Background It is debated whether a low total diversity of the gut microbiota in early childhood is more important than an altered prevalence of particular bacterial species for the increasing incidence of allergic disease. The advent of powerful, cultivation-free molecular methods makes it possible to characterize the total microbiome down to the genus level in large cohorts. Objective We sought to assess microbial diversity and characterize the dominant bacteria in stool during the first year of life in relation to atopic eczema development. Methods Microbial diversity and composition were analyzed with barcoded 16S rDNA 454-pyrosequencing in stool samples at 1 week, 1 month, and 12 months of age in 20 infants with IgE-associated eczema and 20 infants without any allergic manifestation until 2 years of age (ClinicalTrials.gov ID NCT01285830). Results Infants with IgE-associated eczema had a lower diversity of the total microbiota at 1 month (P = .004) and a lower diversity of the bacterial phylum Bacteroidetes and the genus Bacteroides at 1 month (P = .02 and P = .01) and the phylum Proteobacteria at 12 months of age (P = .02). The microbiota was less uniform at 1 month than at 12 months of age, with a high interindividual variability. At 12 months, when the microbiota had stabilized, Proteobacteria, comprising gram-negative organisms, were more abundant in infants without allergic manifestation (Empirical Analysis of Digital Gene Expression in R [edgeR] test: P = .008, q = 0.02). Conclusion Low intestinal microbial diversity during the first month of life was associated with subsequent atopic eczema. / <p>Funding Agencies|BioGaia AB, Stockholm, Sweden||Ekhaga Foundation, the Heart and Lung foundation||Research Council for the South-East Sweden|F2000-106|Olle Engqvist Foundation||Swedish Asthma and Allergy Association||Swedish Research Council||University Hospital of Linkoping||Soderberg Foundation||Vardal Foundation for Health Care Science and Allergy Research, Sweden||BioGaia AB||</p>

Secretion is a fundamental process of bacterial microorganisms. It is responsible for diverse functions such as cell-to-cell communication, nutritional up-take, environmental adaptation, physiological responses, and evasion of the immune system of a host. To accomplish the task of secretion, bacteria have evolved multi-protein complexes, known as secretion apparatuses, which span the bacterial membranes serving as a conduit between the interior of bacteria and the extracellular milieu. Francisella tularensis is a Gram negative bacterium capable of growth inside macrophages. Francisella tularensis causes a rare but severe disease known as tularemia. The Francisella pathogenicity island (FPI) is a circa 30-kb genetic region that harbours genes of unknown function implicated in virulence of this organism. Although many of the FPI-encoded protein products do not appear to have any known homologues, some of the FPI proteins show similarity to proteins involved in type VI secretion (T6S) of other bacteria. T6S systems are newly described bacterial virulence factors evolutionarily related to bacteriophages. We have tested the hypothesis the FPI encodes a secretion system. The FPI-encoded secretion system secretes a novel protein, IglC, into the extracellular milieu during broth growth. Systematic deletion mutagenesis determined the contribution of individual FPI genes to intramacrophage growth and secretion. We further characterized the secretion system by determining the subcellular localization of each FPI protein in the bacterial cell. An interaction between two inner membrane proteins, PdpB and DotU, was observed by co-immunoprecipitation, and the stability of PdpB requires DotU. Similarly, an interaction of IglA and IglB was demonstrated. Biochemical and fluorescence microscopy evidence suggest IglC is secreted into macrophages during intracellular localization of bacteria. Finally, a model of the FPI-encoded secretion system is presented. Our experiments provide biochemical, genetic and microscopy evidence that the FPI encodes a secretion system. The analysis of FPI-encoded secretion provides novel insights that may help us understand the role of FPI-encoded secretion in Francisella intracellular growth and virulence.

