Die genomische und transkriptomische Landschaft der klinischen Isolate von Escherichia coli und Pseudomonas aeruginosa

Large amounts of genomic data have been obtained due to the rapid advances in DNA sequencing technology. With efficient computational platforms, these data can provide many possibilities to improve our knowledge on species evolution and their genetic makeup. The general interest of this thesis is to facilitate studies on important biological questions by attaining the relevant information from transcriptomic and genomic data. The aims of my thesis were i) to develop the pan-genome based RNA-Seq data analysis pipeline in order to analyze ex vivo gene expression profiles of uro-pathogenic Escherichia coli isolates and ii) to create the consensus sequence of the Pseudomonas aeruginosa core genome in order to identify single nucleotide polymorphisms (SNPs) at high accuracy and to find the patho-adaptive mutations in P. aeruginosa clinical isolates. To address these aims I developed and used the pan-genome of E. coli in order to map and analyze the RNA-Seq reads that were associated with an acute urinary tract infection. Whereas the in vivo gene expression profiles of the majority of genes were conserved among the 21 E. coli strains, the specific gene expression profiles of the accessory genome were diverse and reflected phylogenetic relationships. In addition to that, whole genome sequencing data was used to gain insights into the genetic variations of 99 clinical P. aeruginosa isolates. I created the consensus sequence for every core gene based on the most frequent nucleotide. I used it as reference for the identification of SNPs across all clinical isolates. The identified SNPs were classified into clonal-specific, single and phylogenetically independent SNPs. The majority of the SNPs were clonal-dependent and single SNPs. However, I identified a large set of 2,252 genes which had one or more phylogenetically independent non-synonymous mutation. Moreover, the ratio of dN/dS on 3,814 genes revealed that the core genome is not under selection pressure. In summary, this thesis explores pan-genome-based as well as consensus sequence-based approaches on transcriptomic and genomic sequencing data of clinical isolates of E. coli and P. aeruginosa respectively. The results of the thesis contributed to understanding of sequence variations that are selected in the environment of the human host and lead to bacterial adaptation and pathogenicity. This is not only important for the basic scientific research, but also to understand the link between diversity and community structure and function.Aufgrund der schnellen Fortschritte in der DNA-Sequenzierungstechnologie wurden große Mengen genomischer Daten erhalten. Mit effizienten Rechenplattformen können diese Daten viele Möglichkeiten bieten, unser Wissen über die Evolution von Arten und ihren genetischen Aufbau zu verbessern. Das allgemeine Interesse dieser Arbeit ist es, Studien zu wichtigen biologischen Fragen zu ermöglichen, indem relevante Informationen aus transkriptomischen und genomischen Daten gewonnen werden. Ziel meiner Dissertation war es, i) die auf dem Pan-Genom basierende RNA-Seq Datenanalyse Pipeline zu entwickeln, um ex vivo-Genexpressionsprofile von uropathogenen Escherichia coli-Isolaten zu analysieren und ii) die Konsensussequenz des Pseudomonas aeruginosa-Kerns zu erstellen Genom, um einzelne Nukleotidpolymorphismen (SNPs) mit hoher Genauigkeit zu identifizieren und die pathoadaptiven Mutationen in klinischen P. aeruginosa-Isolaten zu finden. Um diese Ziele zu erreichen, entwickelte und verwendete ich das Pan-Genom von E. coli, um die RNA-Seq-Reads abzubilden und zu analysieren, die mit einer akuten Harnwegsinfektion assoziiert waren. Während die in vivo-Genexpressionsprofile der meisten Gene unter den 21 E. coli-Stämmen konserviert waren, waren die spezifischen Genexpressionsprofile des akzessorischen Genoms unterschiedlich und spiegelten phylogenetische Beziehungen wider. Darüber hinaus wurden Daten zur vollständigen Genomsequenzierung verwendet, um Einblicke in die genetischen Variationen von 99 klinischen P. aeruginosa-Isolaten zu erhalten. Ich erstellte die Konsensussequenz für jedes Kerngen basierend auf dem häufigsten Nukleotid. Ich habe es als Referenz für die Identifizierung von SNPs in allen klinischen Isolaten verwendet. Die identifizierten SNPs wurden in klonenspezifische, einzelne und phylogenetisch unabhängige SNPs eingeteilt. Die Mehrheit der SNPs waren klonabhängige und einzelne SNPs. Ich identifizierte jedoch eine große Menge von 2.252 Genen, die eine oder mehrere phylogenetisch unabhängige, nicht synonyme Mutationen aufwiesen. Darüber hinaus zeigte das Verhältnis von dN / dS bei 3.814 Genen, dass das Kerngenom nicht unter Selektionsdruck steht. Zusammenfassend werden in dieser Dissertation Pan-Genom-basierte sowie Consensus-Sequenz-basierte Ansätze zur Transkriptom- und Genom-Sequenzierung von klinischen Isolaten von E. coli bzw. P. aeruginosa untersucht. Die Ergebnisse der Dissertation trugen zum Verständnis von Sequenzvariationen bei, die in der Umgebung des menschlichen Wirts selektiert werden und zu einer bakteriellen Anpassung und Pathogenität führen. Dies ist nicht nur für die wissenschaftliche Grundlagenforschung wichtig, sondern auch, um den Zusammenhang zwischen Vielfalt und Struktur und Funktion der Gemeinschaft zu verstehen

