Orientador: Carlos Henrique Inácio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:01:29Z (GMT). No. of bitstreams: 1 Correa_DanielHenriquedoAmaral_D.pdf: 14618006 bytes, checksum: 0865223d71dcf2f775b71ebcedecc875 (MD5) Previous issue date: 2010 / Resumo: Proteínas enoveladas incorretamente, com freqüência levam à formação de agregados fibrilares contendo extensas estruturas em folha-?, comumente denominadas de fibrilas amilóides. A hipótese acerca da capacidade de formar fibrilas amilóides com estruturas idênticas e ricas em estruturas beta, ser uma propriedade genérica de toda proteína, apoia-se no fato de até mesmo proteínas sem conexão com doenças, como a mioglobina, serem capazes de gerar estruturas fibrilares. Embora várias proteínas sejam intrinsecamente desordenadas, muitas são apropriadamente empacotadas, podendo se desenovelar totalmente ou parcialmente de maneira a expor regiões propensas à agregação, que podem converter o polipeptídeo em fibrilas amilóides. De fato, vários estudos sugerem que intermediários parcialmente enovelados estão envolvidos na fibrilogênese. A apomioglobina (apoMb) de baleia do espermacete é uma proteína bem caracterizada, que forma um intermediário durante o desenovelamento do estado nativo ou após a diluição do estado desenovelado em tampão de enovelamento. A mioglobina é uma proteína altamente solúvel, cujas propriedades do estado nativo não sugerem uma predisposição dessa em formar fibrilas amilóides, corroborado pela organização de sua seqüência de resíduos de aminoácidos em hélices-? bem definidas sem elementos de estrutura em folha-?. Contudo, até a mioglobina forma fibrilas amilóides em certas condições, sugerindo que a capacidade de formar fibrilas seja uma característica comum de toda proteína e, portanto, não estando relacionada a uma estrutura primária específica. Neste projeto, visamos mapear as potenciais interações envolvidas na agregação e formação de fibrilas amilóides em apomioglobinas. Para tanto, apresentamos aqui os estudos dos efeitos de 19 mutantes de apoMb na cinética amiloidogênica da mesma. A indução de fibrilas amilóides foi realizada através da incubação das apoMbs em tampão borato de sódio 50 mM, pH 9 e aquecidas a 65°C. O processo de agregação foi acompanhado por medidas da emissão de fluorescência de Tioflavina T (ThT) e espectroscopia de dicroísmo circular (CD). Outras propriedades morfo-fisicoquímicas das amilóides de apoMb foram também estudadas: energia de ativação da formação de fibrilas, organização estrutural, citotoxicidade, efeito de semeadura, desmontagem por uréia. Nossos resultados mostram que alguns mutantes (7 no total) afetaram a cinética de formação das amilóides, e surpreendentemente, esses efeitos correlacionam-se bem com o efeito que a mutação tem sobre a estabilidade do estado nativo, mas não com o efeito sobre a estabilidade do estado intermediário do enovelamento. As estruturas globais (investigadas por difração de raios-X) das fibrilas formadas pelas ampomioglobinas selvagem e mutantes mostram-se indistinguíveis. Experimentos de citotoxicidade, utilizando um modelo de células neuronais N2A (neuroblastoma de camundongo), e semeadura, confirmam que as diferentes formas de agregados das proteínas são capazes de diminuir a viabilidade celular e de acelerar a formação das fibrilas. Generalizando, nossos resultados suportam a hipótese de que, embora o desenovelamento parcial preceda a formação de fibrilas em apomioglobina, a formação do intermediário de enovelamento não parece ser um passo obrigatório no processo e assim, o enovelamento e a formação de agregados/fibrilas são aparentemente distintos para essa proteína. / Abstract: Protein misfolding usually leads to the formation of fibrillar aggregates with extensive ?-sheet structure, commonly termed amyloid fibrils. The hypothesis that the ability to form ordered ?