Universiẗat, Diss., 2004--Köln.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-10-26T12:28:06Z No. of bitstreams: 1 Dissertação Igor.pdf: 1540006 bytes, checksum: 333c9da080a2bb906e1bfea7082e9aae (MD5) / Made available in DSpace on 2016-10-26T12:28:06Z (GMT). No. of bitstreams: 1 Dissertação Igor.pdf: 1540006 bytes, checksum: 333c9da080a2bb906e1bfea7082e9aae (MD5) / A linfadenite caseosa, causada por Corynebacterium pseudotuberculosis (C.p) é considerada uma doença crônica que acomete caprinos e ovinos, causando perdas econômicas graves no Nordeste do Brasil. Neste trabalho buscou-se avaliar aspectos da resposta imunológica de camundongos BALB/c durante a infecção com duas diferentes linhagens de Corynebacterium pseudotuberculosis (T1 e C57) que apresentam graus de virulência diferentes: a cepa T1 caracteriza-se por apresentar infecções mais brandas, ao passo que a C57 um comprometimento maior dos animais infectados. A infecção experimental de camundongos reproduz os principais aspectos observados em caprinos e ovinos. Para este experimento foram utilizados 15 camundongos da linhagem BALB/c subdivididos em três grupos: grupo controle (n=5), grupo infectado com 104/ml da linhagem T1(n=5) e um grupo infectado com 104/ml da linhagem C57(n=5). Após 70 dias de infecção esses animais foram eutanasiados. Realizou-se também a avaliação morfológica (presença de granulomas e peso do baço). A resposta imune inata foi avaliada através do padrão de migração celular para a cavidade peritoneal, através da quantificação total e diferencial de células presentes nesta cavidade. A avaliação da resposta imune adaptativa foi realizada através da mensuração da população de linfócitos TCD4+ e TCD8+ presentes no baço de cada camundongo BALB/c, através da imunofenotipagem por citometria de fluxo, bem como a mensuração das citocinas IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, IL-22, IL-27, INF-γ e TNF-α no homogenato do baço. A linhagem C57 induziu alterações morfológicas (presença de granulomas e peso do baço) mais acentuadas, que a linhagem T1. Foi possível também identificar que durante a infecção com a linhagem C57 ocorreu um maior direcionamento celular para o local da infecção e um aumento dos linfócitos TCD4+ e TCD8+. Observou-se também que as citocinas do perfil inflamatório IL-1 alfa e IL-6 e regulatório IL-13 e IL-10 apresentaram diferença significativa, demonstrando um aumento no grupo dos animais infectados com a linhagem T1. As citocinas TNF-α e IL-17 são difíceis de serem padronizadas, uma vez que não foi observado a produção detectável destas citocinas. As citocinas IL-2, IL-4, INF-γ, IL-22, IL-21 e IL- 27 não apresentaram diferenças significativas entre os grupos. Pode-se concluir que diferentes linhagens de C.pseudotuberculosis (T1 e C57) podem apresentar graus de virulência diferentes, que por sua vez, podem interferir no processo de estabelecimento da doença. Os resultados encontrados contribuem para uma melhor compreensão do tipo de resposta e dos mecanismos imunológicos envolvidos durante a infecção com diferentes linhagens de C. pseudotuberculosis.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-08-13T16:46:26Z No. of bitstreams: 1 Dissertação_ICS_Heidiane Alves dos Santos.pdf: 1654611 bytes, checksum: d1c1496a80a371c6d05868103f946b63 (MD5) / Made available in DSpace on 2013-08-13T16:46:26Z (GMT). No. of bitstreams: 1 Dissertação_ICS_Heidiane Alves dos Santos.pdf: 1654611 bytes, checksum: d1c1496a80a371c6d05868103f946b63 (MD5) / A linfadenite caseosa é uma doença infecto-contagiosa crônica, de ocorrência mundial que acomete pequenos ruminantes, causando grandes perdas econômicas. Esta doença tem como agente etiológico a bactéria Corynebacterium pseudotuberculosis e caracteriza-se pela formação de granulomas em gânglios linfáticos superficiais, podendo também acometer órgãos e linfonodos internos, como forma de resposta do sistema imune do hospedeiro à penetração deste agente que resiste a ação bactericida das células fagóciticas. Este patógeno se relaciona filogeneticamente ao Mycobacterium tuberculosis. A avaliação da resposta imune é uma importante ferramenta para localização de animais possivelmente infectados, dificultando desta forma a disseminação do agente. Estudos têm demonstrado a importância de citocinas na defesa de patógenos intracelulares facultativos, inclusive para