Diversity and Activity of Roseobacters and Roseophage

Bacteria of the Roseobacter lineage are dominant bacterioplankton in coastal systems and contribute significantly to secondary production in oceanic environments. Generalities of Roseobacter ecology, diversity, and distributions are known, but the intraspecific differences between species and their dynamics over short temporal periods is not well understood. Bacteriophage that infect Roseobacters (‘roseophage’) have the potential to shunt secondary production into the dissolved carbon pool and through the process of infection alter Roseobacter physiology. Despite their significance, little effort was made prior to the onset of this study to characterize roseophage. Using culture dependent and independent approaches, I describe the diversity and activity of Roseobacters and roseophage from two distinct coastal environments. Chapter 2 describes the development of an alternative method to enumerate viruses using epifluorescence microscopy that not only reduces sample processing costs, but also the total volume of sample required. A novel species of the Roseobacter lineage (Marivita roseacus) is proposed in Chapter 3. M. roseacus is unique in its needle-like morphology, forming long, relatively inflexible chains of cells. The Marivita genus is characterized by a distinct ecology, being closely associated with algae, resistant to grazing, and present in numerous marine and saline environments. Chapter 4 details the use of deep-amplicon sequencing (16S rDNA) to describe bacterial succession patterns during a mesocosm algal bloom, revealing the temporal dynamics of ~100 distinct phylotypes. A multivariate analysis showed that temporal portioning amongst the bacterial community was occurring at both high and low taxonomic levels. Chapter 5 details the isolation and genomic characterization of roseophage and describes their ecology using publically available metagenomic databases collected from throughout the world. Four distinct phage were isolated and sequenced including an N4-like strain, a novel Siphoviridae, and two temperate Podoviridae. The two temperate phage were practically identical at the nucleotide level, except for a 3000 bp putative replication module, which showed no homology between the two. Overall, this dissertation suggests that ecological partitioning within the Roseobacter lineage is occurring at and arguably below traditional species level taxonomic classifications and microdiversity amongst closely related marine bacteria is likely the norm rather than the exception

Development of bioinformatic tools to identify and characterize linear protein epitopes

The adaptive immune system produces antibodies to specifically target antigens. Identifying and characterizing epitopes on target antigens enables numerous medical applications such as vaccine design, diagnostic discovery, and therapeutic antibody development. We have developed computational tools that characterize epitopes using large sets of antibody-binding peptides. To identify epitopes in target proteins, we used an approach termed K-mer Tiling of Protein Epitopes (K-TOPE). In this approach, we divided protein sequences into short overlapping subsequences of length k (k-mers). Then, we defined and scored epitopes using each k-mer’s enrichment in the sets of antibody-binding peptides. Using K-TOPE, we accurately identified epitopes for monoclonal and polyclonal antibodies. Next, using 250 specimens, we identified commonly targeted epitopes in nearly 3,000 viral proteins as well as two bacterial proteomes. Importantly, these epitopes agreed with previously reported results. To map antibody binding in epitopes, we developed Multiplexed Epitope Substitution Analysis (MESA). In this approach, target epitopes were divided into short overlapping k-mers. Then, each k-mer was exhaustively substituted with all amino acids. The effects of these substitutions were used to identify amino acid preferences at important binding positions in the epitopes. By applying this method to monoclonal antibodies and multiple sets of specimens, we identified binding motifs which agreed with an alternative computational approach. Finally, K-TOPE and MESA were used to characterize epitopes and antigens in age-related macular degeneration (AMD), herpes simplex virus (HSV), and Chagas disease. We identified 42 AMD-specific epitopes, 30 HSV2-specific epitopes, and 222 Chagas-specific epitopes. Several epitopes were in validated antigens while many were novel. MESA demonstrated that generally only 4-5 positions in these epitopes were important for binding. Future application of these approaches could enhance our understanding of the role that antibodies play in disease progression

