl forte aumento, a livello mondiale, del fenomeno della resistenza agli antibiotici richiede lo sviluppo di nuove strategie antimicrobiche, basate su meccanismi d’azione diversi da quelli degli antibiotici, per combattere le infezioni batteriche. Due delle strategie più promettenti in quest’ottica sono la terapia fotodinamica (PDT) e l’utilizzo di peptidi cationici antimicrobici (CAMPs). Durante il mio dottorato di ricerca ho studiato alcuni aspetti peculiari di entrambe queste strategie, in due linee di studio che alla fine sono state riunite. Nella prima linea di ricerca, focalizzata sulla PDT con porfirine cationiche, ho studiato quale impatto abbiano alcune condizioni sperimentali sull’efficienza di fotosensibilizzazione di batteri, ed ho identificato alcuni dei bersagli molecolari della loro azione su Staphylococcus aureus. Nella seconda linea di ricerca ho studiato il meccanismo d’azione di un particolare CAMP, l’apidaecina 1b, che ho successivamente coniugato con un fotosensibilizzatore allo scopo di creare un nuovo, più efficiente, agente antimicrobico. La PDT utilizza molecole capaci di assorbire la luce visibile, dette fotosensibilizzatori (PS), che, quando illuminate in presenza di ossigeno molecolare, generano specie reattive dell’ossigeno, che hanno un forte effetto citotossico su cellule batteriche precedentemente incubate col PS stesso. Nella prima fase di studio ho valutato l’effetto di alcune condizioni sperimentali sulla capacità del PS di legarsi alla cellula batterica e, di conseguenza, sull’efficienza di fotoinattivazione di batteri. In particolare sono stati valutati gli effetti di lavaggi effettuati dopo l’incubazione dei batteri con il PS, nonché della presenza di diversi cationi nel mezzo di incubazione. In questo studio è stata utilizzata una porfirina dicationica, la cui efficienza nella fotoinattivazione di Staphylococcus aureus ed Escherichia coli è stata misurata mediante irradiamento con dose crescenti di luce blu. L’irradiamento è stato effettuato sia lasciando nella sospensione batterica il PS non legato alle cellule che rimuovendolo mediante uno o quattro lavaggi. Questi lavaggi hanno prodotto effetti completamente opposti nei due microrganismi oggetto di studio: da un lato si è riscontrato un forte aumento dell’efficienza di fotosensibilizzazione di E. coli, dall’altro un decremento di quella di S. aureus. L’aumento dell’efficienza di fotosensibilizzazione in E. coli è probabilmente dovuto al fatto che, nel tempo necessario per effettuare i lavaggi, la frazione di porfirina legata alle cellule batteriche riesce a raggiungere siti cellulari più sensibili alla PDT. Il fatto che si sia utilizzato un tempo di incubazione molto breve (5 minuti) rende molto plausibile questa ipotesi. L’aggiunta di un catione monovalente (Na+) nel mezzo di irradiamento non ha causato alcuna variazione dell’efficienza di fotosensibilizzazione di S. aureus, che invece è stata fortemente incrementata da quella di cationi bivalenti (Ca2+ e Mg2+). Al contrario, la fotosensibilizzazione di E. coli è stata sensibilmente diminuita in presenza di cationi (sia mono che bivalenti), con un effetto più marcato in assenza di lavaggi. In ogni caso, utilizzando questa porfirina dicationica gli effetti prodotti sia dai lavaggi che dalla presenza di cationi sono stati minori di quelli riscontrati in precedenza con altri fotosensibilizzatori. Per l’identificazione di alcune delle proteine che sono bersaglio della PDT è stato scelto un approccio di tipo proteomico, comprendente la separazione con elettroforesi bidimensionale dei lisati batterici e l’identificazione di proteine con tecniche di spettrometria di massa. Al fine di ottenere un’analisi il più possibile dettagliata, sono stati valutati i cambiamenti nel profilo proteomico di S. aureus causati dalla PDT con due diverse porfirine cationiche. Per ciascuna di queste porfirine, che differiscono notevolmente nell’attività fotosensibilizzante, sono stati selezionati, sulla base della differente mortalità indotta in sospensioni di S. aureus, due trattamenti fotodinamici: uno subletale, che consente una sopravvivenza dal 60 all’80% dei batteri, e l’altro più forte, che consente la sopravvivenza di circa l’1% dei batteri. Le mappe bidimensionali ottenute da lisati proteici di batteri non sottoposti a PDT sono quindi state confrontate, mediante l’utilizzo dell’apposito software Proteomweaver, con quelle ottenute da lisati di batteri sottoposti ai