C. pseudotuberculosis, destacando-se o interferon-gama (IFN-) citocina característica de células Th1, que pode está envolvida na proteção à patógenos intracelulares. O presente estudo avaliou aspectos da resposta imunológica de camundongos da linhagem C57Black/6 selvagem e knockout para IFN-gama, durante a infecção por Corynebacterium pseudotuberculosis. Os grupos foram acompanhados ao longo de 7 e 14 dias após infecção intraperitoneal com 107 UFC, durante os quais foram avaliados a disseminação bacteriana, a frequência de granulomas, a variação de peso do baço, o padrão de migração celular para a cavidade peritoneal, além da resposta imune humoral através da dosagem de IgG e subclasses e resposta imune celular através da dosagem de citocinas e imunofenotipagem. Os camundongos knockout para IFN-gama se mostraram mais susceptíveis à infecção por Corynebacterium pseudotuberculosis apresentando maior disseminação bacteriana nos linfonodos mesentéricos, maior frequência de granulomas, aumento considerável no peso do baço, intensa migração celular para a cavidade peritoneal, principalmente neutrófilos e macrófagos e expressão de linfócitos T CD8+ nas células esplênicas, e consequentemente menor expressão de citocinas pró-inflamatórias e regulatórias. Estes achados apontam para a importância do IFN-γ na resposta imune a patógenos intracelulares a exemplo de C. pseudotuberculosis, ao tempo em que demonstra a resposta deficiente do sistema imune dos animais nocauteados à infecção. / Salvador

Made available in DSpace on 2014-09-09T12:30:34Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 63.pdf: 4449689 bytes, checksum: 310b00c4ad34ad5d748ccf5005586e50 (MD5) Previous issue date: 2011 / Além dos quadros de difteria clássica, Corynebacterium diphtheriae tem sido isolado de diversos processos infecciosos invasivos. Escassos são os trabalhos que procuram investigar in vivo os fatores que influenciam o estabelecimento e/ou a disseminação de amostras potencialmente invasoras de C. diphtheriae. Além disso, na literatura encontramos a descrição de inúmeros casos de um patógeno otencialmente perigoso para a população o Corynebacterium ulcerans, que se apresenta como um desafio para os órgãos de Saúde Pública brasileiros que somente recentemente o incluíram na rotina dos esquemas de isolamento e identificação. O presente estudo teve como objetivos gerais investigar processos invasivos desencadeados por amostras de C. diphtheriae e C. ulcerans potencialmente invasivas utilizando modelos experimentais em animais. Além de realizar a pesquisa de C. ulcerans em populações de caninos mantidos em instituição destinada ao abrigo de animais domésticos localizada na região metropolitana do Rio de Janeiro. Neste sentido foram desenvolvidos os seguintes objetivos específicos: (i) Avaliação do potencial artritogênico de cepas de origens diversas de C. diphtheriae e C. ulcerans através do modelo de artrite em murinos; (ii) Monitoramento em diferentes intervalos de tempo de microrganismos viáveis no sangue, pulmões, rins, fígado, baço, cérebro coração e articulações; (iii) Determinação dos níveis séricos de citocinas IL-6 e TNF-α decorrentes do processo infeccioso. Os resultados indicaram o modelo experimental de artrite em camundongos convencionais Swiss Webster infectados por via i.v. como mais adequado para a análise do potencial invasor das amostras. As 13 amostras de C. diphtheriae e C. ulcerans estudadas apresentaram potencial artritogênico em intensidades variadas, independente da capacidade de produção de DNAse, toxina diftérica e fosfolipase D (PLD). Dentre as amostras testadas, as de C... / Corynebacterium diphtheriae has been isolated from several invasive infectious diseases besides diphtheria disease. Few reports attempt to explain in vivo the factors influencing the establishment and / or the spread of potentially invasive strains of C. diphtheriae. Moreover, a large number of cases of a potentially infectious pathogen, Corynebacterium ulcerans, have been described worldwide, which can be seen as a challenge for Brazilian public health agencies which only recently included the isolation and identification of this pathogen in routine microbiological analysis. Therefore, this study aims to investigate general processes caused by invasive strains of C. diphtheriae and C. ulcerans using experimental models in animals. An additional