La legionelosis es una enfermedad respiratoria con origen en sistemas que formen aerosol y que contenga el agente biológico Legionella sp. En las últimas décadas se ha desarrollado en España un marco normativo para su prevención y control. El presente artículo expone la epidemiología de la legionelosis y la importancia del control de la transmisión de la bacteria en la lucha contra la enfermedad. Para ello, se hace patente la revisión de las instalaciones críticas y la inclusión de otras nuevas en la legislación preventiva así como la estimación del riesgo, la mejora en los procesos de diagnóstico y el avance en nuevos protocolos de prevención. / Legionellosis is a respiratory disease originating in systems that produce aerosol and contain Legionella sp. In recent decades, Spain has developed a regulatory framework for prevention and control of legionellosis. This article describes the epidemiology of legionellosis and the importance of controlling the transmission of bacteria in the fight against the disease. In that regard, it becomes clear the role of reviewing critical facilities and the inclusion of new ones in the preventive legislation, the estimation of risk, and the improvement in the diagnostic processes and progress in new prevention protocols.

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.

Legionella pneumophila, o agente etiológico da doença dos Legionários, é conhecida por desencadear a formação de poro em membranas de macrófagos derivados de medula óssea (BMMs) por mecanismos dependentes do sistema de secreção do tipo IV conhecido como Dot/Icm. Neste trabalho, foram utillizados vários mutantes de L. pneumophila em combinação com camundongos nocautes para investigar os fatores bacterianos e do hospedeiro envolvidos na formação de poro em BMMs. Observamos que apesar da atividade do Dot/Icm, a formação de poro não ocorre em BMMs deficientes para caspase-1 e Nlrc4. A formação de poro foi temporalmente associada com a secreção de IL-1b e precedeu a lise celular e a piroptose. A formação de poro foi dependente do Dot/Icm, mas independente de várias proteínas efetoras, da multiplicação bacteriana e da síntese de novo de proteínas. A flagelina, a qual é conhecida em ativar o inflamassoma de Nlrc4, foi necessária para a formação de poro; a bactéria mutante flaA falhou em induzir a permeabilização celular. Consequentemente, a transfecção da flagelina purificada foi suficiente para desencadear a formação de poro independente da infecção. Utilizando 11 diferentes espécies de Legionella, nós observamos alta formação de poro em resposta à L. micdadei, L. bozemanii, L. gratiana, L. jordanis e L. rubrilucens, e essa resposta estava correlacionada com a expressão de flagelina por essas espécies. Além disso, verificamos que as proteínas Asc e Caspase-11 apresentam fenótipo intermediário na formação de poro, sugerindo que outras vias podem estar envolvidas no processo. Observamos também que a formação de poro desencadeada por L. pneumophila difere daquela induzida pelo ATP. Em conjunto, nossos resultados sugerem que a formação de poro não é uma resposta específica de L. pneumophila nem o resultado de dano da membrana induzido pelo Dot/Icm. Ao invés disso, a formação de poro é uma resposta do hospedeiro altamente coordenada, dependente dos componentes do inflamassoma Nlrc4 e caspase-1 e é desencadeada em resposta a bactérias que expressam o sistema de secreção do tipo IV e flagelina. / Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with IL-1b secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis and L. rubrilucens, and this trait correlated with flagellin expression by these species. Furthermore, we found that Asc and Caspase-11 showed an intermediate phenotype in pore formation, suggesting that other pathways may be involved in this process. We also observed that the pore formation triggered by L. pneumophila differs from the pore induced by ATP. Together, the results suggest that pore formation is neither L. pneumophilaspecific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

Legionella pneumophila (Lp) est une bactérie ubiquitaire dans les environnements aqueux et responsable d’une pneumopathie potentiellement sévère : la légionellose. La majorité des souches impliquées appartiennent au sérogroupe 1 (Lp1) et à un sous- groupe spécifique de souches portant un épitope particulier dites mAb3/1+. Cependant, la différence de distribution entre les souches retrouvées dans l’environnement et celles impliquées en clinique n’est pas clairement élucidée. Notre travail a porté sur la détection de deux facteurs de virulence de Lp. Nous avons voulu mettre en évidence l’existence de Phenols Soluble Modulines (PSMs), peptides uniquement décrit chez Staphylocoques et avons ainsi pu démontrer l’activité de peptides prédits par analyse in silico chez Lp capables d’activer la réponse inflammatoire par la