diversi trattamenti fotodinamici. Tutte le proteine delle mappe bidimensionali che, a seguito dell’analisi, hanno mostrato di essere state significativamente modificate dai trattamenti fotodinamici, sono quindi state identificate tramite spettrometria di massa (MALDI-TOF/TOF). Sulla base delle loro funzioni nella cellula, le proteine identificate sono quindi state assegnate a diverse classi funzionali, al fine di scoprire quali funzioni cellulari venissero maggiormente colpite dalla PDT. Tra le 265 proteine globalmente identificate dall’analisi con Proteomweaver, 70 hanno mostrato significative variazioni di intensità dovute ai trattamenti fotodinamici; tra queste, la maggioranza era composta da proteine implicate nella risposta allo stress ossidativo, nel metabolismo energetico e nella captazione di zuccheri. Comparando gli effetti della PDT tra le due porfirine, si è scoperto che i livelli di intensità di molte proteine sono stati modificati da entrambe, ed in genere nella stessa direzione. Particolarmente interessante è stata la scoperta che le tipologie di modifica del profilo proteomico di S. aureus, causate dai trattamenti fotodinamici, non sono compatibili con le risposte ad agenti ossidanti (come per esempio perossidi o superossidi) da parte della cellula batterica; questo suggerisce che la PDT ha come bersagli specifiche proteine. I risultati ottenuti sono di particolare importanza perché, approfondendo la conoscenza del meccanismo d’azione della PDT, potrebbero aiutare nel disegno di nuovi fotosensibilizzatori più efficienti di quelli attualmente in uso. Nella seconda linea di ricerca ho studiato alcune proprietà legate all’attività antimicrobica ed alla capacità di ingresso nella cellula batterica di un peptide cationico antimicrobico, l’apidaecina 1b. I risultati ottenuti da questo studio hanno quindi permesso di utilizzare l’apidaecina stessa come vettore di altre molecole, coniugandola con una porfirina al fine di ottenere un nuovo agente antimicrobico, con un maggior spettro d’azione rispetto ai suoi singoli costituenti. L’apidecina 1b è un piccolo (soli 18 amminoacidi) peptide che viene molto studiato per via di alcune sue particolari capacità, tra cui una buona inibizione della crescita di batteri Gram (-) e, soprattutto, un’eccezionale abilità nell’entrare nelle cellule mediante un meccanismo che non comporta la formazione di pori nelle membrane. Inoltre, è stato dimostrato che l’apidaecina non presenta tossicità per cellule eucariotiche a concentrazioni che sono invece letali per i batteri. Allo scopo di studiare il meccanismo d’azione dell’apidaecina sono stati sintetizzati degli ibridi peptide-peptoide dell’apidaecina stessa, nei quali ognuno dei tre residui di arginina è stato sostituito con residui di N-(3-guanidinopropyl)-glicina, e si sono quindi valutate le MIC (minima concentrazione inibente) di ognuno degli analoghi nei confronti di diversi batteri. Né l’apidaecina né i suoi ibridi peptide-peptoide hanno mostrato attività antimicrobica nei confronti di Staphylococcus aureus e Pseudomonas aeruginosa. Invece, in Escherichia coli si è potuta osservare una lieve diminuzione del valore della MIC, rispetto al peptide naturale, con gli ibridi sostituiti nelle posizioni 4 e 12, mentre l’attività antimicrobica veniva completamente persa nell’ibrido sostituito nella posizione 17. Questi risultati suggeriscono che l’arginina in posizione 17 possa giocare un ruolo particolarmente importante nel meccanismo di traslocazione dell’apidaecina all’interno della cellula. Quindi, sia l’apidaecina che i suoi ibridi peptide-peptoide sono stati marcati mediante legame con una molecola di fluoresceina, allo scopo di monitorarne la capacità di legame con la cellula batterica mediante tecniche di microscopia di fluorescenza e citometria di flusso. Sfortunatamente, la marcatura con la fluoresceina ha causato la perdita dell’attività antimicrobica e di gran parte della capacità di legarsi ed entrare nella cellula batterica sia dell’apidaecina che dei suoi ibridi. Questo effetto è dovuto principalmente alla massa aggiuntiva portata dalla fluoresceina ai peptidi. Tuttavia, si è osservato che, a differenza degli altri, l’ibrido peptide-peptoide con la sostituzione dell’arginina 17 non presentava alcuna capacità di legarsi alle cellule batteriche. Questo risultato ha confermato sia l’importanza di questa arginina per la capacità antimicrobica dell’apidaecina che, in generale, l’importanza del