objective is to conduct a research on C. ulcerans in populations of dogs kept in domestic animal shelter located in the metropolitan region of Rio de Janeiro. Thus, we have proposed the following specific objectives: (i) assess the arthritogenic potential of various C. diphtheriae and C. ulcerans strains by arthritis model in mice, (ii) monitor, at different time intervals, the presence of viable microorganisms in the blood, lungs, kidneys, liver, spleen, brain, heart and joints, (iii) determine serum levels of IL-6 and TNF-α under infectious process. The results indicated that the model of experimental infection in conventional Swiss Webster mice infected by IV route is suitable for the analysis of potential invasive strains. The 13 strains of C. diphtheriae and C. ulcerans showed arthritogenic potential on different levels, regardless of ability to produce DNase, diphtheria toxin and phospholipase D (PLD). Among the samples tested, C. diphtheriae isolated from cases of endocarditis and C. ulcerans isolated from infectious processes in humans and animals showed the greatest arthritogenic potential (> 50%) compared with the samples of C. diphtheriae isolated from classic diphtheria cases and C. ulcerans isolated from asymptomatic dogs. Histopathologic analysis showed the presence of subcutaneous edema, inflammatory reaction, damage to bone and synovial hypertrophy, consistent with a condition of severe arthritis for the samples considered more arthritogenic. Viable bacteria were recovered from the blood, kidneys, liver, spleen and joints. Both species caused polyarthritis, with wrists and ankles more often affected. The samples of C. ulcerans CDC-KC279 and 809 beside polyarthritis, preduced ulcerated lesions at the affected joints and nodose in the tail with extensive necrosis at the inoculation site (tail vein). The correlation between the arthritis and systemic levels of IL-6 and TNF-α was observed for the strains of C. ulcerans, with the exception of strain BR-AD22 that was unable to produce arthritis. In conclusion, our study showed a potential arthritogenic strain-dependent in C. diphtheriae and C. ulcerans indicating differences in virulence and invasive potential of these species independent of the production ability of diphtheria toxin and PLD.

Made available in DSpace on 2015-07-08T12:28:18Z (GMT). No. of bitstreams: 2 4.pdf: 1791166 bytes, checksum: e18bbf1db8c3f87f4338a15297302f69 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Casos de infecções invasivas por corinebactérias, colonizadoras do ambiente e da microbiota humana ou de animais, tem sido crescentes em decorrência de melhor sobrevivência de indivíduos imunocomprometidos. Além da fenotipagem, métodos moleculares tem sido fundamentais na identificação de bastonetes Gram positivos irregulares(BGPI). O presente estudo teve como objetivo a caracterização fenotípica e genotípica de estirpes de corinebactérias isoladas de sítios intravenosos de pacientes internados em um hospital universitário no Rio de Janeiro. Neste sentido, 60 estirpes de microrganismos Gram positivos foram analisadas por metodologia enotípica convencional, sistemas API Coryne e Vitek 2 (bioMérieux) e análise das sequências dos genes 16S rRNA e rpoB, utilizada como metodologia de referência para avaliação dos sistemas fenotípicos. Os dados indicaram o isolamento de Corynebacterium striatum (44,68 %), Corynebacterium amycolatum (31,91 %), Corynebacterium jeikeium (8,51 %), Corynebacterium urealyticum (6,39 %), Corynebacterium diphtheriae (4,26%), Corynebacterium simulans (2,12%) e Corynebacterium minutissimum (2,12%) do sangue de pacientes fazendo o uso ou não de dispositivos invasivos. As espécies predominantes C. striatum e C. amycolatum apresentaram 8 e 9 perfis de resistência aos agentes antimicrobianos, respectivamente. O perfil de resistência com sensibilidade apenas à tetraciclina, linezolida e vancomicina, foi prevalente durante um surto epidêmico de C. striatum ocorrido em 2010. Este mesmo perfil foi observado para C. amycolatum, C. jeikeium e C. urealyticum. A identificação definitiva da maioria das estirpes de C. striatum, C. amycolatum, C. jeikeium, C. simulans e C. minutissimum só foi possível pela genotipagem. Interessantemente, a análise das sequências do gene 16S rRNA permitiu a identificação de outros microrganismos como Abiotrophia defectiva (6,67%), Arthrobacter (1,67%), Brevibacterium (11,67%) e Microbacterium (1,67%). / Cases of invasive infections corynebacteria , colonizing the environment and human and animal microbiota, has been increasing due to better survival of immunocompromised individuals. Besides phenotyping, molecular methods have been of great value in the identification of Gram positive irregular rods (BGPI). The present study aimed to phenotypic and genotypic characterization of isolates from corynebacteria isolated of intravenous sites of patients at a university hospital in Rio de Janeiro. Thus, 60 isolates of Gram positive microrganisms were analyzed by conventional phenotype methodology, and Vitek and API Coryne 2 systems (bioMérieux) and by sequence analysis of 16S rRNA and rpoB genes. The genotypic identification was used as reference methods for evaluation of phenotypic systems. The data indicated the isolation of Corynebacterium striatum (44,68 %), Corynebacterium amycolatum (31,91 %), Corynebacterium jeikeium (8,51%), Corynebacterium urealyticum (6,39 %), Corynebacterium diphtheriae (4,26%), Corynebacterium simulans (2.12%) and Corynebacterium minutissimum (2,12%) from the blood of patients making use or not of invasive devices. The predominant species C. striatum and C. amycolatum presented 8 and 9 profiles of resistance to antimicrobial agents, respectively. The resistance profile with sensitivity just to tetracycline, linezolid and vancomycin, was prevalent during an outbreak of C. striatum occurred in 2010. The same profile was observed for C amycolatum, C. jeikeium and C. urealyticum. The definitive identification of most strains of C. striatum, C. amycolatum, C. jeikeium, C. simulans and C. minutissimum was possible only by genotyping . Interestingly, the sequence analysis of the 16S rRNA gene allowed the identification of other microorganisms such as Abiotrophia defectiva (6,67%), Arthrobacter (1,67 %), Brevibacterium (11.67 %) and Microbacterium (1.67 %). In conclusion, BGPI isolates from invasive infections should not be neglected and sequence analysis of 16S rRNA and rpoB genes can contribute to the definitive identification of the species of Gram positive microorganisms, including corynebacteria involved in these infections.

Submitted by ANA KARLA MIRANDA PRADO null (karlinhamprado@hotmail.com) on 2017-04-24T20:41:26Z No. of bitstreams: 1 DissertaçãoAnaCorrecao final.pdf: 2950511 bytes, checksum: 267c66c96725c6306041b2f73554ba8d (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-26T13:59:46Z (GMT) No. of bitstreams: 1 prado_ak_me_sjrp.pdf: 2950511 bytes, checksum: 267c66c96725c6306041b2f73554ba8d (MD5) / Made available in DSpace on 2017-04-26T13:59:46Z (GMT). No. of bitstreams: 1 prado_ak_me_sjrp.pdf: 2950511 bytes, checksum: 267c66c96725c6306041b2f73554ba8d (MD5) Previous issue date: 2017-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Corynebacterium pseudotuberculosis é uma bactéria gram-positiva causadora da Linfadenite Caseosa (LC) em caprinos e ovinos. A LC é uma doença infectocontagiosa crônica que prejudica a produção de carne, leite e lã em vários países, incluindo o Brasil. Uma vez que o tratamento da doença é muitas vezes inviável e ineficaz, a eliminação dos animais infectados no rebanho tem sido uma das principais medidas de contenção da enfermidade. Vários grupos de pesquisa vem se dedicando ao estudo de C. pseudotuberculosis visando a identificação de fatores moleculares envolvidos na virulência e patogenicidade durante a infecção. Embora alguns destes componentes já tenham sido descritos, como a fosfolipase D, novos estudos são necessários para que seja possível compreender as diversas interações regulatórias que são intrínsecas a esse microrganismo, assim muitos grupos de pesquisas produzem de forma recombinante a enzima transaldolase de C. pseudotuberculosis, envolvida na glicólise e no metabolismo das pentoses-fosfato a fim de buscar inibidores que possam controlar sua ação enzimática. Nesse contexto, os flavonoides são compostos polifenólicos encontrados nas plantas e, em alguns casos, atuam como inibidores de infecções por microrganismos. Assim, a motivação deste trabalho consistiu em identificar e quantificar uma possível interação do flavonoide morina com a enzima transaldolase, a fim de bloquear a atividade de replicação e infecção de LC. Deste modo os objetivos desse estudo foram