voie du NF-?B et sont dotés d’une action cytotoxique. Notre deuxième axe d’étude a porté sur le lipopolysaccharide (LPS) de Lp. Afin de vérifier si la prédominance de certaines souches était liée à un biais diagnostique, nous avons voulu tout d’abord vérifier la sensibilité de 3 tests urinaires diagnostiques envers le LPS extrait de souches de différents sous- groupes de Lp1 et sérogroupes de Lp et avons ainsi pu montrer que ces tests sont capables de détecter tous les LPS de Lp1. La sensibilité envers le LPS des autres sérogroupes est très variable mais reste insuffisante pour permettre leur détection. Nous avons ensuite utilisé ces LPS extraits pour vérifier la réponse immunitaire innée en fonction des souches de Lp1. Ainsi les souches mAb3/1+ activent moins le système immunitaire que les souches mAb3/1-, ce qui pourrait expliquer alors une moins bonne clairance de ces souches permettant leur multiplication à l’origine d’une infection. Au final, notre travail a permis d’étudier deux facteurs de virulence potentiels au sein de Lp, pouvant expliquer partiellement la prédominance de certaines souches en pathologie humaine / Legionella pneumophila (Lp) is a ubiquitous intracellular bacterium found widely in the environment and is the cause of an opportunistic infection named legionellosis. The majority of the strains involved belong to serogroup 1 (Lp1) and to a specific subgroup named mAb3/1+, linked to a specific epitope expressed at the cell membrane. However the distribution difference between the strains found in the environment and the ones involved in pathology is not fully understood. We here studied two virulence factors of Lp. We first demonstrated the existence of Phenols Soluble Modulines (PSMs), smalls peptides that only have been described for Staphylococcus and found that the peptides that were predicted for Lp by in silico analysis were able to activate the innate immune response by NF-?B pathway and were able to have a cytotoxic activity. We also studied the lipopolysaccharide (LPS) of Lp. To found out if the predominance of some strains was linked to a diagnosis biais, we first evaluated the sensitivity of 3 urinary antigens tests against extracted LPS of strains belonging to all the sous-groups of Lp1 and serogroups of Lp. We then demonstrated that those tests are able to detect all LPS of Lp1, independently of mAb3/1 character. The sensitivities of the 3 tests were very variable for the other serogroups of Lp, but were too low to be able to detect those LPS in practice. We then used these extracted LPS to evaluate the innate immune response for different strains of Lp1. We demonstrated that mAb3/1- strains needed lower dose of LPS to activate the innate immune response than mAb3/1+ strains, which could be linked to a better clearance of the bacteria from the host, which doesn’t develop an infection. This work has studied two potentially virulent factors of Lp, which could partially explain the predominance of some strains of Lp in human pathology

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Legionella pneumophila est une bactérie à Gram négatif, intracellulaire facultative, responsable de la légionellose (ou maladie des Légionnaires) chez l'Homme. Les fluoroquinolones et les macrolides sont utilisés en première intention dans le traitement antibiotique de cette maladie. Cependant, les échecs thérapeutiques sont fréquents, et le taux de mortalité demeure élevé (10-15% des cas, plus de 30% chez le patient immunodéprimé). Bien qu'aucune souche de L. pneumophila résistante à ces antibiotiques n'ait été isolée à ce jour, ces échecs peuvent faire évoquer la possibilité d'une sélection in vivo de mutants résistants. Le mécanisme génétique principal d'acquisition de la résistance aux fluoroquinolones correspond à l'accumulation de mutations au niveau des gènes codant pour l'ADN gyrase et la topoisomérase IV ; en particulier celles affectant les codons en positions 83 et 87 du QRDR (quinolone resistance determining region) du gène gyrA entrainent une résistance de haut niveau à ces antibiotiques. Le première aspect de notre projet était d'élaborer un test de PCR en temps réel permettant de détecter chez L. pneumophila des mutants gyrA résistants aux fluoroquinolones et de les différencier des souches sauvages par analyse des températures de fusion des amplifias obtenus. Après optimisation, ce test nommé qPCRgyrALp amplifie spécifiquement une portion du QRDR du gène gyrA de l'espèce L. pneumophila et permet de détecter et de différencier les mutations gyrA83 et gyrA87. Nous avons ensuite utilisé ce test pour la recherche de mutants gyrA directement dans divers prélèvements respiratoires provenant de 82 patients atteints de légionellose, certains en échec thérapeutique après traitement par une fluoroquinolone. Les résultats ont montré pour quatre patients un profil de courbe de fusion semblable