Orientadores: Arnaldo Yoshiteru Kuaye, Kathryn J. Boor / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T23:53:30Z (GMT). No. of bitstreams: 1 Kabuki_DirceYorika_D.pdf: 1158257 bytes, checksum: e37465f074c6973fa67baa13e368f2fa (MD5) Previous issue date: 2004 / Resumo: Os queijos frescais estilo Hispânico são alimentos de alto risco à contaminação por L. monocytogenes e já foram associados à pelo menos dois surtos de listeriose nos Estados Unidos da América. O conhecimento maior das fontes de contaminação por L. monocytogenes no processamento de queijos frescos tipo Hispânico é crítico para permitir o desenvolvimento de estratégias mais eficazes para o controle deste perigo. Neste trabalho, foi realizado um diagnóstico da contaminação por L. monocytogenes em três indústrias processadoras de queijos frescais tipo Hispânico, nos Estados Unidos. Um total de 246 amostras ambientais foi coletado e analisado para L. monocytogenes utilizando-se o método preconizado pela ¿Food and Drug Administration¿ (FDA) e o método ¿Biosynth L. monocytogenes detection system¿ (LMDS). As amostras de queijo, produzidos nestas indústrias (n=111), foram analisadas utilizando-se o método da FDA, modificado pela inclusão dos meios de cultura ¿L. monocytogenes plating medium¿ (LMPM) e ¿Listeria monocytogenes confirmatory plating medium¿ (LMCM) utilizados no método LMDS. L. monocytogenes foi detectada em 6,3% dos queijos e 11,0% das amostras ambientais. Dentre as amostras ambientais, aquelas obtidas de caixa vazada de plástico, dreno e piso apresentaram alta ocorrência de L. monocytogenes, com taxas de 55,6%, 30,0% e 20,6%, respectivamente. Apenas uma indústria apresentou resultados positivos em amostras de queijos e de superfícies que contatavam o alimento. O método da FDA mostrou maior sensibilidade do que o método LMDS para detecção de L. monocytogenes em amostras ambientais, e a inclusão dos meios LMPM e LMCM não melhorou a performance do método do FDA para detecção do patógeno em queijos. A subtipagem molecular, através da análise alélica dos genes de virulência actA e hly e da ribotipagem automatizada, foi utilizada para traçar a contaminação por L. monocytogenes nas indústrias. O ribotipo DUP-1044A, que havia sido previamente associado a surtos de listeriose humana em vários estados nos Estados Unidos em 1998, foi o subtipo mais comumente identificado (20/36 isolados) e foi isolado em 2 estabelecimentos. Este ribotipo se mostrou persistente e amplamente disseminado em uma das indústrias, onde foi também responsável pela contaminação do produto final. Nossa hipótese é que cepas deste ribotipo tenham habilidade específica em permanecer no ambiente de processamento. Apesar dos surtos de listeriose terem sido associados a queijos Hispânicos produzidos com leite não pasteurizado, os resultados obtidos neste trabalho revelam que a permanente contaminação ambiental pode representar outra fonte importante de contaminação do produto final / Abstract: Latin-style fresh cheeses, which have been linked to at least two human listeriosis outbreaks in the US, are considered to be high risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in three Latin-style fresh cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-month period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n=111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. L. monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains and floor samples showed the highest contamination rates with 55.6%, 30.0% and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the US, was the most commonly identified subtype (20/36 isolates) and was isolated from two plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination / Doutorado / Doutor em Tecnologia de Alimentos

Les bactéries du genre Shigella sont responsables de la shigellose, une maladie diarrhéique invasive du colon. L’entrée et la dissémination de Shigella à travers l’épithélium colique sont médiées par un système de sécrétion de type III (SST3) codé par un plasmide de virulence. Au sein de ce plasmide se trouve une région de 30-kb comportant les gènes impliqués dans l’entrée de la bactérie dans les cellules hôtes. Ces gènes sont regroupés en deux loci :le locus ipa-ipg qui code pour les protéines sécrétées et leurs chaperons ainsi que le locus mxi-spa codant pour les composants de l’appareil de sécrétion de type III (AST3), constitué d’un bulbe cytoplasmique, d’un corps basal transmembranaire et d’une aiguille se projetant au niveau extracellulaire. Ce système permet la sécrétion ordonnée et hiérarchique de différentes classes de protéines et la translocation de certaines d’entre elles (appelées effecteurs) dans le cytoplasme de la cellule hôte où elles interfèrent avec les voies de signalisation cellulaires. Avant le contact avec la cellule hôte, l’AST3 est inactif et verrouillé par les protéines IpaB et IpaD formant le complexe d’extrémité.