realizar a expressão, purificação, a caracterização da estrutura secundária e estabilidade térmica por dicroísmo circular e verificação de interação entre transaldolase e morina por espectroscopia de fluorescência da proteína transaldolase. Os resultados da expressão mostram que a proteína transaldolase com 40kDa foi purificada em cromatografia de afinidade seguida de cromatografia de exclusão molecular. A sua estrutura secundária apresentou 74% de alfa hélice, 0% de folha beta, 7,9% de alças e 18,1 % de estruturas aleatórias. A análise da desnaturação térmica mostrou que a temperatura de melting foi de 48°C, indicando que a proteína é estável. A interação entre Morina e Transaldolase apresentou mecanismo de supressão estáticodinâmico, com uma constante de associação moderada e um sítio de interação. A análise termodinâmica mostrou que o processo de interação é espontâneo ∆G<0, endotérmico ∆H>0 e entrópico ∆S<0. Assim, sabendo-se que a proteína Transaldolase é a proteína chave no processo das infecções por Corynebacterium pseudotuberculosis e considerando as propriedades antibacterianas e antiproliferativas do flavonoide morina, sugere-se que este composto possa ser investigado para os seus usos específicos. Sugere-se que a interação da transaldolase com a Morina possa exercer um papel de carreador ou seja, uma forma pela qual a proteína leva a molécula para o local que ela atua, e, assim, a Morina consiga realizar sua função antiproliferativa e bloquear as infecções. / Corynebacterium pseudotuberculosis is a gram-positive bacterium which causes Caseous Lymphadenitis (CL) in small ruminants. CL is a chronic infectious disease which impairs meat, whool and milk production in many countries including Brazil. Once the treatment for CL is not efficient, removing affected animals from herds represents one of the major strategies to prevent the disease from spreading. Many research groups have been looking for molecular components in C. pseudotuberculosis that are involved in virulence and infection, among which phospholipase D stands out as the major one described so far. However, new studies are necessary for the understanding of the microorganism’s biology, including its intrinsic regulation mechanisms. The object of study of this work was the enzyme transaldolase involved in glycolysis and in the metabolism of pentoses-phosphate, necessary for the formation of nucleic acids that the bacterium uses to replicate in order to find inhibitors that can control its enzymatic action. In this context, flavonoids are polyphenolic compounds found in plants and, in some cases, act as inhibitors of infections by microorganisms. Thus, the purpose of this work is to identify and quantify a possible interaction of the flavonoid morin with the enzyme transaldolase, in order to block LC´s replication activity and infection. The objectives of this study included expression and purification of C. pseudotuberculosis´s transaldolase protein, the characterization of the secondary structure and thermal stability by circular dichroism and the study of the interaction between transaldolase and morin by fluorescence spectroscopy. The transaldolase protein, with approximately 40kDa, was purified on affinity chromatography followed by molecular exclusion chromatography. Its secondary structure had 74% of alpha helix, 0% of beta sheet, 7.9% of loops (turn) and 18.1% of random structures. The thermal denaturation analysis showed that the melting temperature is 48 °C, indicating that the protein is stable. The interaction between Morina and Transaldolase presented a static-dynamic suppression mechanism, with a moderate association constant and one interaction site. The thermodynamic analysis showed that the interaction process is spontaneous ΔG <0, endothermic ΔH> 0 and entropic ΔS <0. Thus, with the knowledge that Transaldolase protein is the key protein in the process of Corynebacterium pseudotuberculosis infections and considering the antibacterial and antiproliferative properties of the flavonoid morine, it is suggested that this compound can be investigated for its specific uses. It is suggested that the interaction of transaldolase with Morina may play a role of carrier, that is, a way in which the protein takes the molecule to the place it acts, and thus Morina can perform its antiproliferative function and block the Infections.

