Les bactéries du genre Shigella sont responsables de la dysenterie bacillaire, ou shigellose, chez l'être humain, causant plus de 125 millions d'épisodes et 14 000 morts par an. Cette infection est caractérisée par l'inflammation et la destruction de la muqueuse intestinale. La bactérie utilise un système de sécrétion de type III (SST3) pour manipuler la physiologie des cellules épithéliales intestinales et du système immunitaire favorisant l'invasion de la muqueuse et enrayant la mise en place d'une réponse adaptative efficace. Le SST3 peut être comparé à une seringue moléculaire traversant la paroi bactérienne sous la forme d'anneaux membranaires, contenant une tige interne (MxiI), et d’une aiguille extracellulaire (MxiH). L'assemblage de cette dernière se termine par la mise en place d'un complexe d'extrémité formé par plusieurs copies des protéines IpaD et IpaB. Le SST3 prend en charge différentes classes de substrats à sa base via un complexe protéique comprenant l'ATPase Spa47. Les translocateurs (IpaB et IpaC) sont les premiers substrats à être sécrétés. Ceux-ci sont stockés dans le cytoplasme en complexe avec leur chaperon IpgC et sont recrutés à l'extrémité de l'aiguille lors du contact avec la membrane de la cellule hôte pour y former un pore à l'aide de la protéine IpaD. Ce pore permet l'injection des autres substrats du SST3 (effecteurs) qui vont interférer avec les voies de signalisation cellulaire. Il existe deux classes d'effecteurs, les effecteurs précoces (dont OspD1) stockés au préalable dans le cytoplasme et sécrétés suite au contact cellulaire. Ce contact active l’expression des effecteurs tardifs via un couplage assuré par deux complexes, OspD1-MxiE et translocateurs-IpgC. La sécrétion d’OspD1 et des translocateurs libère leurs partenaires qui agissent comme activateurs transcriptionnels. La régulation de la sécrétion dépend de plusieurs acteurs situés dans les différentes parties du SST3. Le complexe d'extrémité et la protéine MxiC contrôlent la sécrétion aux niveaux extra- et intracellulaires alors que l'aiguille transmettrait le signal de sécrétion entre ces deux complexes. Ce paradigme reste cependant encore peu compris et le mode de fonctionnement du complexe d’extrémité et de la protéine MxiC reste à éclaircir.<p>Nos travaux menés sur la protéine IpaD nous ont permis de mettre en évidence un phénotype de sécrétion intermédiaire. Celui-ci est caractérisé par la sécrétion des translocateurs et des effecteurs précoces, sans toutefois observer de sécrétion d’OspD1 et des effecteurs tardifs, suggérant un mécanisme de discrimination entre OspD1 et les effecteurs précoces. Ce phénotype de sécrétion est similaire à celui induit par l’interaction IpaD-désoxycholate. En effet, les variants d’ipaD restant fonctionnels pour la mise en place du pore provoquent également une augmentation de l’insertion des translocateurs et du pouvoir invasif. Nous avons également identifié la région d’IpaD nécessaire au maintien d’IpaB au niveau du complexe d’extrémité ainsi qu’un rôle de son domaine central dans l’insertion du pore. Nous avons enfin étudié l’effet d’anticorps monoclonaux anti-IpaD. Ces résultats nous ont permis de proposer un modèle de fonctionnement du complexe d’extrémité lors de l’insertion du pore, d’identifier les épitopes conférant une protection in vitro et in vivo ainsi que l’existence d’un polymorphisme qui empêche la liaison de ces anticorps à IpaD provenant d’autres sérotypes.<p>Notre étude sur MxiC a mis en évidence de nouveaux partenaires d’interaction (MxiI et IpgC). Ces résultats montrent que l’interaction MxiC-MxiI est nécessaire pour la régulation de la sécrétion des effecteurs précoces par MxiC. De même, la mutation mxiIQ67A provoque un phénotype similaire à la mutation mxiHK69A, ce qui suggère que le mécanisme de régulation impliquant l’aiguille est similaire pour la tige interne. Enfin, l’interaction renforcée MxiC-Spa47, via IpgC probablement couplée à un translocateur, apporte des pistes quant au rôle de MxiC dans la sécrétion des translocateurs.<p>Les rôles identifiés pour les différents régulateurs de la sécrétion ouvrent de nouvelles pistes pour la compréhension du fonctionnement du SST3. Leurs modes de fonctionnement restent cependant encore flous et nécessitent des études complémentaires.