Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) provoquent des infections sévères chez les personnes atteintes de mucoviscidose. L'infection peut varier d'une forme asymptomatique à une forme plus aiguë pouvant entraîner une pneumonie nécrosante et une septicémie, connue sous le nom de syndrome cepacia. Afin d'étudier les infections causées par le Bcc, nous avons développé un nouveau modèle in vivo, l'embryon de poisson zèbre. Nous avons montré que B. cenocepacia K56-2 pouvait se répliquer dans les macrophages et causer une infection aiguë mortelle pour les embryons. En revanche, B. stabilis LMG14294 induit une infection persistante chez les embryons. Dans cette étude, nous avons montré que les macrophages jouaient un rôle-clé dans la multiplication de K56-2 et dans l'induction d'une réponse inflammatoire MyD88-dépendante, caractérisée par la surexpression des gènes codant pour Cxcl8 (ou IL-8) et l'IL-1b. En l'absence de macrophages, les bactéries sont incapables de se multiplier durant les premières 24h de l'infection, ce qui donne un avantage pour la survie des embryons. L'absence de MyD88 induit aussi l'augmentation de la survie des embryons infectés par K56-2. Mais de manière paradoxale, les bactéries se multiplient mieux chez les embryons myd88-/- mutants que chez les embryons sauvages. Ceci suggère que ce n'est pas le nombre de bactéries qui est important pour l'infection, mais que c'est la réponse inflammatoire excessive causée par cette infection qui entraîne la mort des embryons. Afin d'avoir une vision globale des changements d'expression des gènes de l'hôte durant l'infection, nous avons effectué une expérience de RNAseq. Comme attendu, l'infection aiguë se caractérise par une importante modulation du transcriptome de l'hôte qui augmente avec le temps. A l'opposé, l'infection persistante n'induit que très peu de changements. La réponse immunitaire innée, et en particulier la voie des TLR, ainsi que l'apoptose sont très fortement activées durant une infection aiguë. Pour sa part, B. stabilis module essentiellement les gènes codant pour le système du complément.Le rôle critique des macrophages lors d'une infection par Bcc chez les poissons zèbre est en accord avec les récentes observations cliniques. Ceci suggère que le stade intracellulaire de B. cenocepacia et la réponse inflammatoire qui s'ensuit peuvent être des cibles pour le développement de nouvelles thérapies permettant de lutter contre cette infection. / Bacteria belonging to the Burkholderia cepacia complex (Bcc) can cause chronic infection with periods of acute exacerbation and sometimes fatal necrotizing pneumonia (“cepacia syndrome”) in individuals with cystic fibrosis (CF), and are associated with poor prognosis. Here, we exploited the exciting possibilities for in vivo non-invasive imaging of Bcc infection in transparent zebrafish embryos, with an innate immune system with remarkable similarity to that of humans, and numerous genetic and genomic tools to study the role of host phagocytes and the innate immune response in the pro-inflammatory character of the infection.We show that macrophages play a critical role in intracellular multiplication of B. cenocepacia K56-2 and induction of a MyD88-dependent fatal inflammatory response, characterised by high levels of cxcl8 and il1b expression. Surprisingly, in sharp contrast to the situation found for infections with other pathogens including Mycobacterium marinum and Staphylococcus aureus, in the absence of macrophages, K56-2 survived but was unable to replicate in the first 24 h, which resulted in a significant pro-survival advantage to the host compared to wild type embryos that died within 2 to 3 days. The Toll-like receptor (TLR) pathway is a major arm of the cell-mediated innate immune response with MyD88 as a key adaptor protein involved in the production of pro-inflammatory cytokines. We found that the absence of MyD88 also provided a pro-survival effect to the embryos after infection with K56-2. Paradoxically, the bacteria replicated better in myd88-/- mutant than wild type embryos, suggesting that it is not bacterial burden per se, but the inflammatory response that kills the embryos. Interestingly, cxcl8 and il1b expression were not significantly induced during the first 7 hours in the myd88-/- mutant while a strong induction was seen in control embryos, suggesting that a Myd88-dependent inflammatory response during early macrophage stages significantly contributes to fatal infection.Next, we performed RNAseq to analyse global changes in host gene expression during acute and persistent infection induced by K56-2 and B. stabilis LMG14294 respectively. Whereas acute infection was characterised by strong modulation of host gene expression increasing over time, persistent infection showed modulation of only a small set of genes. TLR and apoptosis signaling pathways were amongst the strongly activated groups during acute infection, in line with the strong inflammatory character of K56-2. During persistent infection, the major differentially expressed gene set concerned genes encoding complement proteins. The critical role for macrophages in Bcc infection in zebrafish is in agreement with recent clinical observations. We suggest that the intracellular stages of B. cenocepacia and the ensuing inflammatory response are essential targets to explore for the development of new therapies to combat this infection.