PCRi praimeridisaini parendamine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Polümeraasi ahelreaktsioon ehk PCR on molekulaarbioloogia meetod, mis võimaldab paljundada spetsiifilist DNA lõiku. Protsess toimub tsükliliselt ja kätkeb endas järgmisi etappe: kaheahelalise DNA järjestuse (sihtmärkjärjestuse) sulatamist kõrgel temperatuuril, kahe spetsiifilise DNA järjestuse (PCRi praimeri) seondumist sihtmärkjärjestusele konkreetsest PCRi katsest sõltuval madalamal temperatuuril (ehk praimerite sulamistemperatuuril Tm) ning seondunud praimerite pikendamist vastavalt spetsiifilisele DNA lõigu järjestusele kindla valgulise ensüümi abil. Paljundatud DNA-d detekteeritakse, kas spetsiifiliselt geelil pikkuse järgi või reaalajas produkti paljundamise käigus tekkiva signaali abil. PCR võimaldab sel viisil tuvastada suvalisest DNA proovist (kliiniline-, veterinaar-, toidu-, keskkonnaproov jne) vaid kindlale liigile iseäralikku DNA järjestust ning seetõttu on tehnoloogia leidnud rakendust erinevates valdkondades erinevate liikide või tüvede tuvastamiseks. Üks olulisemaid eeldusi edukaks PCRi teostamiseks on täpsete ja tundlikke praimerite disainimine (PCRi praimeridisain). PCRi praimeridisain sisaldab endas erinevaid etappe muuhulgas sihtmärkjärjestuse välja valimist (nt kindla järjestuse valimist bakterigenoomist) ning PCRi praimerijärjestuste disaini. Käesolev töö ongi keskendunud PCRi praimeridisaini erinevate etappide parendamisele: laialdaselt kasutusel oleva PCRi praimeridisaini programmi Primer3-e poolt kasutatava praimerite sulamistemperatuuri Tm arvutamise valemi täiustamisele ning sellele, kuidas prokarüootseid liigispetsiifilisi kordusjärjestusi PCRi sihtmärkjärjestustena kasutada ning, millise effekti see PCRi tulemustele annab. Viimase osana sisaldab töö prokarüootsete liigispetsiifiliste kordusjärjestuste iseloomustamist 613 erinevas prokarüootses liigis. Antud töö tulemused aitavad kaasa uute ja paremate molekulaardiagnostika testide loomisele esiteks lihtsustades nende väljatöötamist tänu antud töös leitud ja kirjeldatud PCRi sihtmärkjärjestustele, teiseks tõstes nende töökindlust, kuna liigispetsiifilistele kordusjärjestustele disainitud praimerid on kõrgendatud tundlikkusega ning, kuna parendatud praimeridisaini-programm Primer3 võimaldab disainida praimereid, mis vastavad täpsemalt etteantud molekulaardiagnostika testide tingimustele .Polymerase chain reaction or PCR is a method in molecular biology, which enables amplification of specific DNA regions. It is a cyclic process, that comprises of heat denaturation of double-stranded DNA (target sequence), hybridization of two short oligonucleotides (called PCR primers) to the denaturated target sequence at temperature specific to the PCR reaction (termed melting temperature Tm) and extension of hybridized primers by the enzyme DNA polymerase. The amplified DNA can be identified either specifically on electrophoresis gel by its product length or by monitoring the signal generated through activation of primer attached flourecent labels during the amplification in real-time. Thus, PCR enables identification of the DNA sequence of interest from an arbitrary DNA sample (e.g. clinical, veterinary, food, environmental sample) and therefore it is widely applied in different areas for particular species or strain identification. The design of specific and sensitive PCR primers is very important for the success of PCR. PCR primer design comprises of different steps, among which choice of the target sequence (e.g. certain DNA region in a bacterial genome) and the design of the primer sequences are crucial. Current thesis is focused on the improvement of the different steps of PCR primer design. First, enhancements to widely used PCR primer design program Primer3 are introduced. These enhancements enable to calculate primer melting temperature more accurately (includes modernization of the primer melting temperature formula as well as the auxiliary formulas that enable to take the concentrations of salt ions in the PCR reaction buffer into consideration). Second, we provide a methodology for finding the prokaryotic species-specific repeats and their application as PCR targets in primer design. Also, the positive impact to PCR sensitivity of inclusion of repetitive sequences as PCR targets is ascertained. The last part of this thesis covers the characterization of species-specific repetitive sequences in 613 different prokaryotic species. The results of current thesis facilitate the design of new and more reliable tests to molecular diagnostics because of the followings: first, new PCR target sequences are introduced, second, we have shown that primers designed to species-specific repetitive sequences increase the sensitivity of PCR and third enhanced primer design program enables to design primers that follow more precisely the preset conditions of tests in molecular diagnostics