<p><p><p>Shigella are responsible for bacillary dysentery, or shigellosis, in human beings causing over 125 million episodes and 14 000 deaths per year. This infection is characterized by inflammation and destruction of the intestinal mucosa. The bacteria use a type III secretion system (T3SS) to manipulate the physiology of intestinal epithelial cells and the immune system favoring the invasion of the mucosa and halting the development of an efficient adaptive response. The T3SS can be compared to a molecular syringe that extends from the bacterial cell wall which contains an internal rod (MxiI), and an extracellular needle (MxiH). The assembly of the latter ends with assembly of a tip complex formed by multiple copies of IpaB and IpaD proteins. The T3SS recruits different classes of substrates at its base via a complex comprising the Spa47 ATPase. The translocators (IpaB and IpaC) are the first substrates to be secreted. They are stored in the cytoplasm in complex with their chaperone (IpgC) and are recruited at the needle tip upon contact with host cell membrane to form a pore via IpaD. This pore allows the injection of other substrates of the T3SS (effectors), which will interfere with the cellular signaling pathways. There are two classes of effectors, early effector (including OspD1) stored in the cytoplasm and secreted upon cell contact. This contact activates the expression of late effectors genes through a complex formed by MxiE (blocked by OspD1) and IpgC. Both proteins are released through OspD1 and translocators secretion. Secretion regulation depends on several actors located at different parts of the T3SS. The tip complex and the gatekeeper MxiC regulate secretion at the T3SS tip and base, the needle subunits transmitting a secretion signal between these two complexes. This paradigm, however, is still poorly understood and the operating mode of the tip complex and MxiC remains unclear.<p><p>Our work on IpaD protein allowed us to identify an intermediate secretion phenotype which is characterized by the secretion of translocators and early effector, but no secretion of OspD1 and late effectors, suggesting a discriminating mechanism between early effectors and OspD1. This secretion phenotype is similar to that induced by deoxycholate-IpaD interaction. Indeed, IpaD point mutants responsible for this phenotype cause an increase in the pore insertion and cell invasion. We also identified the region of IpaD necessary to maintain IpaB at the needle tip as well as a role of IpaD central domain in the pore insertion. We finally studied the effect of anti-IpaD monoclonal antibodies. These results allowed us to propose a working model of the tip complex end upon pore insertion, identify epitopes conferring protection in vitro and in vivo as well as the existence of a polymorphism that prevents the binding of these antibodies to IpaD from other serotypes.<p><p>Our MxiC study showed new interaction partners (MxiI and IpgC). These results showed that the MxiC-MxiI interaction is necessary for the regulation of early effectors secretion of by MxiC. Moreover, a mxiIQ67A mutation causes a phenotype similar to the mutation mxiHK69A, suggesting that the regulatory mechanism involving the needle is shared by the inner rod. Finally, the enhanced interaction MxiC-Spa47 through IpgC, probably in complex with a translocator, provides clues for the role of MxiC in translocators secretion.<p><p>The roles identified for the various regulators of secretion open up new avenues for understanding how the T3SS functions. Their ways of working are however still unclear and require further study.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

A Transferência Horizontal de Genes (THG) é um dos principais mecanismos de evolução bacterianos, impactando a evolução de praticamente todas famílias gênicas. Neste trabalho identificamos e avaliamos padrões de possíveis transferências horizontais de genes pertencentes a duas classes funcionais de dois níveis taxonômicos distintos. Caracterizamos a ocorrência e evolução de 45 genes importantes para a fixação de N2 em 479 genomas de Proteobacteria. Identificamos cinco potenciais aquisições de genes ligados a fixação de N2 por linhagens de Proteobacteria, as quais foram identificadas consistentemente em 36 dos genes analisados. Realizamos predições de transferências horizontais dos 45 entre todos os 479 genomas de Proteobacteria e identificamos possíveis enriquecimentos de THG, provavelmente ligados à sinais filogenéticos e ecológicos. Desenvolvemos um pipeline para identificação semi-automática de efetores do Sistema Secretor do Tipo III em Aeromonas, o qual reportou 21 famílias de potenciais efetores presentes em 105 genomas. Entre os 21 efetores identificados 17 foram descritos pela 1º vez em Aeromonas, corroborando a sensibilidade de nosso pipeline. Com o auxílio de nossos colaboradores foram realizados testes de citotoxidade