<p>Chez Shigella, le gatekeeper MxiC séquestre les effecteurs au niveau du cytoplasme bactérien avant la transmission par l’aiguille du signal d’activation de la sécrétion mais les composants intermédiaires liant l’aiguille à MxiC restaient inconnus. Au cours de ce travail, nous avons montré que MxiC forme un complexe avec la sous-unité de la tige interne, MxiI, afin de bloquer l’entrée du canal de sécrétion et que cette interaction est conservée chez Yersinia et Salmonella. Nous démontrons que, suite au contact cellulaire, la dissociation de ce complexe facilite le switch de sécrétion des translocateurs aux effecteurs. Nos résultats révèlent également que MxiC est capable de s’associer au chaperon IpgC afin de réguler la sécrétion des translocateurs. De plus, nous avons identifié les domaines de MxiC engagés dans la régulation du SST3 et rapporté un nouveau rôle de MxiC dans l’échappement aux macrophage impliquant une possible inhibition de la voie apoptotique classique afin de promouvoir une pyroptose. Chez Shigella, IpaD gouverne la composition du complexe d’extrémité et est impliqué dans la régulation de la sécrétion. Nous avons développé une étude phénotypique de ses régions coiled-coil et centrale et montré que la composition du complexe d’extrémité permet de définir à la fois l’état d’inductibilité de l’AST3 et la sécrétion des effecteurs tardifs. Par ailleurs, notre étude fonctionnelle des domaines de MxiC et IpaD suggère que les capacités de Shigella à échapper au macrophage et à insérer un pore de translocation ne sont pas strictement couplées. <p>La dernière partie de ce travail s’est focalisée sur la caractérisation de la protéine Spa13 de Shigella. Nous avons découvert que le défaut de sécrétion du mutant spa13 est dû à l’instabilité de la sous-unité MxiH de l’aiguille et que Spa13 n’est pas sécrété par le SST3. Nos résultats indiquent également un rôle de Spa13 dans l’escorte de chaperons et l’activation de l’appareil d’exportation afin de promouvoir la sécrétion des substrats./Shigella is the causative agent of shigellosis, also known as bacillary dysentery, an invasive disease of the human colonic epithelium. During infection, Shigella uses a type III secretion system (T3SS) to penetrate enterocytes and to disseminate into the colonic epithelium, leading to destruction of the mucosal lining and shigellosis symptoms. Most of the virulence factors of Shigella are encoded by a large plasmid harboring a 30-kb region that is sufficient to promote bacterial entry into host cells. This entry region is organized in two loci, one corresponding to the the ipa-ipg genes encoding the secreted proteins and their cognate chaperones while the other encodes Mxi-Spa proteins that form the type III secretion apparatus (T3SA), consisting of a cytoplasmic bulb, a basal body spanning the bacterial envelope and a hollow needle. The T3SS allows the ordered and hierarchical secretion of effectors by inserting a translocation pore in the host cell membrane through which effector proteins are injected into the cytosol. Before host cell contact, the T3SA is inactive and plugged by the tip complex proteins IpaB and IpaD. <p>In Shigella, the gatekeeper MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle but the molecules involved in linking the needle and MxiC are unknown. We demonstrated that MxiC and the predicted inner-rod component MxiI form a complex plugging the T3SA entry gate and showed that this interaction is conserved in Yersinia and Salmonella. Dissociation of this complex seems to facilitate the switch in secretion from translocators to effectors upon host cell contact. Our results also revealed that MxiC binds to the chaperone IpgC to regulate translocators secretion. Moreover, we identified the domains of MxiC involved in the T3S regulation and reported a new role in macrophage escape by potential inhibition of the classical apoptosis to promote pro-inflammatory pyroptosis. <p>In Shigella, IpaD rules the composition of the tip complex and is involved in secretion control and translocon insertion. We therefore undertook a phenotypic analysis of its coiled-coil and central regions and showed that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Besides, our functional study on MxiC and IpaD domains suggests that Shigella abilities to escape macrophage vacuole and to insert the translocation pore are uncoupled.<p>The last part of this work is related to the characterization of the Spa13 protein of Shigella. We found that the secretion defect of the spa13 mutant is due to the instability of the needle component MxiH and that Spa13 is not a secreted substrat. Our results also support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch to promote substrates secretion. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished