Bacillus anthracis is a soil dwelling microbe with pronounced pathogenic potential. Historically, anthrax has infected livestock and man. In the modern-age, anthrax is a bioterrorism concern with major incidents every decade. While the threat of large scale attacks is currently viewed as unlikely, the threat is consistent and constant. Current methods to defend against such an attack focus on antibiotics and containment of public panic. Antibiotic resistance, while not currently an issue for anthrax, could easily become so with genetically engineered weaponized strains created by rogue states or independent actors. This project evolved from collaborations between the Grose lab and the Robison lab, both housed in the Microbiology and Molecular Biology Department at Brigham Young University in Provo, Utah. Two undergraduates in the Grose lab isolated 23 genetically distinct phage that infect the non-pathogenic Bacillus anthracis Sterne strain. Results from spot testing on a diverse library of 11 fully virulent strains that represent the extant genetic diversity of pathogenic B. anthracis in BYU’s BSL-3 facility give credence to the idea that phage could be useful in containing this pathogen. Phage were isolated from environmental samples using enrichment culture, high titer lysates of isolated phage were created, and differential assays were performed. Experiments to show phage differences included electron microscopy, restriction digests, and spot testing using different isolates of B. anthracis. These data identified several novel phage that could infect a wide variety of virulent B. anthracis isolates. Preliminary results also showed most of these phage to be different both morphologically and genetically.We propose that phage therapy deserves further research, public awareness, and increased understanding for governmental regulatory awareness.

Yes / This paper outlined our method for developing polymer-linked contact lens type materials for rapid detection and differentiation of Gram-positive, Gram-negative bacteria and fungi in infected corneas. It can be applied to both model synthetic or ex-vivo corneal models and has been successfully trialed in an initial efficacy tested animal study. First a hydrogel substrate for the swab material is selected, we have demonstrated selective swabs using a glycerol monomethacrylate hydrogel. Alternatively any commercial material with carboxylic acid functional groups is suitable but risks nonspecific adhesion. This is then functionalised via use of N-hydroxysuccinimide reaction with amine groups on the specified highly branched polymer ligand (either individually gram negative, gram positive or fungal binding polymers or a combination of all three can be employed for desired sensing application). The hydrogel is then cut into swabs suitable for sampling, used, and then the presence of gram positive, game negative and fungi are disclosed by the sequential addition of dyes (fluorescent vancomycin, fluorescein isothiocyanate and calcofluor white). In summary this method presents: Method to produce glycerol monomethacrylate hydrogels to minimize nonspecific binding Methods of attaching pathogen binding highly branched polymers to produce selective hydrogel swabs Method for disclosing bound pathogens to this swab using sequential dye addition

Réalisation d'un outil de base pour la mise en œuvre des systèmes relationnels. Présentation du projet MICROBE, visant à l'implantation d'un SGBD relationnel sur micro-ordinateurs. Aperçu sur l'état de l'art en matière de mémoires relationnelles. Etude des methodes d'accès aux données, en particulier la methode des B*-arbres.