dominio C-terminale sulla capacità di legame e di ingresso nella cellula batterica. Il coniugato (che è stato chiamato T-api), ottenuto dall’unione di una porfirina anionica (monocarbossi-tetrafenil porfirina, cTPP) con il dominio N-terminale dell’apidaecina 1b, non ha mostrato alcuna attività antimicrobica al buio. Tuttavia, in seguito all’irradiamento con luce blu, T-api si è dimostrato un efficiente fotosensibilizzatore, con un’efficienza fortemente dipendente dalle diverse tipologie di batteri considerate. In particolare, il trattamento fotodinamico con T-api è stato estremamente efficace con E. coli e S. aureus, e leggermente meno efficace, ma comunque sufficiente per indurre un’apprezzabile mortalità, in P. aeruginosa. Esperimenti analoghi, eseguiti trattando i batteri con la porfirina oppure l’apidaecina da sole, oppure con le due insieme ma non coniugate, non hanno causato alcuna mortalità in E. coli e P. aeruginosa. In S. aureus, invece, si è riscontrata una marcata mortalità in seguito al trattamento con cTPP e luce, ma comunque minore di quella ottenuta con T-api. Quindi, ho dimostrato che la coniugazione di un peptide antimicrobico con una molecola fotosensibilizzante può portare alla sintesi di agenti antimicrobici estremamente efficaci, e con uno spettro d’azione superiore rispetto a quelli dei suoi singoli componenti.

The increase of multi-resistant bacteria highlights that the golden era of antibiotics is ending and that alternative treatmentsare urgently needed. Phages have been historically used to treat bacterial infections prior to the discovery of antibiotics and have gained renewed interest in the past decade. Despite the advantages of phage therapy over traditional antibiotic usage, a number of concerns persist over their clinical application centring on their efficacy and safety. This thesis presents four papers that focus on the isolation and characterization of phages that target reference strains and drug-resistant strains of E. coli as well as their infection dynamics and kinetics. In Paper I, six of thirty isolated phages were selected to be characterized for their growth parameters and host range using two commonly used methods. The study showed that the host range (an important selection criteria for phages) of the phages can change based on the assessment method and that the lysis efficiency of phages is host-dependent. The study suggests that standardised methods to assess the host range and lytic activity of phages are required to reduce result variability between research groups. Paper II investigated a rare phage with C3 morphotype from the Podoviridae family and characterised it via genomic, proteomic, morphologic and phylogenetic analysis. The study revealed previously unseen aspects including the formation of a honeycomb structure comprised of phage head during DNA packaging, the possible contractile nature of the tail and the 280 million year co-evolution between the major head protein and the scaffolding protein. Paper III highlights the need to take the immune system into consideration when designing phage therapeutics. In the study, four purified structurally distinct phages (selected from the three main phage families) were exposed to human cells (HT-29 and Caco-2 immortalised intestinal epithelial cell lines and donor-derived peripheral blood mononuclear cells) and the immunogenicity of the phages determined. Phage immunogenicity was shown to vary in a concentration and phage dependent manner with SU63 (a Myoviridae) being the most immunogenic phage and SU32 (a Siphoviridae) the least immunogenic. In the presence of human cells and a suitable host, phages were shown to maintain their killing efficacy as well as the ability to proliferate. Paper IV studies the infection dynamics of an experimental two-phage cocktail against a single bacterial host in vitro and in silico. However, in silico analysis and in vitro analysis produced conflicting results, in which mathematical modelling predicted the complete clearance of bacteria for all treatment scenarios whereas experimental results showed a 1-3log10 reduction in bacterial content. Practical experiments also showed increased anti-bacterial activity when the time between the additions of each phage was varied. This discrepancy suggests that the current mathematical model is unsuitable due to the inability to account for discrete variables such as interference.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

 