para efetores identificados in silico, e apenas quatro não inibiram o crescimento de Saccharomyces cerevisiae. Por fim, desenvolvemos um método para agrupamento de famílias gênicas com histórias evolutivas similares que não requer a reconstrução de árvores filogenéticas, aumentando a eficiência computacional. Aplicamos o método desenvolvido para reconstrução da filogenia de Aeromonas, o qual mostrou-se compatível com dados presentes na literatura. / Horizontal Gene Transfer (HGT) is one of main mechanisms of bacterial evolution, affecting virtually all gene families. In this document we identified and assessed putative horizontal transfers of genes from two functional classes from two distinct taxonomic levels. We characterized the distribution and evolution of 45 genes important to N2 fixation among 479 Proteobacteria genomes. We identified five potential distinct acquisitions of such genes by Proteobacteria lineages. The distinct origins are consistently identified in 36 out of the 45 assessed genes. We computed possible horizontal transfers of the 45 genes among the 479 Proteobacteria genomes, and we identified enrichments of HGT, likely related to phylogenetic and ecological signals. We developed a semi-automated pipeline to identify effectors of the Type III Secretion System within Aeromonas, which reported 21 putative effector families distributed among 105 genomes. Among the 21 likely effectors 17 have been described in Aeromonas for the first time, highlighting the sensibility of our pipeline. Our colaborators performed cytotoxicity tests for the 21 likely effector families identified by in silico analysis, and only four did not inhibited Saccharomyces cerevisiae growth. Lastly, we developed a method to cluster gene families according to shared evolutionary history, without the requirement of phylogenetic tree reconstruction, increasing computational efficiency. We applied this proposed method during Aeromonas phylogenetic reconstruction, and it showed up compatible with data available on the literature.

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

<p> The plant cytoskeleton is commonly considered a vital component of cell growth and development; however, it also plays a critical role in plant immunity. During plant immunity, the cytoskeleton orchestrates rapid and precise immune-associated processes. For instance, the cytoskeleton mobilizes and orients the movement of organelles, proteins, and chemical signaling. To counter plant immunity, bacterial pathogens deliver virulence proteins, known as T3Es (type III effectors), into plant cells through a needle-like apparatus called the type III secretion system (T3SS). A novel T3E, called RipU, interacts with the cytoskeleton. Data has shown that RipU co-localizes with cytoskeletal markers in tobacco leaves. Ectopic expression of RipU can suppress PTI responses like ROS bursts or seedling growth inhibition. Tomato plants inoculated with <em>Rs</em> K60 lacking RipU showed less wilting and root colonization, suggesting that RipU plays a role in pathogenesis and virulence. Furthermore, inducible expression of RipU in Arabidopsis dramatically alters plant development. These plants have wavy roots, branching root hairs, and underdeveloped true leaves. Our results suggest that by targeting the cytoskeleton, RipU contributes to <em>Rs</em> K60s pathogenicity and virulence. </p>

The Type III Secretion (T3S) system is a system utilized by many pathogenic bacteria to inject proteins into host cells during an infection. Effector proteins enter the host cell by passing through the proteinaceous T3S translocon, which forms a pore on the host cell membrane. Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the T3S system, and very little is known about how the P. aeruginosa translocon forms. The proteins PopB and PopD are believed to assemble into the P. aeruginosa translocon. A pore-forming heterocomplex of PopB and PopD has been reconstituted in model membranes, however this heterocomplex has not been assessed in its relation to the translocon formed on the host cell. The interaction of this heterocomplex with other T3S system components was measured to determine if this complex acts similarly to the translocon. Initial assays that can be used to compare the molecular weight of the translocon isolated from eukaryotic cells after P. aeruginosa contact to the calculated molecular weight of the heterocomplex were developed as well. This study provides insight into how the PopB:PopD heterocomplex formed in model membranes relates to the translocon formed during a P. aeruginosa infection.