-rich amyloid fibers with identical structures is a generic property of proteins is supported by the fact that even proteins with no connection to disease, such as myoglobin, are able to generate fibrillar structures. Although several proteins are intrinsically disordered, many are properly packed and should unfold totally or partially exposing aggregation-prone regions that can convert the polypeptide into amyloid fibrils. Actually, several studies suggest that partially folded intermediates are involved in fibrillogenesis. Sperm whale apomyoglobin (apoMb) is a well-characterized protein that forms an intermediate after either unfolding from the native state or dilution of the unfolded protein in a folding buffer. Mb is a highly soluble protein whose native state properties do not suggest a predisposition to form amyloid fibrils, corroborated by its amino acid residues sequence organization in well-defined ?-helices with no ?-sheet elements. However, even Mb forms amyloid fibrils under certain conditions, suggesting that the ability to form fibrils is a common feature of all proteins and is not related to a specific primary structure. In this work, we aim to map potential interactions involved in apomioglobin aggregation and fibrils formation. To such aim, we present here the studies of effects on amiloidogenic kinetics of 19 apoMb mutants. The induction of amyloid fibrils was performed by incubating apoMb proteins on 50 mM sodium borate buffer at pH 9 and heat to 65°C. The aggregation process was monitored both by thioflavin T (ThT) emitted fluorescence and circular dichroism (CD) spectroscopy measurements. Other morph-physicochemical properties of apoMb amyloids were also studied: activation energy of fibril formation, structure organization, cytotoxicity, seeding effect, disassembly by urea. Our results show that some mutants (7 in total) affect the amyloid formation kinetics, and surprisingly, these effects are well correlated with the effect that the mutation has on the stability of the native state but not with the effect on the stability of the folding intermediate. The overall structures, probed by X-ray diffraction, of fibers formed by mutants and wild-type apomyoglobin are indistinguishable. Cytotoxicity experiments, using a neuronal cell line model N2A (murine neuroblastoma), and seeding experiments, confirm that different aggregated forms of proteins are capable of decreasing the cell viability and to speed up the formation of fibrils. Generally, our results support the hypothesis that although partially unfolding precedes fibril formation in apomyoglobin, formation of the folding intermediate is not an obligatory step in the process and thus folding and aggregation/fibril formation are apparently distinct in this protein. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

As fosfolipases A2 (PLA2s, ou fosfatidil-acil hidrolases EC 3.1.1.4) catalisam especificamente a hidrólise das ligações ácido-éster na posição sn-2 de glicerofosfolipídios liberando, como produto da catálise, ácidos graxos e lisofosfolipídio. São encontradas em plantas, mamíferos e em veneno de animais vertebrados e invertebrados e estão envolvidas em uma ampla variedade de processos fisiológicos. A fosfolipase A2 secretada humana do grupo IIA (hsPLA2 gIIA) é uma proteína de fase aguda da resposta imunológica, pois sua expressão é induzida por endotoxinas e citocinas via processos autócrinos e/ou parácrinos durante processos inflamatórios de relevância clínica. A hsPLA2 gIIA mostra efeito bactericida contra infecção por Staphylococcus aureus, e tem marcada preferência por fosfolipídios aniônicos tais como fosfatidilglicerol (PG) encontrados em membranas bacterianas. Uma grande variedade de inibidores de PLA2 do grupo IIA foi descrita na literatura, incluindo substâncias polianiônicas que atuam contra os efeitos inflamatórios destas enzimas. Suramina é um derivado de naftiluréia polissulfonado que recentemente mostrou ligação com os resíduos catiônicos no sítio de reconhecimento interfacial de Bothropstoxina-I (BthTX-I), uma PLA2-Lys49 isolada do veneno de Bothrops jararacussu, inibindo a atividade miotóxica da proteína. Devido ao tipo de interação diferenciada da suramina com BthTX-I em relação aos inibidores competitivos de PLA2, nós avaliamos a especificidade de ligação da suramina na hsPLA2 gIIA como um modelo para estudar este novo tipo de inibidor de PLA2s. O efeito da suramina nas atividades biológicas e de membranas