The interactions between Medicago truncatula and Phytophthora medicaginis were examined using skl, a mutant blocked in ethylene perception, and a range of wild accessions of this plant species. P. medicaginis infection of M. truncatula plants resulted in compatible responses, whereas the mutant genotype was found to be hyper-susceptible to the pathogen. Phytophthora reproduction and colonization rates of Medicago tissues supported this conclusion. Infection of skl with different pathogens reinforced this observation. Ethylene production in infected A17 and skl roots showed reduced ethylene evolution in the mutant and suggested that a positive feedback loop, known as autocatalytic ethylene production, amplified the ethylene signal. To complement the study, expression analyses of defense response genes in this interaction were studied by real time RTPCR of Phytophthora-infected and mock-infected roots. The genes analyzed were PAL, CHS, IFR, ACC oxidase, GST, and PR10. The sequences needed for the analysis were found through the scrutiny of the M. truncatula EST database employing phylogenetics and bio-informatics tools. In A17 all the genes studied were up-regulated, although the specific gene expression patterns differed. The comparison of gene expression between A17 and skl genotypes allowed the differentiation between ethylene-dependent and ethylene-independent responses. Discrete results showed that ACC oxidase homologues were downregulated in the ethylene perception mutant, corroborating the ethylene observations. However, the expression of genes involved in the phenylpropanoid metabolism was increased in skl relative to A17, suggestive of an antagonism between the ethylene perception pathway and the regulation of the phenylpropanoid pathway. This result implied that Medicago phytoalexins accumulate in the disease interaction, but raised questions about their role in resistance to Phytophthora infection. This study establishes a link between mechanisms that regulate symbiotic infection and the regulation of disease resistance to Oomycete pathogens, especially P. medicaginis. The results served to identify a series of Phytophthora-induced genes, which remain pathogen-responsive even in the absence of a functional ethylene perception pathway. While it is possible that the products of these genes are involved in resistance to P. medicaginis, the present results demonstrate that ethylene perception is required for resistance.

Endophytes are organisms that live inside plants without causing disease and include microbes that benefit their hosts by aiding in nutrient acquisition and pathogen control. This thesis concerns the endophytes of the genus Zea which includes modern maize (Zea mays L.). Beginning 9,000 years ago, maize was domesticated from wild grasses in Mexico (teosintes), bred into diverse varieties and moved to new soils throughout the Americas. The impact of these long-term changes on the associated endophytic communities has not been examined. Furthermore, today, maize is routinely transplanted around the world to facilitate breeding, but the short-term impact of switching soils on endophyte composition is not known. I attempted to answer the first question by surveying the bacterial endophytes that inhabit 14 diverse ancestral, ancient and modern Zea genotypes. To answer the second question, three extreme Zea genotypes, ancestral, intermediate and modern, were grown side by side on two extreme soils that span the tropical-to-temperate migration route of maize. Endophyte populations from seeds, roots and shoots were DNA fingerprinted using terminal restriction length polymorphism (TRFLP) of 16S rDNA. To understand microbial functions, bacteria were cultured and tested for >13 in vitro traits including nitrogen fixation, phosphate solubilization, plant hormone production and antibiosis. Relationships between endophyte communities were analyzed using principle component analysis (PCA) and Sᴓrensen’s similarity index. The results show that different Zea tissues and genotypes have diverse endophytic communities. The community composition of seed endophytes correlates with host phylogeny suggesting that as humans bred maize, they inadvertently impacted its microbial inhabitants, though the change was gradual. Soil swapping and growth on sterile sand confirm that shoot and root endophyte communities in juvenile plants are primarily inherited. However, a given maize genotype can also select and take up the same microbes (based on TRFLP) from geographically diverse soils. These latter results show that the endophyte communities of Zea plants are significantly buffered from the short-term effects of migration. A few microbes and microbial traits are conserved across all Zea genotypes and soil treatments, suggestive of a core taxonomic and functional microbiota for this agriculturally important genus. / OMAFRA New Directions, Ontario Research Fund, Canadian Foundation for Innovation