No description available.

No description available.

O músculo cricofaríngeo desempenha um importante papel na deglutição. Acredita-se que seu comportamento elástico seja dependente não apenas do componente muscular, mas também do tecido conectivo intramuscular. O objetivo desse estudo foi analisar a presença e a distribuição do colágeno total, colágenos tipo I e III, fibras elásticas, fibronectina e versican no endomísio do músculo cricofaríngeo em cadáveres de diferentes idades. Vinte e sete músculos foram obtidos mediante autópsia de indivíduos de ambos os sexos com idades entre 28 e 92 anos. Foram realizadas colorações histoquímicas, Método Picrossírius e Método Resorcina-fuccina com oxidação prévia pela oxona, e imunoistoquímicas, para colágeno tipo I, tipo III, fibronectina e versican. A medida dos elementos estudados foi feita por meio de um sistema de análise de imagens que incluía um microscópio, conectado a um computador por meio de uma câmera de vídeo. Foi utilizado o software Image pro Plus, versão 4.1. Para cada caso, quinze imagens não sobrepostas de cada coloração no aumento de 400x foram analisadas. A área de marcação positiva dentro do endomísio do músculo cricofaríngeo foi determinada por um padrão de cor específico para cada coloração. A área de cada elemento da matriz extracelular foi expressa como porcentagem da área total do estudada. Os dados foram expressos em medianas e intervalos interquartílicos. A correlação entre idade e os diferentes elementos da matriz extracelular foi realizada por meio da correlação de Spearman. O teste de Mann-Whitney para distribuição não paramétrica foi utilizado para comparar as áreas porcentuais e os indivíduos de diferente sexo. Todos os testes foram realizados pelo software SPPS versão 13.0 e foi admitido um calor de significância com p < 0,05. O colágeno foi o elemento mais abundante dentre os estudados. Encontrou-se fibras elásticas longitudinais à fibra muscular, finas fibras transversais entre as fibras musculares e espessamento as fibras elásticas nos pólos da fibra muscular. Foi encontrada uma grande variação no conteúdo de fibronectina e versican entre os casos. Não foi evidenciada diferença estatisticamente significativa entre os elementos estudados, o sexo e a idade. Os resultados sugerem que a presença e distribuição desses elementos são importantes para a função do músculo cricofaríngeo e para a manutenção da homeostase. A porcentagem de colágeno encontrada é condizente com a característica esfinctérica do músculo e o arranjo de fibras elásticas contribui para o comportamento elástico e a capacidade de rapidamente reassumir a posição tônica após a abertura durante as deglutições. As variações da quantidade de fibronectina e versican, podem ser resultante da susceptibilidade a fatores agressores. A ausência de alterações com a idade pode significar que o músculo não esteja sujeito às mesmas alterações decorrentes da idade que outros músculos esqueléticos / The cricopharyngeus muscle is thought to play an important role in swallowing and related activities. Its elastic behavior is likely to depend not only on its muscular components, but also on the intramuscular connective tissue. Our objective is to analyze the presence and distribution of total collagen, type I and III collagen, elastic fibers, fibronectin and versican in cricopharyngeus muscle endomysium in adults of a wide age range. Twenty-seven cricopharyngeus muscles obtained from male and female cadavers (age range, 28-92 years-old) were analyzed with the Picrosirius method, oxidized Weigert resorcin-fuchisin, immunohistochemistry. Quantification of stained areas in the cricopharyngeus endomysium with different techniques was performed by an image analysis system connected to a light microscope. The correlation between age and the density of different extracellular