München, Techn. Univ., Diss., 2004.

Made available in DSpace on 2016-05-11T12:56:37Z (GMT). No. of bitstreams: 2 rhaissa_vieira_ioc_mest_2016.pdf: 3046248 bytes, checksum: d61e9828a7069c1879b0fd074610f11a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Burkholderia cenocepacia é uma bactéria gram-negativa associada a infecções pulmonares oportunistas acometendo portadores de fibrose cística, doença granulomatosa crônica ou que apresentem algum tipo de imunodeficiência, podendo resultar em declínio da função pulmonar e no quadro séptico conhecido como síndrome cepacia. Por mecanismos de escape B. cenocepacia promove atraso na maturação do fagolisossomo, enquanto fatores envolvidos na resistência a ROS desempenham papel na sobrevivência intracelular. Dados de nosso laboratório mostram que cultivos in vitro de macrófagos peritoneais de camundongos e da linhagem RAW 264.7 estimulados com IFN\03B3 e LPS, quando desafiados com B. cenocepacia, apresentam reduzidos níveis de NOx nos sobrenadantes. Assim, o objetivo do presente trabalho foi avaliar a resposta efetora de macrófagos alveolares de camundongo da linhagem AMJ2-C11 na infecção por B. cenocepacia. Além disso, avaliamos a infecção de camundongos imunocompetentes C57BL/6 ou geneticamente deficientes para a enzima óxido nítrico sintase induzível (iNOS/NOS2). Nossos resultados indicam que B. cenocepacia é capaz de desativar mecanismos efetores da resposta clássica de macrófagos, redirecionando para o perfil alternativo de ativação. Observamos em cultivos de macrófagos AMJ2-C11 expostos à B. cenocepacia (i) concentrações reduzidas de NOx e concentrações aumentadas de ureia em sobrenadantes, (ii) aumento da expressão da enzima arginase e baixa expressão de iNOS/NOS2 nestas células, (iii) menor frequência de células expressando MHC-I e moléculas coestimuladoras (CD80, CD86 e CD40), e por fim (iv) baixas concentrações de TNF nos sobrenadantes Semelhante ao encontrado nos sobrenadantes dos cultivos na presença de B. cenocepacia, os lavados pleurais de animais infectados pela bactéria apresentam aumento na produção de ureia em detrimento da produção de NOx. Em 72 horas de infecção, contagem de CFU encontra-se aumentada nos animais deficientes em iNOS/NOS2, enquanto é controlada em animais C57BL/6 imunocompetentes. Todos os camundongos infectados apresentaram perda de peso nas primeiras 72 horas de infecção. Animais C57BL/6 recuperaram peso corporal, apresentando ao término da análise ganho ponderal semelhante aos não infectados. Por outro lado, animais inos-/- infectados acumularam perda ponderal no decorrer do tempo analisado, apresentando em torno de 40% de perda de massa corpórea e apresentaram 100% de mortalidade em 13 dias pós-infecção, ao passo que não houveram mortalidade em animais controles (NI). Os pulmões dos animais inos-/- infectados apresentam alterações histológicas agudas, comparados tanto aos controles não infectados, e mais intensas que os animais C57BL/6 infectados. Em conjunto nossos dados sugerem que a infecção por B. cenocepacia interfere no balanço das vias óxido nítrico sintase induzível e arginase, favorecendo esta última, o que poderia promover a atenuação da resposta inflamatória e a persistência da infecção / Abstract: Burkholderia cenocepacia is a gram-negative bacteria associated with opportunistic lung infections affecting patients with cystic fibrosis, chronic granulomatous disease or immunodeficiencies, which can result in decreased pulmonary function and sepsis known as cepacia syndrome. By escape mechanisms B. cenocepacia promotes delay in maturation of phagolysosome, since cell factors involved in ROS resistance play a role in intracellular survival. Data from our laboratory show that cultures in vitro of mouse peritoneal macrophages and RAW 264.7 cell line stimulated with LPS and IFN\F067\F02C when challenged with B. cenocepacia present reduced NOx levels in the supernatants. The aim of the present work was to evaluate the effector response of mouse alveolar macrophages of the cell lineage AMJ2-C11 challenged by infection with B. cenocepacia. In addition, we evaluated the infection of mice immunocompetent (C57BL/6) or genetically deficient for the enzyme inducible nitric oxide synthase (iNOS/NOS2). Our results indicate that B. cenocepacia can disable effector mechanisms of classical response of macrophages, redirecting to the alternative profile of activation. We observed in AMJ2-C11 macrophage exposed to B.cenocepacia (i) reduced concentrations of NOx and increased concentrations of urea in supernatants, (ii) increased expression of arginase and low expression of iNOS/NOS2 in these cells, (iii) lower frequency of cells expressing MHC-I and costimulatory molecules (CD80, CD86, CD40), and finally (iv) lower TNF concentrations in the supernatants Akin the findings in supernatants of AMJ2-C11 macrophage exposed to B. cenocepacia, pleural washes recovered from infected mice show increased production of urea, while NOx was barely detected. At 