The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

A bactéria Xylella fastidiosa 9a5c é o agente causal da clorose variegada dos citros (CVC) e responsável por grandes perdas econômicas na citricultura. O genoma de X. fastidiosa 9a5c foi totalmente seqüenciado e a sua análise permitiu identificar vários genes possivelmente envolvidos na patogenicidade/virulência da bactéria. Como os sintomas da CVC desenvolvemse um longo tempo após a infecção da planta pela bactéria e a severidade da doença têm sido associada à altas temperaturas, é possível que a expressão de fatores de patogenicidade/virulência seja dependente de densidade celular e/ou estresses térmicos. Desta forma, o crescimento in vitro da X. fastidiosa foi monitorado através da absorbância de suspensões bacterianas à 600nm (A600), número de unidades formadoras de colônias (UFC) e viabilidade celular, durante 16 dias de cultivo em meio PW modificado líquido, à 28 e 32ºC. Proteínas extracelulares foram extraídas e analisadas através da eletroforese bidimensional em gel de poliacrilamida (2D-PAGE). Bioensaios foram utilizados para verificar a produção de moléculas sinais por X. fastidiosa. Os resultados obtidos mostraram que o crescimento de X. fastidiosa à 32ºC, com base na A600, não diferiu do crescimento à 28ºC. No entanto, com base no número de UFCs e viabilidade celular, o crescimento de X. fastidiosa diferiu em função da temperatura, tempos de incubação e a interação entre esses fatores. X. fastidiosa produziu maior número de proteínas extracelulares à 32 do que à 28ºC, mostrando que a secreção de proteínas em X. fastidiosa é regulada pela temperatura de incubação. Várias proteínas extracelulares produzidas por X. fastidiosa à 28 e 32ºC são moduladas durante o crescimento da bactéria. A maior parte das proteínas extracelulares produzidas por X. fastidiosa são proteínas ácidas e de massa molecular aparente entre 20-60 kDa. X. fastidiosa não sintetizou moléculas de lactonas de homoserina aciladas (LHAs) reconhecidas pelo sistema repórter utilizado, mas sintetizou uma molécula extracelular em meio de cultivo, semelhante ao DSF produzido por X. campestris pv. campestris, capaz de restaurar a atividade da endoglucanase através do sistema repórter utilizado. A concentração desta molécula em meio PW modificado foi dependente da densidade de células de X. fastidiosa no meio.
The bacterium Xylella fastidiosa is the causal agent of the citrus variegated chlorosis (CVC) and is responsible for significant economic losses in citriculture. The genome of X. fastidiosa has been completely sequenced and revealed several genes probably involved in pathogenicity/virulence. Since CVC symptoms are develop a long time after infection of plant by the bacterium and the severity of the disease has been associated with high temperatures, it’s possible that the expression of pathogenicity/virulence factors is dependent on cellular density and/or temperature stresses. Thus, the growth of X. fastidiosa in modified liquid PW medium was measured based on the absorbance of suspensions at (A600), number of colony-forming units (CFU) and cellular viability, during 16 days at 28 and 32ºC. Extracellular proteins were extracted and analysed by two-dimensional gel electrophoresis (2D-PAGE). Bioassays were used to determine whether X. fastidiosa produces signal molecules involved in quorum perception. The results showed that temperatures of 28 and 32ºC did not affect the growth of the bacterium, based on A600. Temperatures of 28 and 32ºC, incubation times and the interaction of both factors affected bacterial growth based on CFU numbers and the cellular viability. X. fastidiosa produced higher number of extracellular proteins at 32 than at 28ºC, showing that protein secretion is dependent on growth temperature. Several extracellular proteins produced by X. fastidiosa at 28 and 32ºC were modulated the bacterial growth. Most of the extracellular proteins produced by X. fastidiosa were acidic with apparent molecular mass within 20-60 kDa. X. fastidiosa did not synthesize N-acyl homoserine lactone (AHL) recognizes by the reporter system used. However, it synthesized an extracellular molecule in modified PW medium, similar to DSF produced by X. campestris pv. campestris, which is able to restore endoglucanase activity by the reporter system. The concentration of this extracellular molecule produced by X. fastidiosa was dependent on cellular density.