Recovery and characterization of viral diversity from aquatic short- and long-read metagenomes

Viruses are the most abundant biological entities in marine ecosystems and play an essential role in global biogeochemical cycles. They have important ecological functions as drivers of bacterial populations through lytic infections and contribute to bacterial genetic diversification. Unfortunately, their study is severely limited by the difficulty to culture and isolate them in lab conditions. Culture-independent techniques such as metagenomics can complement culture-based approaches to capture more phage diversity. However, the vast majority of viral sequences recovered through these methods are uncharacterized and therefore do not provide any information about their interactions with the bacterial community, a phenomenon that has been named “viral dark matter”. In this thesis, several bioinformatic techniques are applied to both short- and long-read metagenomic datasets to recover biological information from marine viral sequences contained therein. A pipeline for recovering viral sequences based on a reference genome was developed and applied to the study of myophages infecting the alphaproteobacterial SAR11 clade, one of the most abundant bacterioplankton groups in surface marine and freshwater ecosystems. We were able to recover 22 new genomes which include the first genomes of myophages infecting LD12, the SAR11 freshwater clade. These sequences are underrepresented in datasets derived from the viral fraction, suggesting a bias of either technical or biological nature. Surprisingly, this family of phages code for an operon which resembles the secretion system type VIII operon in Escherichia coli. The function of this phage operon is still unknown. Next, a long-read dataset from the Mediterranean Sea was explored for viral contigs to contrast phage recovery between long- and short-read datasets. The analysis revealed that while long-read assemblies resulted in viral sequences of better quality, there was a sizable amount of intra-clade viral diversity that was not included in the assemblies. This viral diversity only found in long reads is even greater than previously thought. This untapped diversity could aid biotechnological efforts as evidenced by the discovery of new endolysins. Finally, a tool (Random Forest Assignment of Hosts, or RaFAH) for assigning hosts to phage sequences obtained from metagenomic datasets was created. The tool is based on a machine learning tool trained with phage protein clusters generated de novo. Benchmarking shows that RaFAH is on par with other state-of-the-art classifiers and is able to classify phage contigs at the level of Kingdom, which makes it the first classifier to accurately detect Archaea viruses from metagenomic samples. A feature importance analysis reveals that the protein clusters with the most predictive power are those involved in host recognition.Los bacteriófagos (”fagos”) son los organismos más abundantes en los ecosistemas marinos y tienen un papel esencial en los ciclos biogeoquímicos globales. Asimismo, influencian la evolución de las poblaciones bacterianas que infectan y contribuyen a la diversificación del acervo genético bacteriano. Desgraciadamente, su estudio se ve limitado por la dificultad de cultivar y aislar estos organismos en el laboratorio. El uso de técnicas que no requieren cultivo, como la metagenómica, pueden complementar el cultivo en laboratorio para recuperar una mayor diversidad de fagos. Sin embargo, la inmensa mayoría de secuencias virales recuperadas mediante metagenómica no pueden ser caracterizadas, por lo que no proporcionan ninguna información sobre sus interacciones con la comunidad bacteriana, un fenómeno que se ha nombrado “materia oscura viral”. En esta tesis se han utilizado múltiples procesos bioinformáticos en colecciones de metagenomas de lectura corta y larga para caracterizar las secuencias virales que contienen. Se ha desarrollado un procedimiento para recuperar secuencias virales a partir de un genoma de referencia y se ha aplicado al estudio de miofagos que infectan al clado SAR11 de las Alfaproteobacteria, uno de los grupos de bacterioplankton más abundantes en agua dulce y agua salada de superficie. Se consiguió recuperar 22 nuevos genomas que incluyen el primer genoma que infecta LD12, el subclado de SAR11 de agua dulce. Estos genomas están poco representados en colecciones obtenidas de la fracción viral, lo que sugiere que las afecta un sesgo técnico o biológico. Sorprendentemente, esta familia de fagos contiene un operón similar al sistema de secreción tipo VIII de Escherichia coli. La función de este operón es aún desconocida. Asimismo, se contrastó la recuperación de secuencias víricas entre colecciones de lectura corta y larga utilizando colecciones obtenidas en el mar Mediterráneo. Los resultados muestran que aunque los ensamblajes derivados de las lecturas largas producen secuencias virales de mejor calidad, en el proceso se pierde una gran cantidad de diversidad intraclado. Esta diversidad es mucho mayor de la recuperada con lecturas cortas, y podría explotarse para aplicaciones biotecnológicas, como el descubrimiento de nuevas endolisinas. Finalmente, se desarrolló un programa (Random Forest Assignment of Hosts, o RaFAH) para asignar hospedadores a secuencias virales obtenidas de colecciones metagenómicas. El programa se basa en el uso de algoritmos de machine learning entrenados con grupos de proteínas creados de novo. RaFAH muestra un rendimiento similar a otros clasificadores de secuencias y es capaz de clasificar secuencias víricas al nivel taxonómico de Reino, siendo así el primer clasificador capaz de detectar fagos que infectan arqueas con precisión. El análisis de importancia de rasgo revela que los grupos de proteínas con mayor poder predictivo son aquellos involucrados en el reconocimiento del hospedador