72 hours of infection, CFU counts were increased in pleural washes of iNOS/NOS2-deficient mice, while bacteria growth was controlled in immunocompetent mice. All infected mice showed initial weight loss. After 72 hours of infection, C57BL/6 mice recovered body weight, showing similar weight gain to uninfected mice. On the other hand, B. cenocepacia-infected inos-/- mice accumulated body weight loss (~ 40%) during the course of infection, showing 100% of mortality at 13 days post-infection, whereas none of the control mice showed mortality. The lungs of B. cenocepacia-infected inos-/- mice showed acute histological alterations in comparison with uninfected controls, and more intense abnormalities when compared to infected C57BL/6 mice. Taken together our data suggest that the infection by B. cenocepacia interferes with the balance of the inducible nitric oxide synthase and arginase, favoring the latter, which could promote the attenuation of the inflammatory response and the persistence of infection / 2017-05-09

Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) peuvent causer des infections pulmonaires dévastatrices chez les patients atteints de mucoviscidose. Les bactéries Bcc sont capables de survivre et se multiplier dans les macrophages in vitro. Des études cliniques ont confirmé que ces bactéries opportunistes se localisent au niveau des cellules phagocytaires et ne forment pas des biofilms dans les poumons des patients infectés comme on le croyait généralement. En utilisant le modèle d'infection zebrafish, nous avons établi précédemment que les macrophages constituent un site clé pour la réplication bactérienne et le développement de l'infection aiguë mortelle. Dans la présente étude, nous avons étudié le rôle des stades intracellulaires de B. cenocepacia en développant des nouvelles lignées transgéniques reportrices pour les études d’imagerie en temps réel, nous avons étudié le rôle de l'autophagie in vivo et analysé le profil « transcriptomique » des macrophages lors de l'infection par B. cenocepacia chez le poisson zèbre.En accord avec les études in vitro, nous avons constaté que la protéine « Microtubule-associated protein 1A/1B-light chain 3 » (Lc3), protéine clé dans l’autophagie, a été recrutée au niveau des vacuoles contenant B.cenocepacia K56-2. Nous avons observé en temps réel que les bactéries étaient capables de se répliquer dans ces organelles. Cependant la modulation de l'autophagie par voie génétique et pharmacologique n'a pas changé de manière significative le profil de réplication de B.cenocepacia K56-2 lors de l'infection. En dépit de la charge bactérienne inchangée, une baisse de l’autophagie était reliée avec une augmentation de la mortalité par rapport aux embryons de type sauvage. Ceci suggère une corrélation entre l'autophagie et l'inflammation, et nous proposons que la capacité de B. cenocepacia à arrêter la maturation des (auto) phagosomes et la présence de « cross-talk » entre l’autophagie d’une part et l’inflammasome de l’autre jouent un rôle important dans les réactions inflammatoires observées. Pour approfondir les connaissances sur le rôle des macrophages lors de l'infection aiguë, nous avons déterminé le profil transcriptomique des macrophages isolés à partir d'embryons de poisson zèbre infectés par B. cenocepacia après l'infection. Notre analyse bio-informatique a montré que la plupart des gènes surexprimés sont impliqués dans la signalisation de la réponse immunitaire, tandis que la plupart des gènes sous exprimés sont associés à la transcription et à la traduction. Nous avons confirmé l’activation de l’expression de tnfa dans les macrophages, et nous avons constaté que l'expression des cytokines cxcl8 et Il1b, induite par l'infection, ne dépendait pas de la signalisation par le récepteur TNFa, TNFRSF1A.Nos résultats contribuent à une meilleure compréhension de l'interaction entre B. cenocepacia et les macrophages in vivo, et peuvent contribuer à l’identification de nouvelles cibles pour le développement de thérapies anti-infectieuses pour lutter contre ces bactéries intracellulaires. / Opportunistic bacteria belonging to Burkholderia cepacia complex (Bcc) can cause devastating pulmonary infections in cystic fibrosis patients. These bacteria can survive and replicate in macrophages in vitro. Clinical evidence confirmed that the bacteria localize in phagocytic cells, and do not form biofilms in the lungs of infected patients as generally believed. Using a zebrafish infection model we established previously that macrophages are a critical site for bacterial replication and development of acute fatal infection. In the present study, we further explored the role of the intracellular stages of B. cenocepacia by developing new transgenic reporter lines for real time imaging of subcellular trafficking, studying in detail the role of autophagy in vivo, and performing host transcriptome analysis of FACS-sorted macrophages from zebrafish larvae infected with B. cenocepacia.In agreement