à celui du mutant gyrA83. Le séquençage du QRDR de gyrA à partir de ces prélèvements respiratoires a confirmé cette mutation chez deux patients. L'utilisation de la technique de séquençage à haut débit a permis de quantifier ces mutants gyrA83 chez ces deux patients, permettant de montrer un remplacement progressif in vivo de la population de L. pneumophila sensible aux fluoroquinolones par une population résistante à ces antibiotiques. Le deuxième aspect de notre travail a été de développer des tests de PCR quantitative en temps réel (qPCR) permettant de quantifier la charge bactérienne à L. pneumophila dans les prélèvements cliniques des patients infectés, avant et au cours du traitement antibiotique, dans la but de prédire l'évolution clinique et le pronostic final de ces patients. Nous avons utilisé deux tests de qPCR, ciblant soit le gène codant pour l'ARNr16s (qPCR16S) soit le gène mip (qPCRmip) dans des prélèvements respiratoires de 116 patients atteints de légionellose. Chez certains patients, nous avons pu déterminer la cinétique de la charge bactérienne au cours du temps, alors que les patients recevaient une antibiothérapie adaptée. Les premières cinétiques recueillies montrent la possibilité de différencier les patients qui répondent rapidement au traitement antibiotique et évoluent favorablement au cours de la 1ère semaine d'hospitalisation, de ceux qui présentent une réponse modeste au traitement et nécessitent une hospitalisation prolongée, voire décèdent. La PCR en temps réel semble donc représenter un outil pronostique d'intérêt au cours de la légionellose. Le type de cinétique observé chez un patient donné semble pouvoir prédire l'évolution des patients et la nécessité d'ajuster le traitement antibiotique. / Legionella pneumophila is a Gram- negative, facultative intracellular bacterium responsible for legionellosis (or Legionnaires' disease ) in humans. The fluoroquinolones and the macrolides are used as first-line antibiotic treatment of this disease. However, treatment failures are common, and the mortality rates remain high (10-15 % of cases, more than 30% in immunocompromised patients). Although L. pneumophila strain resistant to these antibiotics have never been isolated, treatment failures may suggest the possibility of in vivo selection of resistant mutants. The main genetic mechanisms associated with acquired resistance to fluoroquinolones correspond to the accumulation of mutations in the genes encoding DNA gyrase and topoisomerase IV, especially those affecting codons 83 and 87 of the QRDR (quinolone resistance determining region) of the gyrA gene, which are associated with high level resistance to these antibiotics. The first aspect of our project was to develop a real-time PCR test to detect gyrA QRDR mutants and differentiate them from wild-type strains of L. pneumophila by analysis of melting temperatures of the amplified DNA. After optimization, the qPCRgyrALp test specifically amplified a portion of the gyrA QRDR of L. pneumophila and could detect and differentiate gyrA83 and gyrA87 mutations. Then, we checked the presence of gyrA mutants directly in respiratory samples collected in 82 legionellosis patients, including some after treatment failure with a fluoroquinolone. For four patients, results corresponded to a melting curve profile similar to that of the gyrA83 mutant. Amplification and sequencing of the gyrA QRDR directly from these respiratory samples confirmed this mutation in two patients. The use of high-throughput sequencing technology allowed us to quantify the gyrA83 mutants in these two patients, allowing demonstration of in vivo gradual replacement of the fluoroquinolones susceptible population of L. pneumophila by a resistant one. The second aspect of our work was to develop quantitative real-time PCR tests offering the possibility to quantify the L. pneumophila bacterial load in respiratory specimens before and during antibiotic treatment, in order to predict the clinical course and the final prognosis of these patients. We used two qPCR tests, either targeting the gene encoding 16S rRNA (qPCR16S ) or the mip gene (qPCRmip ) in respiratory samples from 116 patients with Legionnaires' disease. In some patients, we determined the kinetics of bacterial loads over time, while patients received appropriate antibiotic therapy. The kinetics we observed allowed differentiation of patients who respond quickly to antibiotic treatment and were released from hospital within the first week following admission, from those with a modest response to treatment and requiring prolonged hospitalization or finally died. Thus, our real-time PCR tests seem to be good prognostic tools for evaluation of legionellosis prognosis. The type of kinetics observed in a given patient may allow the clinician to predict the evolution of patients and the need to adjust the antibiotic treatment.