Integrative cell biology

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Bioinformatica en Biotecnología y BiomedicinaClave Programa: DBICódigo Línea: 7Las proteínas son la clave para entender la biología celular. La determinación de su rol y función nos ayuda a descubrir las características de los procesos moleculares en la base de la vida. Las técnicas de alto rendimiento han permitido a los científicos acumular una gran cantidad de datos sobre secuencias de ADN de miles de organismos diferentes. La función de las proteínas codificadas en estas porciones de ADN se determina por métodos de anotación manuales o automáticos, utilizando experimentos computacionales y biológicos para obtener una descripción coherente. Aunque la revisión manual de estas predicciones finalmente produce las anotaciones más fiables, este enfoque no es factible con la tasa actual de secuencias depositadas en las bases de datos biológicas. Esto afecta el conocimiento de la biología de varios organismos. Los esfuerzos de revisión manual se centran principalmente en la caracterización de organismos modelo En consecuencia, las bases de datos donde se reúne la información abarcan grandes cantidades de datos para un subconjunto específico de organismos. Actualmente, solo los grandes consorcios pueden generar estos recursos web, mientras que otros grupos que investigan organismos recientemente secuenciados carecen de los medios y recursos para lograr una anotación de proteoma más completa. Además, la gran mayoría del software para anotación de proteínas se enfoca solo en algunos aspectos de la función de una proteína; por lo tanto, falta información complementaria que podría derivarse de otras fuentes, tanto in silico como in vivo. El objetivo de esta tesis es desarrollar un nuevo enfoque para la anotación de funciones de proteínas que aborde los problemas mencionados anteriormente, incluidas nuevas herramientas y recursos para mejorar el estado actual en el ámbito de la predicción de la función, para así aplicarlo a organismos no modelos. Lo llamamos ¿Integrative Cell Biology¿ (ICB) o Biología Celular Integrativa. ICB se basa en la integración de varias fuentes de datos, incluyendo características de secuencia y estructura. De esta forma podemos obtener una anotación más amplia que proporciona al usuario una descripción más completa de una proteína. ICB también es capaz de visualizar múltiples proteínas de una manera fácil y rápida a través de un navegador web. Probamos el enfoque Integrative Cell Biology con una ¿pipeline¿ computacional resultante para caracterizar 39 proteomas del superfilo bacteriano Planctomycetes-Verrucomicrobia-Chlamydia (PVC). Además de su relevancia en varios campos, sus proteomas tienen un bajo porcentaje de proteínas anotadas, y solo unas pocas se han caracterizado experimentalmente. Sus propiedades fueron determinadas por observaciones experimentales, mientras que las secuencias que las codifican son en su mayoría desconocidas. Al aplicar el pipeline ICB, aumentamos drásticamente la cantidad de anotaciones de sus proteomas, abordando cuestiones biológicas sobre su comportamiento. Con el fin de hacer que nuestros hallazgos estén disponibles para la comunidad de investigación de PVC, creamos PVCbase, una plataforma única para examinar los resultados de ICB a través de DataTables, realizar búsquedas de secuencia basadas en homología y visualizar las características de la estructura secundaria de las proteínas. Para demostrar aún más las capacidades de ICB, analizamos tres Planctomicetos recientemente secuenciados asociados al entorno de macroalgas. Los genomas de Rubripirellula obstinata LF1, Roseimaritima ulvae UC8 y Mariniblastus fucicola FC18 se ensamblaron, se anotaron utilizando ICB, y se caracterizaron adicionalmente comparándolo con Planctomyces de otros ambientes. Posteriormente se complementaron sus rutas metabólicas y se evaluó su identidad a través de la filogenia. Tras los análisis pudo verse que algunas proteínas están involucradas en la interacción con los hospedadores de algas, incluidas algunas de tamaño extraordinario que merecen un análisis posterior. Se creó una versión de contenedor Docker de ICB que agiliza la instalación y el uso de pipelines, permitiendo que los grupos de investigación con intereses compartidos creen una plataforma similar a PVCbase. La salida de DataTables y la diversidad de herramientas incluidas permiten una transición fluida de secuencias a anotaciones de proteínas fácilmente navegables. Estos recursos crean entornos compartidos para analizar grandes conjuntos de proteínas, con poco o ningún conocimiento de codificación requerido. El concepto de Biología Celular Integrativa y sus recursos derivados contribuyen al campo de la predicción de la función de la proteína y proporcionan una solución en el caso de organismos mal anotados o recién secuenciados. PVCbase ha sido utilizado por varios grupos de investigación en microbiología de PVC (16 universidades de 14 países hasta agosto de 2018) y su base de usuarios se beneficiará de la adición de proteomas y de los análisis. Integrar varias fuentes de información para evaluar la función de la proteína es una posible solución a la inconsistencia y falta de fiabilidad de las herramientas de predicción. Al utilizar ICB, podemos responder preguntas que no podrían abordarse por otros medios. En el futuro, nuevas fuentes de información implementadas en ICB ampliarán nuestro conocimiento de varias características desconocidas de varios organismos.Universidad Pablo de Olavide de Sevilla. Escuela de DoctoradoPostprin

Annotation Error in Public Databases: Misannotation of Molecular Function in Enzyme Superfamilies

Due to the rapid release of new data from genome sequencing projects, the majority of protein sequences in public databases have not been experimentally characterized; rather, sequences are annotated using computational analysis. The level of misannotation and the types of misannotation in large public databases are currently unknown and have not been analyzed in depth. We have investigated the misannotation levels for molecular function in four public protein sequence databases (UniProtKB/Swiss-Prot, GenBank NR, UniProtKB/TrEMBL, and KEGG) for a model set of 37 enzyme families for which extensive experimental information is available. The manually curated database Swiss-Prot shows the lowest annotation error levels (close to 0% for most families); the two other protein sequence databases (GenBank NR and TrEMBL) and the protein sequences in the KEGG pathways database exhibit similar and surprisingly high levels of misannotation that average 5%–63% across the six superfamilies studied. For 10 of the 37 families examined, the level of misannotation in one or more of these databases is >80%. Examination of the NR database over time shows that misannotation has increased from 1993 to 2005. The types of misannotation that were found fall into several categories, most associated with “overprediction” of molecular function. These results suggest that misannotation in enzyme superfamilies containing multiple families that catalyze different reactions is a larger problem than has been recognized. Strategies are suggested for addressing some of the systematic problems contributing to these high levels of misannotation

Characterisation of three bacteriophages infecting serovars of salmonella enterica