with in vitro studies, we found that the autophagy related protein Microtubule-associated protein 1A/1B-light chain 3 (Lc3) was recruited to B. cenocepacia K56-2-containing vacuoles. Although not critical, using real time confocal microscopy, we observed that the bacteria were able to replicate in such organelles. Both genetic and pharmacological modulation of autophagy did not significantly change the replication profile of B. cenocepacia K56-2 during infection. However, reduction in autophagy resulted in more rapid embryo death compared to wild type embryos. This suggests an inverse correlation between autophagy and fatal inflammation, and we hypothesize that the ability of B. cenocepacia to arrest maturation of (auto)phagosomes, and cross talk between autophagy and inflammasome signaling pathways during B. cenocepacia infection play an important role in the observed inflammatory responses. This study further describes the host transcriptome profile of macrophages isolated from infected zebrafish embryos. Our bio-informatics analysis showed that most of the genes up-regulated during infection were involved in immune response signaling, while the major group of down-regulated genes was associated with transcription and translation. We experimentally confirmed rapidly increased expression of tnfa in macrophages, and found that infection-induced expression of the cytokines cxcl8 and il1b did not depend on signalling through the Tnfa receptor, Tnfrsf1a.Our results contribute to a better understanding of the interaction between B. cenocepacia and macrophages in vivo, and the zebrafish may help finding new targets for development of anti-infectious therapies to combat these intracellular bacteria.

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Le complexe Burkholderia cepacia (Bcc) englobe 17 espèces retrouvées dans les infections pulmonaires d'individus atteints de mucoviscidose. Les bactéries de ce complexe sont présentes dans les sols, la rhizosphère de grandes cultures, les eaux usées et peuvent également être rencontrées dans le cadre d'infections nosocomiales. En France, les espèces B. multivorans et B. cenocepacia (Bcen) sont les espèces majoritaires au niveau des infections de patients atteints de mucoviscidose. Divers clones épidémiques ont été décrits au sein de l’espèce Bcen dont le clone ET12 associé au "syndrome cepacia". En 2004, une épidémie nosocomiale impliquant un clone du Bcc est survenue dans un hôpital de l’Ain. Durant ce travail, l’origine de ce clone (B&B), sa classification au sein du Bcc et certains phénomènes génétiques liés à son émergence ont été étudiés. Cela a permis d’identifier ce clone comme appartenant à l’espèce Bcen et une forte proximité de celui-ci avec la lignée ET12. L’étude des facteurs transcriptionnels de la famille σ70 au sein du Bcc a mis en évidence une structure génétique similaire entre la lignée ET12 et ce clone, mais différente de celle observée chez les autres espèces du Bcc. L’analyse d’éléments génétiques répétés de la famille des séquences d’insertion (IS) a cependant permis d’observer une organisation génomique distincte de la lignée ET12. Celle-ci a été reliée à des phénomènes d’instabilité génétique notamment à des phénomènes d’acquisition d’éléments génétiques mobiles de type îlot génomique. L’ensemble de ce travail a permis de caractériser un ensemble de phénomènes génétiques pouvant expliquer l’émergence de clones épidémiques tels que le clone B&B. / The Burkholderia cepacia complex (Bcc) comprises 17 species found in lung infections of individuals with cystic fibrosis. The bacteria of this complex are present in the soil, the rhizosphere of field crops, wastewater and may also be encountered in nosocomial infections. In France, the B. multivorans and B. cenocepacia species are the major species in infections of cystic fibrosis patients. Various epidemic clones have been described within the B. cenocepacia species whose ET12 clone associated with "cepacia syndrome". In 2004, a nosocomial outbreak involving a clone of Bcc occurred in a French hospital. During this outbreak, origin of this clone (B&B clone), its classification within the Bcc and several genetic events associated with its emergence have been studied. These investigations have identified this clone as belonging to the species B. cenocepacia with a strong proximity with the ET12 lineage. The study of transcriptional factors of σ70 family within the Bcc has revealed a similar genetic structure between the ET12 lineage and this clone, but different from that observed in other species of Bcc. Analysis of genetic elements repeated family of insertion sequences (IS), however, allowed to observe a distinct genomic organization of the ET12 lineage. It has been linked to phenomen of genetic instability including acquisition of mobile genetic elements like genomic island (GI). All of this work has helped to characterize a set of genetic events may explain the emergence of epidemic clones such as clone B&B.