Imbalanced gut microbiota fuels hepatocellular carcinoma development by shaping the hepatic inflammatory microenvironment

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and therapeutic options for advanced HCC are limited. Here, we observe that intestinal dysbiosis affects antitumor immune surveillance and drives liver disease progression towards cancer. Dysbiotic microbiota, as seen in Nlrp6(-/-) mice, induces a Toll-like receptor 4 dependent expansion of hepatic monocytic myeloid-derived suppressor cells (mMDSC) and suppression of T-cell abundance. This phenotype is transmissible via fecal microbiota transfer and reversible upon antibiotic treatment, pointing to the high plasticity of the tumor microenvironment. While loss of Akkermansia muciniphila correlates with mMDSC abundance, its reintroduction restores intestinal barrier function and strongly reduces liver inflammation and fibrosis. Cirrhosis patients display increased bacterial abundance in hepatic tissue, which induces pronounced transcriptional changes, including activation of fibro-inflammatory pathways as well as circuits mediating cancer immunosuppression. This study demonstrates that gut microbiota closely shapes the hepatic inflammatory microenvironment opening approaches for cancer prevention and therapy. Steatohepatitis is a chronic hepatic inflammation associated with increased risk of hepatocellular carcinoma progression. Here the authors show that intestinal dysbiosis in mice lacking the inflammasome sensor molecule NLRP6 aggravates steatohepatitis and accelerates liver cancer progression, a process that can be delayed by antibiotic treatment.Peer reviewe

Dynamic properties of sands under cyclic torsional shear

Dynamic properties of soils have to be well understood in order to assure stability and acceptable performance of soil structures under seismic and wave loadings. It has been found the two important dynamic properties - shear modulus and damping factor are complex functions of many variables. In order to study the influence of various factors on shear modulus and damping factor, drained cyclic torsion shear tests were carried out in the hollow cylinder torsion device using medium Ottawa A S T M C-109 sand. Effects of shear strain amplitude, stress history, effective mean normal stress (σ'm = l/3(σ-'1 +σ'2 + σ'3)), principal effective stress ratio (R = σ1/σ3), intermediate principal stress parameter (b = σ2 —σ3)/(σ1 — σ-3)), void ratio, number of cycles of loading are some of the factors studied in this thesis. During the application of cyclic shear stress σ'm ,R and b were kept constant at pre-selected values for each test. This technique allows to study the effect cr'm, R and b independently. For example, the effect of R on dynamic properties can be isolated by a series of tests on specimens that have identical σ'm and b but different levels of R and all parameters σ'm, R and b are held constant during cyclic shear application. It is shown that shear modulus increases with number of cycles of a constant amplitude cyclic shear stress when the induced shear strain is higher than a certain threshold value. The damping, however, decreases with number of cycles even at strain amplitudes less than this threshold value. There is also a threshold value of shear strain below which zero volumetric strain occurs due to cyclic shear loading, and hence no pore pressure would develop if cyclic loading was undrained. Effects of stage testing and small strain history on dynamic properties is shown to be insignificant. With decrease of void ratio, shear modulus increases and damping factor decreases. It is shown that for a given b, the void ratio factor F(e) = (2.17 —e)² ( l + e), collapses the modulus degradation curves obtained at different void ratios in to a single curve. For a given initial stress state and shear strain amplitude, shear modulus obtained at different R levels do not show any significant difference when R < 3. Damping factors, however, seems to be unaffected by the change in R at all R levels. When R < 3, shear moduli in triaxial extension condition (b = 1) are found to be less than those in triaxial compression condition (b = 0) and damping factors for b = 1 are higher than those for b = 0. Both triaxial compression and extension state of loadings yielded same values shear modulus and damping factors at large amplitude of shear strain at R = 3. Test results indicate that when b < 1, the dynamic properties are independent of intermediate principal stress. Effects of stress history due to decrease in R from 3 to 2, is significant only in the small strain range, and as the strain level increase, the effects of stress history diminishes.Applied Science, Faculty ofCivil Engineering, Department ofGraduat