A collection of three newly isolated Salmonella bacteriophages, vB_SenS-Ent1, vB_SenS-Ent2 and vB_SenS-Ent3 was established. These bacteriophages were characterised by electron microscopy, host range, sensitivity to restriction enzymes and profiles of structural proteins on SDS-PAGE gels. The complete genome sequences of each bacteriophage were established to greater than 30x coverage and bioinformatics analysis identified the functions of a number of coding sequences. Since the last update of virus taxonomy by the ICTV a number of additional genome sequences for bacteriophages infecting the genus Salmonella have been reported in the literature. To date, all but one of the Siphoviridae comprising the Salmonella bacteriophages with fully sequenced genomes remain unclassified by the ICTV. Comparative genomic analysis reveals that a number of these phages form a coherent group within the Siphoviridae and supports the establishment of a new genus, the “Setp3likeviruses”. The proposed genus includes 5 bacteriophages infecting Salmonella; SETP3, vB_SenS-Ent1, SE2, wksl3 and SS3e, and 5 infecting Escherichia; K1G, K1H, K1ind1, K1ind2 and K1ind3. This group share identical virion morphology, have terminally redundant, circularly permuted genomes ranging between 42-45 kb in size and are characterised by high nucleotide sequence similarity, shared homologous proteins and conservation of gene order.Bioluminescent bacterial reporters, transformed to express the luxCDABE operon of Photorhabdus luminescens, were used to establish the activity of the vB_SenS-Ent bacteriophages in microtitre broth lysis assays, efficacy as biological control agents for the removal of Salmonella in contaminated foods and for spatial measurements of plaque expansion in agar overlays

Comparative genomics for studying the proteomes of mucosal microorganisms

A tremendous number of microorganisms are known to interact with their animal hosts. The outcome of the interactions between microbes and their animal hosts range from modulating the maintenance of homeostasis to the establishment of processes leading to pathogenesis. Of the numerous species known to inhabit humans, the great majority live on mucosal surfaces which are highly defended. Despite their importance in human health, little is known about the molecular and cellular basis of most host-microbe interactions across the tremendous diversity of mucosal-adapted microorganisms. The ever-increasing availability of genome sequence data allows systematic comparative genomics studies to identify proteins with potential important molecular functions at the host-microbe interface. In this study, a genome-wide analysis was performed on 3,021,490 protein sequences derived from 867 complete microbial genome sequences across the three domains of cellular life. The ability of microbes to thrive successfully in a mucosal environment was examined in relation to functional genomics data from a range of publicly available databases. Particular emphasis was placed on the extracytoplasmic proteins of microorganisms that thrive on human mucosal surfaces. These proteins form the interface between the complex host-microbe and microbe-microbe interactions. The large amounts of data involved, combined with the numerous analytical techniques that need to be performed makes the study intractable with conventional bioinformatics. The lack of habitat annotations for microorganisms further compounds the problem of identifying the microbial extracytoplasmic proteins playing important roles in the mucosal environments. In order to address these problems, a distributed high throughput computational workflow was developed, and a system for mining biomedical literature was trained to automatically identify microorganisms’ habitats. The workflow integrated existing bioinformatics tools to identify and characterise protein-targeting signals, cell surface-anchoring features, protein domains and protein families. This study successfully demonstrated a large-scale comparative genomics approach utilising a system called Microbase to harness Grid and Cloud computing technologies. A number of conserved protein domains and families that are significantly associated with a speiii iv cific set of mucosa-inhabiting microorganisms were identified. These conserved protein regions of which their functions were either characterised or unknown, were quite narrow in their coverage of taxa distribution, with only a few protein domains more widely distributed, suggesting that mucosal microorganisms evolved different solutions in their strategies and mechanisms for their survival in the host mucosal environments. Metabolic and biological processes common to many mucosal microorganisms included: carbohydrate and amino acid metabolisms, signal transduction, adhesion to host tissues or contents in mucosal environments (e.g. food remnants, mucins), and resistance to host defence mechanisms. Invasive or virulence factors were also identified in pathogenic strains. Several extracytoplasmic protein families were shared among prominent bacterial members of gut microbiota and microbial eukaryotes known to thrive in the same environment, suggesting that the ability of microbes to adapt to particular niches can be influenced by lateral gene transfer. A large number of conserved regions or protein families that potentially play important roles in the mucosa-microbe interactions were revealed by this study. Several of these candidates were proteins of unknown function. The identified candidates were subjected to more detailed computational analysis providing hypothesis for their function that will be tested experimentally in order to contribute to our understanding of the complex host-microbe interactions. Among the candidates of unknown function, a novel M60-like domain was identified. The domain was deposited in the Pfam database with accession number PF13402. The M60-like domain is shared amongst a broad range of mucosal microorganisms as well as their vertebrate hosts. Bioinformatics analyses of the M60-like domain suggested a potential catalytic function of the conserved motif as gluzincins metalloproteases. Targeting signals were detected across microbial M60-likecontaining proteins. Mucosa-related carbohydrate-binding modules (CBMs), CBM32 was also identified on several proteins containing M60-like domains encoded by known mucosal commensals and pathogens. The co-occurrence of the CBMs and M60-like domain, as well as annotated potential peptidase function unveiled a new functional context for the CBM, which is typically connected with carbohydrate processing enzymes but not proteases. The CBM domains linked with members of different protease families are likely to enable these proteases to bind to specific glycoproteins from host animals further highlighting the importance of proteases and CBMs (CBM32 and CBM5_12) in host-microbe interactions.EThOS - Electronic Theses Online ServiceMedical School, Newcastle UniversityGBUnited Kingdo

Between adaptation and virulence:A proteomics view on Staphylococcus aureus infections

Staphylococcus aureus is one of the commonly encountered bacteria of the human microbiome. Although mostly a seemingly harmless commensal microbe, S. aureus can act as an invasive pathogen with seriously devastating effects on its host’s health and wellbeing. A wide range of infections caused by this bacterium has been reported to affect diverse parts of the human body, including the skin, soft tissues and bones, as well as important organs like the heart, kidneys and lungs. Particularly, S. aureus is infamous for being a major causative agent of respiratory tract infections that may escalate up to necrotizing pneumonia. Due to its clinical relevance, this pathogen has been intensively studied for many years. Nonetheless, further research in this field is still needed, because of the high capacity of S. aureus to evolve drug resistance, its high genomic plasticity and adaptability and, not in the last place, the plethora of niches within the human body where it can thrive and survive. In this regard, there are still many uncertainties concerning the specific adaptations carried out by S. aureus during colonization and infection of the human body, the transition between both stages, and upon the invasion of different types of host cells. To shed more light on some of these adaptations, the research described in this thesis has employed in vitro models of infection that mimic particular conditions during the infectious process with special focus on the lung epithelium. The adaptations displayed by S. aureus were monitored using advanced proteomics. Furthermore, the analyses documented in this thesis included S. aureus strains with diverse backgrounds and epidemiology to take into account the genetic diversity encountered in this species.Staphylococcus aureus ist eines der am häufigsten vorkommenden Bakterien im menschlichen Mikrobiom. Auch wenn sich S.aureus meist eher harmlos verhält, kann diese Mikrobe als invasiver Krankheitserreger bedrohliche Auswirkungen auf die Gesundheit und das Wohlbefinden von Patienten haben. Ein breites Spektrum an Infektionen, die durch dieses Bakterium ausgelöst werden, kann verschiedenste Körperteile des Menschens wie Haut, Weichteile und Knochen, sowie Lebenswichtige Organe wie das Herz, die Nieren und die Lunge betreffen. S. aureus ist insbesondere als ein Haupterreger von Atemwegsinfekten bekannt, welche sich bis zu einer nekrotisierenden Lungenentzündung entwickeln können. Aufgrund seiner klinischen Bedeutung wird dieser Kranksheitserrger bereits seit Jahren intensiv untersucht. Dennoch ist eine Erforschung von S. aureus wegen dessen Fähigkeiten Antibiotikaresistenzen zu entwickeln, der hohen genomischen Plastizität und Anpassungsfähigkeit und nicht zuletzt, der Vielzahl an Nischen im menschlichen Körper, wo es gedeihen und überleben kann, weiter notwendig. Die spezifischen Anpassungen von S. aureus während der Besiedlung und Kolonisierung des menschlichen Körpers, während des Übergangs zwischen diesen beiden Phasen sowie nach Eindringen in verschiedene Wirtszelltypen sind bisher nur unvollständig aufgeklärt. Um im Rahmen dieser Doktorabeit einige Einblicke in diese Anpassungen zu erhalten, wurden in vitro Infektionsmodelle eingesetzt, die bestimmte Bedingungen des Infektionsprozesses speziell im Lungenepithel widerspiegeln. Diese Anpassungen von S. aureus wurden mittels moderner Proteomanalysen untersucht. Weiterhin wurden im Rahmen dieser Arbeit S. aureus Stämme verschiedenen Ursprungs in die Analysen einbezogen, um auch die genetische Vielfalt dieser Spezies zu berücksichtigen