The availability and assimilation of NH₄⁺ plays an integral role in the growth of microorganisms and the production of amino acids by these organisms. This study investigated the dissociation of NH₄⁺in aqueous solution, its availability and effect on the enzymes of NH₄⁺ assimilation and its influence on lysine production by Corynebacterium glutamicum.In aqueous solution the extent of dissociation of NH₄C1, {NH₄)₂S0₄ and (NH₄)₂HP0₄ increases with decreasing concentration. A model is proposed for the dissociation of these molecules. It is believed that at very low concentrations, dissociation to NH₃ plus the respective counter-ions occurs. At these low concentrations the NH₃ acts as the substrate for glutamine synthetase. At the higher concentrations dissociation is to NH₄⁺ which is the substrate for glutamate dehydrogenase. At these higher concentrations the enzyme activities obtained for glutamate dehydrogenase, at equivalent concentrations of the above ammonium salts, were different when based on the total concentration of NH₄⁺, and similar when based on the concentration of free NH₄⁺. L-Iysine occurs in the +1 ionic form, at pH 7,2. The lysine which is produced during fermentation associates with the anionic counter-ion of the ammonium salt used. The concentration of the free NH₄⁺ in the media appears to affect both the rate of lysine synthesis as well as the yield. The lysine fermentation occurs in two stages; a growth (or replicative) phase, during which very little lysine is produced, and a lysine synthesis (or maturation) phase. During the lysine synthesis phase there is no cell replication, however an increase in the mass of the biomass produced is apparent. Evidence is provided for the possible concomitant synthesis of the the cell wall polymer, glycerol teichoic acid, and lysine. On the basis of this evidence, a nucleotide balance is proposed for lysine and teichoic acid synthesis. The replicative phase and the maturation phase have to be effectively separated to obtain optimal lysine yields and titres. It is believed that teichoic acid synthesis during the replicative phase must be kept to a minimum for optimal yields and titres to be obtained, and on completion of the cell wall and therefore teichoic acid synthesis, lysine synthesis ceases. As the production of lysine appears to be affected by the NH₄⁺ concentration in the culture media, it is proposed that a futile cycle may exist around the transport and assimilation of the NH₄⁺. If the fermentations are run at low free NH₄⁺ concentrations, it was shown that lysine yields of 0,66, on the glucose utilised, are attainable during the fermentation.

Studies were evaluated on the effects of known growth factors on the growth and carotenogenesis of Corynebacterium species strain 7ElC. The complex medium, Tryptic Soy Broth,was found to stimulate growth and production of more pigment in the light and in the dark than did a mineral salts-glucose medium. A complete amino acid mixture added to LSG enhanced carotenogenesis in the dark in Corynebacterium 7ElC, while B-vitamins retarded carotenogenesis. No absolute requirement for one or more amino acids was found,indicating a multiple amino acid requirement. The fewest amino acids found to stimulate carotenogenesis in the dark were a combination of those in the Serine and Histidine families which include serine, glycine, cysteine, and histidine.

L'objectif de cette thèse est l'étude du métabolisme de Corynebacterium glutamicum, et de ses potentialités, au cours de procédés aéro-anaérobies. Après une première phase avec apport d'oxygène pour permettre la croissance bactérienne, une phase anaérobie est induite par arrêt de l'aération et réduction de la vitesse d'agitation. Dans ces conditions, le lactate est le principal métabolite produit. La synthèse de ce dernier a été améliorée en jouant, essentiellement, sur le moment de la transition entre les 2 phases. C. glutamicum 2262 peut ainsi produire 27 g/l de lactate en mode discontinu et 55 g/l en mode semi-continu, suite à un arrêt de l'aération lorsque la concentration en biomasse est d'environ 2,6 g/l. Afin d'exploiter la voie de synthèse d'acide lactique chez C. glutamicum pour la production d'éthanol, les gènes PDC et ADH de Zymomonas mobilis ont été exprimés sous le contrôle du promoteur ldhA endogène de C. glutamicum 2262 et d'une souche de C. glutamicum 2262 sans ldhA. Bien que les productivités en éthanol de ces souches aient été relativement faibles, la suppression de ldhA a entraîné des augmentations de la concentration en éthanol d'environ 15 fois. Une stratégie similaire a été utilisée pour la production d'itaconate. Comme dans le cas de l'éthanol, la concentration en itaconate obtenue est demeurée très faible malgré des essais d'amélioration du procédé de mise en oeuvre de la souche productrice d'itaconate / The objective of this work is the study of Corynebacterium glutamicum metabolism, and of