Rhodobacter capsulatus is a versatile organism capable of growing on different growth conditions including photofermentation in the presence of carbon source, aerobic respiration, anaerobic respiration in the presence of an external electron acceptor such as DMSO. The photofermentative growth of R.capsulatus results in hydrogen production which stands out as an environmentally harmless method to produce hydrogen and accepted as one of the most promising process. Due to the serious problems such as as global climate change and environmental pollution caused by the fossil fuels, there is an increasing requirement for a clean and sustainable energy source. Furtherrmore, the ability of R.capsulatus to fix nitrogen, to use solar energy makes it a model to study various aspects of its metabolism. Thus the goal of this study is to increase the potential in biohydrogen production with the photofermentative bacteria and to investigate the proteins playing roles in different growth modes of the bacteria. In the present study, protein profiles of Rhodobacter capsulatus grown on respiratory, anaerobic respiratory and photofermentative growth modes were obtained. LC-MS/MS system is used to analyze the proteome as a high throughput technique. Physiological analysis such as HPLC for the analysis of the carbon source consumption, GC and analysis of pigments were carried out to state the environmental conditions. As a result, total of 460 proteins were identified with 17 proteins being unique to particular growth condition. Ratios of the proteins in different growth conditions were compared and important proteins were highlighted.

La proteine de la membrane externe, p64k (64 kda), du meningocoque neisseria meningitidis est une des proteines vaccinantes potentielles, envisagees dans la preparation d'un vaccin anti-meningite contre le serogroupe b. Cette proteine (599 residus) appartient a la famille des flavoproteines : elle possede un domaine dihydrolipoamide deshydrogenase (dldh, 482 residus) relie par un segment flexible (81 residus) a un domaine lipoique (86 residus) du cote de son extremite n-terminale. La p64k est l'une des premieres dldh caracterisee avec un domaine lipoique. La connaissance de sa structure tridimensionnelle, par la diffraction des rayons x, doit permettre de localiser les regions impliquees dans la reponse immunitaire. Les methodes du remplacement moleculaire et du remplacement isomorphe ont ete employees parallelement pour parvenir a la resolution de la structure. Un premier modele partiel du domaine dldh a ete determine par remplacement moleculaire. Le probleme des phases a ete resolu par la methode du remplacement isomorphe en utilisant le tamm (compose a 4 atomes de mercure) a 0,6 mm et le xenon a l'etat gazeux (13 bar) pour obtenir des derives lourds. Ces donnees ont valide le premier modele du domaine dldh obtenu remplacement moleculaire, puis ont permis de l'ameliorer et d'achever sa construction. Ce modele a ete affine a 2,7 a de resolution. Les donnees de diffraction enregistrees sur un cristal congele a une temperature de 140\c ont permis d'affiner le modele du domaine dldh a 2,23 a de resolution. Le domaine dldh adopte un repliement similaire a celui des dldh de structures connues. Le centre catalytique, situe a l'interface de l'homodimere, est forme par le fad, un pont disulfure intrahelical (cys161-cys166) et un residu histidine (his572*). La structure du domaine dldh en presence du nad + a ete determine a une resolution de 2,6 a. Le determinant antigenique majeur du domaine dldh a ete localise dans ce modele. Il est situe a la surface de la proteine et est constitue d'une boucle de 10 residus flanquee de deux brins. Un complexe de type antigene-anticorps entre le fragment fab d'un anticorps monoclonal et le domaine lipoique a ete prepare pour etendre l'etude structurale au domaine lipoique de la p64k. Les premiers essais de cristallisation de ce complexe antigene-anticorps ont ete debutes.