Legionella pneumophila, o agente etiológico da doença dos Legionários, é conhecida por desencadear a formação de poro em membranas de macrófagos derivados de medula óssea (BMMs) por mecanismos dependentes do sistema de secreção do tipo IV conhecido como Dot/Icm. Neste trabalho, foram utillizados vários mutantes de L. pneumophila em combinação com camundongos nocautes para investigar os fatores bacterianos e do hospedeiro envolvidos na formação de poro em BMMs. Observamos que apesar da atividade do Dot/Icm, a formação de poro não ocorre em BMMs deficientes para caspase-1 e Nlrc4. A formação de poro foi temporalmente associada com a secreção de IL-1b e precedeu a lise celular e a piroptose. A formação de poro foi dependente do Dot/Icm, mas independente de várias proteínas efetoras, da multiplicação bacteriana e da síntese de novo de proteínas. A flagelina, a qual é conhecida em ativar o inflamassoma de Nlrc4, foi necessária para a formação de poro; a bactéria mutante flaA falhou em induzir a permeabilização celular. Consequentemente, a transfecção da flagelina purificada foi suficiente para desencadear a formação de poro independente da infecção. Utilizando 11 diferentes espécies de Legionella, nós observamos alta formação de poro em resposta à L. micdadei, L. bozemanii, L. gratiana, L. jordanis e L. rubrilucens, e essa resposta estava correlacionada com a expressão de flagelina por essas espécies. Além disso, verificamos que as proteínas Asc e Caspase-11 apresentam fenótipo intermediário na formação de poro, sugerindo que outras vias podem estar envolvidas no processo. Observamos também que a formação de poro desencadeada por L. pneumophila difere daquela induzida pelo ATP. Em conjunto, nossos resultados sugerem que a formação de poro não é uma resposta específica de L. pneumophila nem o resultado de dano da membrana induzido pelo Dot/Icm. Ao invés disso, a formação de poro é uma resposta do hospedeiro altamente coordenada, dependente dos componentes do inflamassoma Nlrc4 e caspase-1 e é desencadeada em resposta a bactérias que expressam o sistema de secreção do tipo IV e flagelina. / Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with IL-1b secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis and L. rubrilucens, and this trait correlated with flagellin expression by these species. Furthermore, we found that Asc and Caspase-11 showed an intermediate phenotype in pore formation, suggesting that other pathways may be involved in this process. We also observed that the pore formation triggered by L. pneumophila differs from the pore induced by ATP. Together, the results suggest that pore formation is neither L. pneumophilaspecific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.

L'objectif global de ma thèse a été d'étudier un mécanisme cellulaire qui intervient dans la traduction des protéines : la trans-traduction. Nous avons étudié le rôle de ce mécanisme chez notre modèle d'étude, la bactérie pathogène de l'homme Legionella pneumophila, l'agent étiologique de la Légionellose. Ce travail a été réalisé sous forme de deux axes majeurs. D'abord, nous avons démontré l'essentialité de ce mécanisme pour la viabilité de cette bactérie et pour sa capacité à se multiplier intracellulairement dans des cellules eucaryotes. Puis nous avons évalué l'efficacité d'un nouvel antibiotique décrit comme étant un inhibiteur de la trans-traduction, pour le traitement de la Légionellose. Ces deux axes ont été complétés par un troisième axe qui visait la mise en place de la technique de Tn-seq chez L. pneumophila et l'archée Pyrococcus furiosus. Cet axe a permis l'ouverture de mes travaux à la recherche d'autres mécanismes essentiels dont nous ignorons le potentiel comme cible antibiotique, et à l'étude des mécanismes de transfert de gène horizontaux / The global objective of my thesis work was the study of a cellular mechanism involved in protein translation: trans-translation. We studied the role of that mechanism in a model organism, the human bacterial pathogen Legionella pneumophila that causes Legionnaire's disease. This work was performed under two principal axes. First, we demonstrated the essentiality of this mechanism for the growth in vitro and the intracellular multiplication of this bacterium in eukaryotic cells. Then, we assessed the efficiency of a new antibiotic compound described as an inhibitor of trans-translation against the etiologic agents of Legionnaire's disease. These two axes were then completed by a third axis, which aimed at implementing the Tn-seq technique in L. pneumophila and the archaea Pyrococcus furiosus. This approach allowed to open my work to the reseach of other essential mechanisms that could be used as antibiotic targets, and to the study of a mechanism of horizontal gene transfer

Les circuits de refroidissement des centrales nucléaires en bord de rivière sélectionnent par leur mode de fonctionnement des micro-organismes à caractère thermophile, parmi lesquels le micro-organisme pathogène, Legionella pneumophila. Pour lutter contre le développement de ce genre bactérien, un traitement de désinfection de l’eau des circuits de refroidissement à la monochloramine peut être employé. Pour participer à la maitrise des risques sanitaires et environnementaux liés à la modification physico-chimique et microbiologique de l’eau naturelle prélevée, EDF s’est engagé dans une démarche d’amélioration des connaissances sur l’écologie de Legionella pneumophila dans les circuits de refroidissement et des liens que cette espèce bactérienne entretient avec son environnement (physico-chimique et microbiologique) favorisant ou non sa prolifération. Ainsi, la diversité et la dynamique des Legionella pneumophila cultivables ont été déterminées dans les quatre centrales nucléaires en bord de Loire pendant un an et leurs liens avec leur environnement physico-chimique et microbiologique ont été étudiés. Cette étude a mis en évidence une forte diversité des sous-populations de Legionella pneumophila et une apparente dynamique qui semble être liée à l’évolution d’un nombre restreint de sous-populations. Les sous-populations de Legionella pneumophila semblent entretenir des relations souche-spécifiques avec les paramètres biotiques et présenter des sensibilités différentes aux variations physico-chimiques du milieu. La conception des circuits de refroidissement pourrait impacter la communauté de légionelles. L’utilisation de la monochloramine perturbe fortement l’écosystème mais ne sélectionne pas de populations tolérantes au biocide. / The cooling circuits of nuclear power plants, by their mode of operating, can select thermophilic microorganisms including the pathogenic organism Legionella pneumophila. To control the development of this species, a disinfection treatment of water cooling systems with monochloramine can be used. To participate in the management of health and environmental risks associated with the physico-chemical and microbiological modification of water collected from the river, EDF is committed to a process of increasing knowledge about the ecology of Legionella pneumophila in cooling circuits and its links with its environment (physical, chemical and microbiological) supporting or not their proliferation. Thus, diversity and dynamics of culturable Legionella pneumophila were determined in the four nuclear power plants along the Loire for a year and their links with physico-chemical and microbiological parameters were studied. This study revealed a high diversity of Legionella pneumophila subpopulations and their dynamic seems to be related to the evolution of a small number of subpopulations. Legionella subpopulations seem to maintain strain-specific relationships with biotic parameters and present different sensitivities to physico-chemical variations. The design of cooling circuits could impact the Legionella pneumophila community. The use of monochloramine severely disrupts the ecosystem but does not select biocide tolerant subpopulations.