Dans son environnement la plante est confrontée à une variété de microorganismes bénéfiques, neutres et pathogènes, qui sont fortement dépendants des ressources carbonées qu’elle libère dans le sol. Le transport de sucres, processus clé de la physiologie de la plante, est essentiel pour les interactions plantes-microorganismes et leur devenir. Au cours de l'évolution, les plantes ont acquis des mécanismes leur permettant de percevoir les signaux microbiens du milieu extérieur, et aboutissant à la transduction d’un signal spécifique puis à des réponses biologiques adaptées (défense versus mutualisme) à la stratégie du microorganisme. Ces réponses assurent la survie et le développement des plantes. Mes travaux de thèse, menés avec un système « d’interaction » simplifié, contribuent à une meilleure compréhension des mécanismes sous-jacents au déterminisme des interactions plantes-microorganismes. Ce système a permis d’étudier, sur des suspensions cellulaires de N. tabacum, les réponses cellulaires précoces déclenchées suite à la perception de molécules microbiennes provenant de microorganismes à stratégie pathogène avirulent ou à stratégie mutualiste. Nous avons mesuré des évènements de signalisation et des flux de sucres induits en réponse à ces molécules microbiennes. Nos résultats ont mis en évidence que les chitotétrasaccharides (CO4), sécrétés par les champignons mycorhiziens à arbuscules dans les stades pré-symbiotiques de l’interaction, mobilisent les mêmes événements de signalisation précoce (H2O2 dépendant de la protéine rbohD, Ca2+ cytosolique, activation de MAPK) que la cryptogéine, un éliciteur des réactions de défense ; mais avec des réponses différentes en terme d’intensité et de cinétique. Les CO4 et la cryptogéine ont par ailleurs montré des impacts distincts sur les flux de sucres et l’expression de transporteurs impliqués. En complément nous avons montré un effet de la modification de la dynamique membranaire associée à la clathrine sur des évènements de signalisation déclenchés par la cryptogéine, ainsi que dans les flux entrants de sucres et l’expression de gènes de transporteurs de sucres. Enfin, l’analyse in silico de l’interactome de transporteurs de sucres chez la plante modèle A. thaliana, nous a permis d’apporter des connaissances supplémentaires quant aux évènements de régulations des transporteurs de sucres et l’identification de protéines régulatrices putatives en interaction avec ces derniers. L’ensemble de ces travaux ouvrent la voie à de nouvelles recherches visant à élucider les mécanismes cellulaires et moléculaires impliqués dans la mise en place des interactions entre plantes et microorganismes. / In their natural environment plants are in close interaction with beneficial, neutral, or pathogenic microbes, which are highly dependent on carbon resources exuded by plant roots. Sugar transport, which is a key process of plant physiology, is essential to support the fate of plant-microbe interactions. During evolution, plants have acquired the ability to perceive microbial molecules, initiating specific signal transduction cascades and leading to adapted response for microbe lifestyles (avirulent, virulent, or benefic). Plant survival will depend on the nature of the induced mechanisms. My PhD work, carried out on a simplified experimental system, contributes to the understanding of mechanisms underlying the determinism of plant-microbe interactions. We used Nicotiana tabacum cells in suspension exposed to microbial molecules derived from mutualistic or avirulent microbes. Using such a simplified system, we analyzed elements of the early signaling cascade and sugar fluxes. We have shown that CO4, which is originating from AMF, initiate early signaling components (rbohD-dependent H2O2, cytosolic Ca2+, MAPK activation) as cryptogein, a defense elicitor, but with distinct profile and amplitude. Those two molecules (CO4 and cryptogein) are responsible of different effects on sugar fluxes and the expression of the underlying sugar transporter genes. In addition, we presented an impact of the alteration of clathrin-mediated process on early signaling events triggered by cryptogein, as well as inward sugar fluxes and expression of sugar transporter genes. Finally, in silico analyses of sugar transporter interactome in Arabidopsis thaliana has provided some possible regulation mechanisms through the identification of new candidate proteins involved in sugar transporter regulation. These information open new perspectives towards a better understanding of the cellular and molecular mechanisms involved in plant-microbe interactions.

No description available.