matrix proteins was tested using Spearman test. T-tests for independent samples were used to analyze the influence of gender and smoking habit on the fractional areas of extracellular matrix. Collagens had the highest density among the analyzed components. Elastic fibers surrounded each muscle cell, longitudinal to their long axis, associated to traversing fibers, forming a fiber network embedding muscle cells. There was a wide variation on fibronectin and versican content among cases. There were no statistical significance for analysis made between those components of extracellular matrix and age andgender. Our findings suggest that presence and distribution of these extracellular matrix components are important to cricopharyngeus muscle homeostasis. The elastic fibers arrangement can contribute for the cricopharyngeus muscle elastic behavior and ability to rapidly reassume its tonic position after opening during swallows. Variations in the expression of fibronectin and versican can beresultant of its injury susceptibility. The absence of changes on extracellular components during aging could mean that cricopharyngeus muscle is not susceptible to similar age changes as other skeletal muscles

A sepse envolve mecanismos complexos de respostas imunológicas e inflamatórias, e o papel do NF- B é essencial. A diminuição da NO sintase endotelial (eNOS) durante a sepse contribui com a disfunção endotelial. A eritropoietina (EPO) é uma citocina protetora de diversos tecidos durante o estresse. Investigamos o papel da EPO na injúria renal aguda (IRA) induzida pela sepse usando o modelo de ligadura e punção do ceco (LPC). Ratos Wistar foram divididos em três grupos: controle; LPC e LPC+EPO (EPO, 4.000UI/kg, administrada 24h e 1h antes da cirurgia). Com a finalidade de estudar os efeitos precoces e tardios da EPO sobre a IRA induzida pela sepse realizamos três etapas de experimentos: Primeira etapa: 24 horas após LPC; Segunda etapa: 48 horas após LPC; Terceira etapa: análise de sobrevida. No estudo precoce o grupo LPC+EPO apresentou clearance de inulina significativamente maior que o grupo LPC. Recuperou os níveis de hematócrito na sepse, melhorou a pressão arterial e a acidose metabólica. No estudo tardio o grupo LPC+EPO apresentou clearance de creatinina significativamente maior que o grupo LPC. Nesta fase tardia a EPO recuperou os níveis de eNOS, suprimiu a infiltração de macrófagos no tecido renal e inibiu a ativação do NF- B. A EPO protege a função renal e aumenta a sobrevida neste modelo de sepse. A proteção da EPO na sepse é dependente, em parte, da inibição do NF- B e do aumento da expressão de eNOS / The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which nuclear factor-kappa B (NF- B ) activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury (AKI) using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three groups: control (sham-operated); CLP-only; and CLP+EPO. The EPO (4000 IU/kg BW, i.p.) was administered 24 h and 1 h before CLP. To study the early and late effects of EPO on sepsis-induced AKI, we performed experiments at 24 h and 48 h after CLP/sham operation, and we plotted the survival curves. At post-procedure hour 24, CLP+EPO rats presented significantly higher inulin clearance than did CLP-only rats; EPO treatment restored hematocrit levels, as well as mean arterial pressure and metabolic balance. At post-procedure hour 48, CLP+EPO rats presented significantly higher creatinine clearance than did CLP-only rats; EPO treatment restored eNOS levels, suppressed macrophage infiltration, and inhibited NF-B activation,thereby increasing survival. In conclusion, EPO protects renal function and increases survival in this model of sepsis-induced AKI. This protection is dependent on eNOS activation and is partly due to inhibition of the inflammatory response via downregulation of NF- B