Liquefaction of sands under multi-axial loading

A fundamental study of the undrained behaviour of sands under multi-axial loading is presented. The study was performed by using the hollow cylinder torsional (HCT) device. The HCT is the only device that permits a soil specimen to be subjected to multi-axial loading with controlled variations in the magnitudes of the three principal stresses and the direction of the major principal stress with the vertical deposition direction. The main objective of the study was to assess the effects of principal stress magnitude, directions and their rotation on sand liquefaction. This is achieved by a systematic study of static and cyclic undrained behaviour of reconstituted loose sand. Shear loading is carried out under strain control. Only such loading permits the needed capture of post peak strain softening characteristics of loose sands. Undesirable runaway strains are inevitable in stress controlled loading modes. In addition to the investigations in the hollow cylinder torsional device, sand behaviour in simple shear as well as under the triaxial conditions was also assessed as reference for comparisons with that under multi-axial stresses. The investigations were carried out using two sands - Fraser River sand and Syncrude sand. Sand specimens were reconstituted by water pluviation, which is considered to duplicate the fabric ofin-situ fluvial and hydraulic fill deposits. Independence of the effective stress path and stress-strain characteristics from the total stress path under fixed principal stress directions and constant value of intermediate principal stress parameter is illustrated. The undrained response of loose sand is highly dependent on the loading direction, implying inherent anisotropy. The friction angle mobilized at phase transformation or steady state is a unique material property, independent of the mode of loading static or cyclic, direction of principal stresses, intermediate principal stress level, consolidation history and the stress and void ratio state prior to undrained shear. There is no unique relationship between steady state or phase transformation strength and void ratio that is independent of the stress path, implying that a unique steady state line does not exist for a sand. The influence of intermediate principal stress, on undrained response is small when the intermediate principal stress parameter, that reflects value of this stress relative to the major and the minor values, is less than about 0.5. At constant values of other parameters increasing confining stress and decreasing relative density under multi-axial loading promote a higher degree of contractive response. The history of principal stress directions during principal stress rotation does not seem to have any appreciable effect on the peak and steady state or phase transformation strength. These strengths are apparently controlled by the peak value of major principal stress inclination experienced during shearing with respect to vertical direction. Principal stresses undergo continuous rotation from 0 to about ±45° in simple shear deformation. A simultaneous change in intermediate principal stress occurs as the major principal stress rotates. The maximum shear stress and maximum shear strain in conventional simple shear deformation approximately equals the horizontal shear stress and shear strain respectively. For a given initial stress and void ratio state, the number of cycles to liquefaction is smaller under cyclic triaxial than under similar 90° jump rotation that do not invoke the weakest triaxial extension loading mode during shear. For a given direction of principal stresses, if the sand is contractive under static loading, it would also be contractive under cyclic loading, provided that the cyclic deviator stress amplitude is higher than the steady state or phase transformation strength in static loading.Applied Science, Faculty ofCivil Engineering, Department ofGraduat

Sequence-Structure Similarity: Do Sequentially Identical Peptide Fragments have Similar Three-Dimensional Structures?

The rapidly growing structure databases enhance the probability of finding identical sequences sharing structural similarity. Structure prediction methods are being used extensively to abridge the gap between known protein sequences and the solved structures which is essential to understand its specific biochemical and cellular functions. In this work, we plan to study the ambiguity between sequence-structure relationships and examine if sequentially identical peptide fragments adopt similar three-dimensional structures. Fragments of varying lengths (five to ten residues) were used to observe the behavior of sequence and its three-dimensional structures. The STAMP program was used to superpose the three-dimensional structures and the two parameters (Sequence Structure Similarity Score (Sc) and Root Mean Square Deviation value) were employed to classify them into three categories: similar, intermediate and dissimilar structures. Furthermore, the same approach was carried out on all the three-dimensional protein structures solved in the two organisms, Mycobacterium tuberculosis and Plasmodium falciparum to validate our results

Removable denture is a risk indicator for peri-implantitis and facilitates expansion of specific periodontopathogens: a cross-sectional study

Abstract Background The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis. Methods During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified. Results 16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (p =  &lt; 0.001) and implant level (p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present. Conclusions Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation. </jats:sec

Removable denture is a risk indicator for peri-implantitis and facilitates expansion of specific periodontopathogens: a cross-sectional study.

Background: The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis. Methods: During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified. Results: 16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (p = < 0.001) and implant level (p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present. Conclusions: Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation

Single-Nucleotide Polymorphism-Based Genetic Diversity Analysis of Clinical Pseudomonas aeruginosa Isolates

Abstract Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Because different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here, we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single-nucleotide polymorphisms that are the basis for branching of the phylogenetic tree. We also identified single-nucleotide polymorphisms that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. Although our results demonstrate that the Pseudomonas aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.</jats:p