its potentialities, during an aero-anaerobic process. After a first phase during which the oxygen was supplied to favor the bacterial growth, the anaerobic phase was induced by the stopping of the oxygen supply and the decreasing of the agitation speed. In these culture conditions, lactate was the main metabolite produced. The production of this organic acid has been increased by modifying the transition time between the aerobic and the anaerobic phases. C. glutamicum 2262 was able to produce up to 27 g/l lactate during a batch process and up to 55 g/l during a fed batch process. To exploit the lactic acid synthesis pathway of C. glutamicum for ethanol production, the PDC and ADH genes from Zymomonas mobilis were expressed under the control of the endogenous promoter of ldhA, in the wild-type strain and in a ldhA-disrupted strain of C. glutamicum 2262. Although the ethanol productivities of these engineered strains were relatively low, the depletion of ldhA resulted in the increases of ethanol final concentration up to 15 times. A similar strategy was applied for the production of itaconate. As previously for the ethanol production, the final concentration of itaconate remained very low despite of some modifications of the process

En raison de leur contribution à la virulence bactérienne, les lipoprotéines et les membres de la voie de biogenèse des lipoprotéines représentent des cibles prometteuses pour la recherche de nouveaux antibiotiques. À la suite de translocation à travers la membrane interne la future lipoprotéine ancrée dans la membrane par l’intermédiaire de son peptide signal, va subir en premier lieu l’addition de sn-1,2-diacylglyceryle sur la fonction sulfhydryle de la future cystéine N-terminale de la lipoprotéine mature. Cette modification est catalysée par Lgt (prolipoprotéin diacylglycérol transférase) avant même que le peptide signal de la lipoprotéine ne soit clivé par Lsp (lipoprotéine signal peptidase). L’action de la peptidase permet de libérer l’amine terminale de la cystéine qui pourra alors, chez les bactéries à Gram-négatif, être acylée par Lnt (lipoprotéin aminoacyl transférase). La présence d’un apolipoprotéine N-acyltransférase (Ppm2-Ms) impliquées dans la N-acylation de LppX a récemment été montrée chez M. smegmatis. Ppm2-Ms fait partie de l'opéron ppm dans laquelle ppm1, une synthase polyprénol-monophosphomannose, a été révélée essentielle dans la synthèse lipoglycans mais dont la fonction dans la biosynthèse des lipoprotéines est totalement inconnue. Afin de clarifier le rôle de l'opéron ppm dans la biosynthèse des lipoprotéines, nous avons étudié les modifications post-traductionnelles de deux modèles (lipoprotéines AmyE et LppX) dans les mutants Δppm1 et Δppm2 chez C. glutamicum.Nos résultats montrent que les deux lipoprotéines modèles sont ancrées dans la membrane et que leurs extrémités N-terminales sont N-acylés par Ppm2-Cg. Le peptide N-teminal acylé de LppX a été également modifié par des groupements d'hexose. Cette O-glycosylation est localisée dans le peptide N-terminal de LppX mais absente dans le mutant Δppm1. Tandis compromise en l'absence de Cg-PPM2, O-glycosylation LppX pourrait être rétabli lorsque Cg-PPM1, Cg-PPM2 ou l'homologue Mt-ppm1 de M. tuberculosis a été surexprimée. Ensemble, ces résultats montrent pour la première fois que Ppm1-Cg (Ppm synthase) et Ppm2-Cg (Lnt) fonctionnent dans une voie de biosynthèse commune dans laquelle la glycosylation et la N-acylation des lipoprotéines sont étroitement couplés / Due to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), cleaved off their signal peptides by lipoprotein signal peptidase (Lsp) and, in Gram-negative bacteria, further triacylated by lipoprotein N-acyl transferase (Lnt). The existence of an active apolipoprotein N-acyltransferase (Ms-Ppm2) involved in the N-acylation of LppX was recently reported in M. smegmatis. Ms-Ppm2 is part of the ppm operon in which Ppm1, a polyprenol-monophosphomannose synthase, has been shown to be essential in lipoglycans synthesis but whose function in lipoprotein biosynthesis is completely unknown. In order to clarify the role of the ppm operon in lipoprotein biosynthesis, we investigated the post-translational modifications of two model lipoproteins (AmyE and LppX) in C. glutamicum ∆ppm1 and ∆ppm2 mutants. Our results show that both proteins are anchored into the membrane and that their N-termini are N-acylated by Cg-Ppm2. The acylated Ntermina peptide of LppX was also found to be modified by hexose moieties. This O-glycosylation is localized in the N-terminal peptide of LppX and disappeared in the ∆ppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX Oglycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed. Together, these results show for the first time that Cg-Ppm1 (Ppm synthase) and Cg-Ppm2 (Lnt) operate in a common biosynthetic pathway in which lipoprotein N-acylation and glycosylation are tightly coupled.