Afin d’étudier l’écologie microbienne, il est nécessaire, dans un premier temps, de déterminer quels micro-organismes sont présents dans un milieu et à quel instant. Ces informations sont requises pour pouvoir ensuite développer des outils permettant de prédire l’assemblage des communautés et les fonctions que celles-ci peuvent contenir. Cependant, la multitude des processus entrant en jeu dans la structure et la composition des communautés microbiennes, rendent leur étude complexe. Parmi les nombreux processus à étudier, il est notamment question de l’échelle temporelle à prendre en compte pour comprendre l’assemblage des communautés microbiennes. En effet, les événements historiques conditionnent la composition et la biodiversité des futures communautés microbiennes. Pourtant, dans les sols, peu d’études se sont intéressées à l’impact des événements historiques dans l’assemblage des communautés microbiennes. Par conséquent, l’objectif de cette thèse était de quantifier comment les différentes histoires du sol ont influencé la structure et biodiversité des communautés bactériennes et oomycètes associées aux plantes hôtes des Brassicaceae à travers le temps. Les rotations de cultures de Brassicaceae sont de plus en plus courantes dans le monde et ont démontré des avantages pour les cultures concernées, telles que la rétention de l’humidité du sol ou la suppression de certains agents pathogènes des plantes. En revanche, l’impact des rotations de cultures de Brassicaceae sur la structure et biodiversité des communautés microbiennes résidentes est peu connu. Ainsi, des terrains agricoles des prairies canadiennes ayant des expériences de rotations de cultures en cours ont été utilisés pour modéliser l’impact des histoires de sol précédemment établies sur les futures communautés microbiennes. Les communautés microbiennes des racines, de la rhizosphère, et du sol éloigné des racines des Brassicaceae ont été étudiées grâce aux métabarcodes d’ARNr 16S ou ITS. La PCR quantitative et des méthodes phylogénétiques ont été utilisées pour améliorer l’analyse des communautés microbiennes. Cette thèse illustre comment différentes histories de sol établies par les cultures de l’année précédente ont continué à structurer les communautés microbiennes de la rhizosphère tout au long de la saison de croissance, à différents stades de croissance, jusqu’à un an après leur établissement. Cependant, le phénomène de rétroactions entre plantes et micro-organismes a permis de masquer cet héritage dans la rhizosphere de différentes espèces hôtes de Brassicacea pour lesquelles des communautés bactériennes phylogénétiquement similaires ont été retrouvées malgré diverses histoires du sol. Nos résultats montrent également que les différentes espèces hôtes de Brassicacea n’avaient pas d’impact sur la structure des communautés d’oomycètes et que le stress hydrique limitait également cette structuration pour les communautés bactériennes. Dans ces deux cas, l’effet de l’histoire du sol était donc encore visible sur la structure les communautés microbiennes durant l’année subséquente. Les découvertes selon lesquelles différentes histoires de sol persistent jusqu'à un an, même en présence de nouvelles plantes hôtes, et qu’elles peuvent continuer à façonner les communautés microbiennes ont des implications importantes pour la gestion agricole et les recherches futures sur les composants physiques de l'histoire du sol. Comprendre comment l'histoire du sol est impliquée dans la structure et la biodiversité des communautés microbiennes à travers le temps est une limitation de l'écologie microbienne et est nécessaire pour utiliser les technologies microbiennes à l'avenir pour une agriculture durable et dans toute la société. / A fundamental task of microbial ecology is determining which organisms are present, and when, in order to improve the predictive models of community assembly and functions. However, the heterogeneity of community assembly processes that underlie how microbial communities are formed and structured are makes assembly of taxonomic and functional profiles difficult. One reason for this challenge is the compounding effect temporal scales have on microbial communities. For example, historical events have been shown to condition future microbial community composition and biodiversity. Yet, how historical events structure microbial communities in the soil has not been well tested. Therefore, the objective of this thesis was to quantify how different soil histories influenced the structure and biodiversity of bacterial and oomycete communities associated with Brassicaceae host plants through time. Brassicaceae crop rotations are increasingly common globally, and have demonstrated benefits for the crops involved, such as retaining soil moisture, or suppressing certain plant pathogens. In contrast, there is a lack of knowledge surrounding how Brassicaceae crop rotations impact the structure and biodiversity of resident microbial communities. As such, on-going agricultural field experiments with crop rotations on the Canadian prairies were used to model how previously established soil histories impacted future microbial communities. The Brassicaceae microbial communities were inferred from the roots, rhizosphere and bulk soil using 16S rRNA or ITS metabarcodes. Quantitative PCR and phylogenetic methods were used to improve the analysis of the microbial communities. This thesis illustrates how different soil histories established by the previous year’s crops continued to structure the microbial rhizosphere communities throughout the growing season, at various growth stages, and up to a year after being established. However, active plant-soil microbial feedback allowed different Brassicaceae host species to mask the soil history in the rhizosphere and derive phylogenetically similar bacterial communities from these diverse soil histories. Furthermore, host plants were unable to structure the oomycete communities, and lost the ability to structure the bacterial rhizosphere communities under water stress. In both circumstances, the soil history continued to structure the microbial communities. The findings that different soil histories persist for up to a year, even in the presence of new host plants, and can continue to shape microbial communities has important implications for agricultural management and future research on the physical components of soil history. Understanding how soil history is involved in the structure and biodiversity of microbial communities through time is a limitation in microbial ecology and is required for employing microbial technologies in the future for sustainable agriculture and throughout society.

Background: Intestinal inflammation elicited by environmental determinants including dietary proteins and microbes is implicated in type 1 diabetes (T1D) pathogenesis. Also, intrinsic pancreatic abnormalities could precede classic insulitis, contributing to T1D. Materials and Methods: Spontaneous rat T1D models were used for in situ analyses of gut and pancreas to explore novel disease pathways using immunohistochemistry and detailed morphometry, gene expression studies, and molecular screening analyses. Results: In BBdp rats, feeding a cereal diet stimulated T1D under germ-free or specific pathogen-free (SPF) conditions compared with a protective hydrolyzed casein (HC) diet. Cereal-induced T1D was paralleled by increased gut T cell infiltration and TH1-associated pro-inflammatory transcription. HC-fed rats displayed an increased number of anti-inflammatory CD163+ M2 macrophages compared with cereal-fed rats. Cereal-associated promotion of T1D in Lewis diabetes-prone (LEW-DP) rats, a different rat model, similarly featured gut T cell infiltration in conjunction with decreased immunoregulation. The Camp gene was induced in diet-protected HC-fed BBdp rats. Camp encodes the cathelicidin antimicrobial peptide (CAMP), a pleiotropic immunomodulatory host defence factor. Intestinal CAMP was enriched in CD163+ M2 macrophages and could represent a novel marker of these tolerogenic innate immune cells. CAMP expression was also discovered in pancreatic lymph nodes (PLN) and islets, indicating a novel role for this factor in target tissue homeostasis. There was a positive correlation between pancreatic CAMP and total islet number. Also, islet-associated CAMP+ cells were increased in rats with islet inflammation, suggesting upregulation in parallel with insulitis. Exogenous CAMP/LL-37 injections increased the abundance of T1D-protective probiotic bacteria and promoted islet neogenesis in BBdp rats. A prospective partial pancreatectomy (PPx) study was performed to obtain pre-diabetic pancreas biopsies from iii pre-insulitic BBdp rats. The number of endothelium-associated CD68+ macrophages was increased in pre-diabetic pancreata, indicating that perivascular inflammation was an early lesion in the animals. In addition, pre-diabetic pancreata featured enhanced regenerative Reg3a and Reg3b gene expression, indicating abnormal islet expansion preceding insulitis. Conclusions: Small intestinal inflammation paired with deficits in local immunoregulation parallels T1D development. CAMP represents a novel factor in T1D that could have several pleiotropic functions including regulation of commensal microbes, intestinal homeostasis, and pancreatic homeostasis. In addition, target tissue abnormalities precede insulitis and T1D. This research focused on the integrative biology of T1D pathogenesis in spontaneous rat models. This work provides a novel working model that incorporates key roles for gut lumen antigens, intestinal immunity, and the role of islets and altered regenerative capacity in T1D. This research could lead to new therapeutic opportunities for T1D treatment.

No description available.