artificiais da hsPLA2 gIIA foi avaliado. A suramina aboliu tanto a atividade hidrolítica da hsPLA2 gIIA quanto a atividade de danificação de membranas artificiais Ca2+ independente. Embora a suramina não tenha inibido a atividade bactericida da hsPLA2 gIIA contra a linhagem Micrococcus luteus, a ativação de macrófagos foi abolida pela mesma de maneira dependente de hidrólise. Além disso, técnicas de simulação de dinâmica molecular, calorimetria de titulação isotérmica e mutagênese sítio dirigida foram utilizadas para mapear os sítios de ligação da suramina na proteína. A interação da suramina com a hsPLA2 gIIA resultou de interações eletrostáticas entre grupos sulfonados com cadeias laterais de aminoácidos da região do sítio ativo e dos resíduos em torno das posições 15 e 116 localizados, respectivamente, na N- e Cterminal. Portanto, estes resultados permitem sugerir que a suramina pode atuar como inibidor de sPLA2s / Suramin is a polysulphonated napthylurea used as an antiprotozoal drug that presents inhibitory activity against a broad range of enzymes. We have evaluated the effect of suramin against the artificial and biological activities of the secreted human group IIA phospholipase A2 (hsPLA2 gIIA), a protein involved in inflammatory processes. To map the suramin binding sites on the hsPLA2 gIIA, proteins with mutations in the active site region and in the protein surface that makes contact with the phospholipids membrane were expressed in E. coli and refolded from inclusion bodies. The activation of macrophage cell line RAW 264.7 by hsPLA2 gIIA was monitored by nitric oxide release, and bactericidal activity of the protein against Micrococcus luteus was evaluated by colony counting and by flow cytometry. The hydrolytic activity of the hsPLA2 gIIA against lipossomes composed of a mixture of dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) was inhibited by a concentration of 100 nM suramin. The activation of macrophages by hsPLA2 gIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA2 gIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 M. The affinity of interaction of the suramin with hsPLA2 gIIA was evaluated by suramine fluorescence and the mutants K15A, K38A, R54A and K123A presented a reduced affinity. The binding of the suramin/hsPLA2 gIIA complex was investigated by molecular dynamics simulations, which indicated two conformations of the bound inhibitor, which involve cationic amino-acid side chains in the active-site region and residues around positions 15 and 116 located in the N- and C-termini respectively in the substrate recognition surface. These results were correlated with isothermal titration calorimetry data, which demonstrated 2.7 suramin-binding sites on the hsPLA2 gIIA. These results suggested that suramin represents a novel class of phospholipase A2 inhibitor

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_simone_simionatto.pdf: 333806 bytes, checksum: 6d2a7ca25216004656318115c514c721 (MD5) Previous issue date: 2008-10-10 / Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), a respiratory disease responsible for significant economic losses. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. Besides, their preparation is expensive because of fastidiously growth of M. hyopneumoniae in vitro. Therefore, the development of alternatives for EP prophylaxis is important for improving health conditions of pigs. Recombinant DNA technology can be used to overcome problems with conventional vaccines. Nevertheless, because of the use of TGA and not TGG to code for tryptophan, translation of mycoplasmal genes terminates prematurely when cloned in other bacteria, such as Escherichia coli. Because of this, the number of antigenic proteins characterized to date in vaccine formulations or immunodiagnostic tests is still restricted. In this work, 63 genetic fragments were selected, which correspond to 48 sequences coding (CDS) of M. hyopneumoniae. First, an overlap extension-PCR method for site-directed mutagenesis of M. hyopneumoniae genes was standardized, aiming at substituting TGA codon by TGG. With this improved method, site-directed mutagenesis was successfully achieved in 14 M. hyopneumoniae genes. Other selected genes were amplified by PCR and cloned into Champion pET200D/TOPO®. A total of 59 genetic fragments were efficiently cloned. From these, 49 had their proteins expressed in E. coli and 35 were purified by affinity chromatography using Ni- Sepharose columns (HisTrap ). Immunogenic and antigenic properties of these proteins were analyzed. For this, the proteins were tested against sera from hyperimmune and from convalescent pigs through ELISA (enzyme-linked immunosorbent assay) and Western blot. Nineteen recombinant proteins were specifically recognized by convalescent pig sera indicates they are expressed during infection. Immune humoral response against recombinant proteins was evaluated in BALB/c mice by ELISA. The results showed that antigens induce variable levels of antibodies, which allows inferring the immunogenicity of each antigen. Sera from inoculated mice with twenty recombinant proteins were able to recognize the native protein by ELISA. This study allowed identifying and characterizing new immunogenic proteins according to their potential for use in diagnostic tests and/or vaccine. These data represent an important contribution for the development of more efficient serological tests and a subunit vaccine for controlling M. hyopneumoniae infections in pigs. / Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína (PES), uma doença respiratória responsável por significativas perdas econômicas. Vacinas comerciais são mundialmente utilizadas no controle desta doença, porém proporcionam apenas proteção parcial. Além disso, são caras devido ao crescimento fastidioso do M. hyopneumoniae in vitro. Desta forma, o desenvolvimento de alternativas para a profilaxia da PES é fundamental para a melhoria da sanidade dos suínos. A tecnologia do DNA recombinante pode ser usada para superar problemas com as vacinas convencionais. No entanto, pela utilização de códons TGA e não TGG para codificar o amino ácido triptofano, a tradução de genes de micoplasmas termina prematuramente quando clonados e expressos em outras bactérias, como Escherichia coli. Devido a isso, o número de proteínas antigênicas de M. hyopneumoniae caracterizadas e testadas como vacinas ou em testes de imunodiagnóstico ainda é restrito. Neste trabalho, 63 fragmentos gênicos foram selecionados, os quais correspondem a 48 seqüências codificadoras (CDS) de M. hyopneumoniae. Num primeiro momento, foi padronizado um método de PCR de sobreposição para mutagênese sítio-dirigida de genes de M. hyopneumoniae, objetivando a substituição do códon TGA por TGG, na seqüência do DNA. Com esse método, a mutagênese sítio-dirigida foi realizada com sucesso em 14 genes de M. hyopneumoniae. Os demais genes selecionados foram amplificados por PCR e clonados no vetor Champion pET200D/TOPO®. No total, 59 fragmentos gênicos foram clonados eficientemente. Destes, 49 tiveram suas proteínas expressas em E. coli e 35 foram purificadas por cromatografia de afinidade em coluna de Ni- Sepharose (HisTrap ). As propriedades antigênicas e imunogênicas destas proteínas foram analisadas. Para isso, as proteínas foram testadas contra soro de suínos convalescentes e soro hiperimune através de ELISA (enzyme-linked immunosorbent assay) e Western blot. Dezenove proteínas recombinantes foram reconhecidas especificamente por soro de suínos convalescentes, indicando que elas são expressas durante a infecção. Resposta imune humoral contra as proteínas recombinantes foi avaliada em camundongos BALB/c através de ELISA. Os resultados demonstraram que os antígenos induzem um nível variável de anticorpos, o que nos permite inferir quanto à capacidade imunogênica de cada antígeno. O soro dos camundongos inoculados com 20 proteínas foi capaz de reconhecer as proteínas nativas através de ELISA. Este estudo permitiu identificar e caracterizar novas proteínas imunogênicas de acordo com o seu potencial para uso em testes de diagnóstico e/ou vacina. Estes dados representam uma importante contribuição para o desenvolvimento de testes sorológicos mais efecientes e vacinas de subunidade para o controle da infecção causada por M. hyopneumoniae em suínos.

Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

Abstract Osteoporosis is a disease characterized by abnormalities in the amount and architectural arrangement of bone tissue, which leads to impaired skeletal strength and increased susceptibility to fractures. Type 5 tartrate-resistant acid phosphatase (TRACP, AcP5) has been suggested to participate directly in bone resorption. In this study, baculovirus expression vector system in insect cells was used to gain large amounts of recombinant type 5 acid phosphatase for structure determination, structure-based functional studies and production of monoclonal antibodies. Active and inactive forms of the enzyme were separated from each other by cation-exchange chromatography, and characterized. The enzyme was crystallized and the three-dimensional structure was determined. Based on the three-dimensional structure of the active site five different enzyme variants were constructed, produced in insect cells, and purified. The wild type enzyme and the mutated forms were characterized, and their kinetic parameters were determined. The importance of amino acids that were expected to be essential for the acid phosphatase activity was confirmed. The acid phosphatase activity and reactive oxygen species generating activity of this dual enzyme proved to exploit different amino acids in their reaction mechanisms. Further studies are needed to clarify the physiological substrates of TRACP in vivo. The findings of this study could form a base for construction of inhibitors for TRACP that could be useful therapeutic agents for osteoporosis and related bone disorders.

La détection de molécules odorantes représente un enjeu important dans divers domaines tels que l’industrie alimentaire, le diagnostic médical et la sécurité du territoire, par exemple. En effet, les odorants, présents par milliers dans notre environnement, véhiculent de nombreuses informations, via leur nature chimique ou leur concentration. Notre système olfactif est capable de discriminer des milliers de molécules différentes via des mécanismes biochimiques impliquant l’association de nombreux partenaires protéiques et un codage combinatoire de l’information. Ces biomolécules, qui englobent notamment les récepteurs olfactifs et les protéines de liaison aux odorants (OBP), constituent une source intéressante d’éléments de détection pour la conception de biocapteurs. Les OBP sont de petites protéines solubles présentent dans le mucus nasal à des concentrations de l’ordre du millimolaire. Leur poche de liaison hydrophobe leur confère la capacité de lier de façon réversible les molécules odorantes. Leur robustesse et leur manipulation aisée en font de bonnes candidates pour l’élaboration de biocapteurs. Au cours de ce travail, nous nous sommes intéressés à la détection de molécules odorantes en associant des OBP comme biorécepteur et l’électrochimie comme méthode de transduction. Par une méthode de mutagenèse dirigée, nous avons montré qu’en modifiant un seul des acides aminés dans la poche de liaison de deux OBP de rat (rOBP2 et rOBP3), il était possible de moduler leurs affinités envers les odorants. En parallèle, nous avons décrit la détection qualitative et quantitative de molécules volatiles à partir d’OBP. Nous avons montré que rOBP3 lie la 2-méthyl-1,4-naphtoquinone (MNQ), une sonde électrochimique. La quantité de MNQ déplacée de la poche de liaison de rOBP3 par la 3-isobutyl-2-méthoxypyrazine (IBMP), un odorant modèle, a été mesurée par voltammétrie cyclique à vagues carrées. Nous avons déterminé les constantes de dissociation des complexes rOBP3 / MNQ et rOBP3 / IBMP. Les valeurs mesurées par électrochimie ont été confirmées par compétition avec une sonde fluorescente et par titration calorimétrique isotherme. En combinant cette nouvelle méthode analytique à des variants de rOBP3 qui présentent des profils de liaison différents et complémentaires, nous avons détecté sélectivement chacun des constituants d’un mélange ternaire d’odorants. Ces travaux, qui allient ingénierie des OBP et électrochimie, offrent des perspectives intéressantes dans le domaine des nez électroniques. / The detection of odorant molecules has become an important challenge in different research area, such as the food industry, medical diagnostics and homeland security. Indeed, the thousands of odorants in our environment provide information on their chemical nature or their concentration. Human olfactory system is capable of discriminating thousands of different molecules thanks to biochemical mechanisms involving multiple protein receptor partners and a combinatorial coding. These biomolecules that include olfactory receptors and odorant-binding proteins (OBP) represent an interesting source of detectors for the design of biosensors. OBPs are small soluble proteins present in nasal mucus at millimolar concentrations. Their hydrophobic binding pocket gives them the ability to reversibly bind odorant molecules. OBPs are robust and easy to produce and are thus good candidates for the design of biosensors. In this work, we focused on the detection of odorant molecules associating OBPs as a bioreceptor and electrochemistry as a transduction method. Using site-directed mutagenesis, we have shown that by substituting a single amino acid in the binding pocket of two rat OBPs (rOBP2 and rOBP3), it is possible to modulate their binding affinities towards odorants. In parallel, we described a qualitative and quantitative method for the detection of volatile molecules using OBPs. We have shown that rOBP3 binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe. The amount of MNQ displaced from the binding pocket of rOBP3 by the model odorant 3-isobutyl-2-methoxypyrazine (IBMP), was measured using square-wave voltammetry. We determined the dissociation constants of the rOBP3 / MNQ and rOBP3 / IBMP complexes. These values measured by electrochemistry were confirmed by a competitive fluorescent assay and isothermal titration calorimetry. By combining this new analytical method to rOBP3 variants with different and complementary binding profiles, we were able to selectively detect each of the components of a ternary mixture of odorants. This work, that combines the engineering of OBPs and electrochemistry, offers us interesting perspectives in the field of electronic noses.

A major factor in the success of Staphylococcus aureus as a pathogen is its vast arsenal of virulence determinants and, more importantly, the tight and precisely- timed regulation of these factors. Here we investigate the product of the S. aureus gene, SAUSA300_1984, encoding a putative transmembrane protein. This as yet uncharacterized protein belongs to the Abi (abortive infection) family, which are commonly annotated as CAAX-proteases, and are significantly understudied in prokaryotes. In S. aureus the disruption of SAUSA300_1984 results in a drastic reduction of proteolytic and hemolytic activity, as well as diminished pigmentation. This phenotype appears to be mediated through reduced agr expression, as determined by qPCR analysis. Importantly, known regulators of agr, such as CodY, MgrA, and ArlR, demonstrate no significant changes in transcription upon 1984 disruption, whilst major alterations were observed for downstream effectors of agr, such as sarS, RNAIII, rot and hla. Complementation and site-directed mutagenesis of 1984 demonstrated that proteolytic activity (via conserved EE residues) was not required for this phenotype, suggesting a potential protein-protein interaction mechanism of interaction. Proteome analysis of the 1984 mutant confirmed a number of our transcriptional observations, such as an increased abundance of Rot and surface associated proteins, as well as a marked decrease in Agr-system proteins levels, with the most striking being AgrB. Virulence profiling revealed a decreased ability of the 1984 mutant to evade constituents of the innate immune response, and impaired survival during murine models of infection. Given that SAUSA300_1984 is encoded 3 genes downstream of RNAIII, our current working hypothesis is that this Abi protein functions to control agr activity through communication with membrane components of this system, potentially via interaction with AgrB. Confirming this, and determining the upstream effectors of this regulatory system are studies currently ongoing in our laboratory.

Chez certains pathogènes, les flavohémoglobines (flavoHbs) jouent un rôle important dans le système de défense des microorganismes en leur conférant une résistance contre le stress nitrosant généré par les cellules du système immunitaire. Dans la mesure où ces protéines sont absentes chez l’homme, plusieurs études ont été réalisées en vue de comprendre le fonctionnement des flavoHbs au niveau moléculaire en les considérant comme des cibles attractives potentielles pour des inhibiteurs sélectifs. L’inactivation de ces enzymes chez les pathogènes entraîne une perte de virulence. Des composés chimiques (dérivés azolés) ont été identifiés comme étant capables d’inhiber leur activité enzymatique et d’affecter les facteurs de virulence bactérienne. Si ces composés sont très utilisés dans le traitement d’infections fongiques cutanées invasives, leur activité antibactérienne potentielle n’est pas encore exploitée.Typiquement, les flavoHbs sont composées de trois domaines : un domaine à hème de type globine, un domaine de site de fixation d’une flavine et un domaine de fixation du substrat de l’enzyme, le NADH. Les structures cristallographiques des flavoHbs montrent que ce sont des protéines capables d'adopter des conformations différentes selon la nature et la présence de ligand de l’hème mais aussi selon l’organisme d’origine. Ces différentes conformations se traduisent par une mobilité d’un seul des trois domaines sans modification majeure des deux autres domaines. Nous avons entrepris, dans ce travail, l’étude de la flavoHb d’une bactérie pathogène, Staphyloccocus aureus (pathogenic bacteria), peu étudiée dans la littérature et sa comparaison avec celle issue de Ralstonia eutropha (bactérie non pathogène), mieux caractérisée, afin de tenter d’apporter des informations nouvelles voire plus spécifiques de leur fonctionnement chez les pathogènes.Le travail présenté dans ce manuscrit décrit tout d’abord la caractérisation fonctionnelle et biochimique de la flavoHb de S.aureus, et approfondi celle de R. eutropha. Du fait de l’absence de structure cristallographique de la flavoHb de S. aureus, nous avons construit un modèle structural théorique en se basant sur l’homologie de séquence forte entre la séquence d’acide aminée de S. aureus et celle de Saccharomyces cerevisiae dont la structure 3D a été récemment déterminée. Dans l’hypothèse où le mécanisme enzymatique des flavoHbs implique une transition entre différentes conformations, les flavoHbs de R. eutropha et S. aureus ont été modifiées génétiquement au niveau du résidu phénylalanine suggéré par l’analyse structurale comme pouvant jouer un rôle dans la transition entre les différentes conformations. Les conséquences de la mutation de Phe396 en Ser ou Ala ont été analysées au regard des activités catalytiques et des propriétés biochimiques de l’enzyme mais aussi des propriétés des transferts d’électrons intramoléculaires entre les groupements prosthétiques (hème et flavine) par différentes techniques spectroscopiques. Ce travail a permis de montrer que l’effet de la substitution du résidu Phe sur les propriétés enzymatiques diffère selon la flavoHb considérée et révèle dans certains cas un site de fixation de substrat autre que le substrat natif. L’effet d’inhibiteurs (dérivés azolés) mais aussi des quinones et de composés aromatiques nitrés sur le fonctionnement des protéines natives et modifiées génétiquement ont été également étudiés dans ce travail, ces derniers composés pouvant agir en tant que "substrats subversifs" pour les flavoHbs, transformant leurs fonctions de protection en fonctions cytotoxiques.L’ensemble de ce travail a montré que, malgré des structures et des séquences protéiques très voisines, les flavoHbs issues de bactéries différentes sont susceptibles de se comporter différemment vis-à-vis de l’introduction d’une même mutation, de l’effet d’un même inhibiteur et peuvent le cas échéant présenter aussi des cycles catalytiques sensiblement différents. / For some pathogens, flavohemoglobins (FlavoHbs) play an important role in the defense of microorganisms by conferring them a resistance against nitrosative stress generated by the immune system cells. Since these protein are absent in human, several studies have been conducted in order to understand the functioning of these proteins at the molecular level, considering them as potential attractive targets for selective inhibitors. Their inactivation results a loss of virulence. Chemical compounds such as azole derivatives have been identified as being capable of inhibiting their enzymatic function and thereby affecting the bacterial virulence factors. If these compounds are widely used in the treatment of invasive fungal skin infections, their potential antibacterial activity is not yet operated.Typically, flavoHbs are composed of three domains: an N-terminal globin domain which harbors a single heme b and a C-terminal ferredoxin reductase-like FAD- and NAD-binding module. The crystallographic structures of flavoHbs show that these proteins are able to adopt different conformations depending on the nature and the presence of heme ligand but also depending on different organisms they are coming from. The different conformations are characterized by a rigid body motion of one of the three domains. We have undertaken in this work, the study of the Staphyloccocus aureus flavoHb, poorly studied in the literature, and compared it to the Ralstonia eutropha flavoHb (non pathogenic bacteria) to provide new and more specific information on behaviors of these proteins derived from pathogens.This PhD work describes first the functional and biochemical characterization of the S. aureus flavoHb. Due to the lack of its crystallographic structure, a theoretical structural model was designed based on the strong sequence homology between the amino acid sequence of S. aureus and Saccharomyces cerevisiae for which the 3D structure was recently determined. We hypothesized that the relative movement of protein domains could be a way to regulate its enzymatic activity by controlling the substrate accessibility. The Phe396 residue (nomenclature according to R. eutropha), suggested by the structural data to play an important role in the enzyme functioning, but also in the equilibrium between different conformations, was substituted by the Ala or Serine amino acids. The effects of the punctual mutations were investigated with respect to the enzyme functioning and biochemical properties. The mutations effect on the internal electron transfer between flavoHbs cofactors (heme and FAD) was also studied by spectroscopic techniques. This work showed that the impact of Phe substitution on the enzymatic properties is different, depending the considered flavoHb and revealed an additional binding site for other substrate than the native one. The effects of azole derivative inhibitors and also quinones and nitroaromatic compounds have been studied on native and genetically modified enzymes functioning. The latter compounds may act as the ‘subversive substrates’ for flavoHb, converting its protective functions into the cytotoxic ones.Taken as a whole, this work showed that, although the protein structures and sequences are similar, the flavoHbs from different bacteria may behave differently towards an identical mutation, an identical inhibitor and could display different catalytic cycles.

Unlike most other host-specific mycoplasmas, Mycoplasma gallinarum exists as a commensal with a host range including most poultry as well as some mammals. This property of M. gallinarum may reflect unique mechanisms for its colonization and persistence in hosts. Whereas M. gallinarum shows leucine and arginine aminopeptidase activity, the genes encoding the enzymes had not been cloned and characterized. We identified an aminopeptidase gene (APN) by oligonucleotide hybridization to a genomic library of M. gallinarum in lambda ZAPII bacteriophage. Nucleotide sequence analysis of overlapping phage clones identified a 1,362 bp open reading frame (ORF) encoding a putative leucine aminopeptidase gene. Database searches indicate that this ORF has 68% nucleotide identity and 51% amino acid identity with the M. salivarium leucine aminopeptidase gene. The active sites of the leucine aminopeptidases in other eukaryotes and prokaryotes were conserved in the cloned aminopeptidase gene. Northern-blot hybridization analysis showed that this ORF is expressed as a 1.5 kb transcript. Southern-blot hybridization analysis demonstrated this gene was present as a single copy in M. gallinarum. Characterization of the leucine aminopeptidase demonstrated that it is a metallo-aminopeptidase (EC 3.4.11.1) and is located in the cytoplasm with a weak interaction with the cell membrane. The subcellular location was further confirmed by immunoblotting with polyclonal anti-recombinant APN serum and M. gallinarum Triton-114 partitions. Immunoblotting results with sera from three chickens experimentally infected with M. gallinarum showed that there were very few proteins in M. gallinarum exposed to the host immune responses and that leucine aminopeptidase was not able to stimulate production of specific humoral antibody. Our results suggest that this leucine aminopeptidase play a role in nutrition supply for the host adaptation of M. gallinarum and that the enzyme was not strongly immunogenic.

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