No description available.

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第25215号 / 人健博第121号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 澤本 伸克, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Pathogen-containing droplets expelled into the air as infected individuals speak, cough, sneeze, or laugh may infect proximal secondary hosts. Facemasks are an effective, low-cost method to help prevent airborne transmission mediated by pathogen-laden droplets in the environment. To understand the testing of facemask materials to protect against airborne pathogens, we followed the American Society for Testing and Materials (ASTM) standard F2101-12 and built an in-house test platform. However, the standard lacks clear guidelines. To address this, we built an aerosol platform at McMaster University to generate and test droplets containing viable bacteria, allowing us to assess the filtration performance of different facemask materials. We created optimized procedures to ensure the generation and sampling of infectious aerosols are consistent and reliable. The operation, maintenance, and sterilization of the platform were also detailed. Airborne disease transmission is a complex phenomenon influenced by multiple factors. Conditions such as muco-obstructive airway disorders can increase the viscosity and solute content of airway lining fluid. Therefore, individual variability in airway mucus composition and initial droplet size may influence transmission dynamics. Thirdly, environmental conditions such as temperature and humidity can change the aerosol microenvironment and alter encapsulated pathogen viability. Evidence suggests infectious aerosols originate from the breakup of the airway lining fluid, and the site of origin within the airways can affect the size of pathogen-laden droplets. Using the bacteriophage Phi6 as a stand-in for the SARS-CoV-2 virus, we examined how droplet size, mucus composition, pathogen load, and environmental conditions (temperature and humidity) affect pathogen survival. Our findings may provide insight into the dynamics of airborne transmission and can help improve strategies to reduce the spread of respiratory infections. Lastly, we adapted the platform to conduct in vitro lung exposure studies of deposition, safety, and efficacy of bactericidal agents. The human airway epithelial cell line, Calu-3, cultured in an air-liquid interface system, was used to recapitulate the physiological characteristics of the respiratory mucosa. Pseudomonas aeruginosa biofilms cultured for 24 hours were placed within the impactor and exposed to aerosolized bactericidal agents. Preliminary results show that our cell-integrated exposure platform can assess in vitro safety, efficacy, and dosage of inhaled therapeutics. This setup can potentially help gather pre-clinical data to support in vivo studies of treatments for respiratory diseases. This platform has significant potential for expanding aerosol research at McMaster University to evaluate the effectiveness of personal protective equipment, study how infectious aerosols spread, and explore inhaled delivery of therapeutic agents to the lungs. These findings contribute to our understanding of airborne transmission and may inform strategies to reduce the spread of infections. / Thesis / Doctor of Philosophy (PhD) / Many respiratory infections are spread through the air by pathogen-containing droplets released from the airways of infected individuals as they breathe, talk, cough, sneeze, or laugh. Facemasks are an easy-to-adopt infection control strategy to guard against airborne infectious agents. An aerosol platform was built at McMaster University amidst the coronavirus pandemic to perform facemask material filtration tests using information from the American Society for Materials and Testing standard F2101-19. However, we soon identified ambiguous aspects of the standard that can potentially prevent the reliable operation of the test platform and the gathering of reproducible test results. The first objective was to modify the test setup and develop precise procedures to address ambiguities of the standard. Modifications of the platform and detailed procedures ensured more accurate and consistent testing of facemask materials. Next, we used the platform to study how long pathogens stay viable within aerosol droplets. Droplets containing the bacteriophage Phi6, a surrogate for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were used to study how droplet size, mucus composition, and environmental conditions (like temperature and humidity) affect pathogen survival. Lastly, we adapted our aerosol platform to investigate the inhaled delivery of antibacterial agents to target lung infections. Using cultures of human airway epithelial cells and Pseudomonas aeruginosa biofilms, we tested the antibacterial efficacy of aerosolized antibiotics and bacteriophages (viruses that kill bacteria). In summary, this thesis work has developed an aerosol platform for conducting reliable material filtration efficiency tests, probing virus stability within airborne droplets, and modelling inhaled drug delivery to the lungs.

Anticorpos são moléculas de grande interesse científico e farmacêutico, principalmente, devido a sua alta especificidade contra antígenos determinados. Atualmente, anticorpos monoclonais estão entre os medicamentos (biofármacos) mais vendidos do mundo. São utilizados para o tratamento das mais diversas doenças, como câncer, retinopatias, doenças inflamatórias e do sistema imune, entre outras. Nos últimos 30 anos, as tecnologias para a obtenção de anticorpos monoclonais evoluíram muito, desde a tecnologia do hibridoma, até os processos de humanização de anticorpos murinos. Entre os métodos mais utilizados para a produção de anticorpos humanos, destaca-se a tecnologia do Phage Display. Nesta técnica, os genes que codificam as regiões variáveis de imunoglobulinas são inseridos no genoma de um bacteriófago, resultando na produção de partículas virais híbridas que contém fragmentos de anticorpos em fusão com uma das proteínas do capsídeo viral. Neste trabalho, desenvolvemos novos vetores para a apresentação de fragmentos ScFv em fusão com duas proteínas das proteínas do capsídeo viral, a pIII e pVIII. Os oligonucleotídeos utilizados para amplificar os genes de imunoglobulinas foram redesenhados e para minimizar a perda do repertório durante a produção da biblioteca, avaliamos em bancos de dados enzimas de restrição que não apresentam sítios de restrição nas sequencias gênicas. Esses sítios de restrição foram utilizados para construir as regiões de clonagem do vetor Phagemid. Outra etapa crítica na produção de bibliotecas de anticorpos é a reação do PCR de overlap, que pode restringir a diversidade de anticorpos e resultar na produção de amplicons codificando anticorpos truncados. Por isso, nossos vetores foram desenhados para permitir a clonagem direta das regiões variáveis das imunoglobulinas humanas ou murinas, sem a necessidade do PCR de overlap. Nossa expectativa, é que estes novos reagentes serão mais efetivos para a produção de novas bibliotecas de anticorpos pelo sistema do Phage Display. / Antibodies are molecules of great scientific and pharmaceutical interest, mainly because of their high specificity against certain antigens. Currently, monoclonal antibodies are among the best selling drugs (biopharmaceuticals) in the world. They are used for the treatment of the most diverse disorders, such as cancer, retinopathies, inflammatory and immune system diseases, among others. In the past 30 years, technologies for obtaining monoclonal antibodies has greatly evolved from hybridoma technology to the humanization processes of murine antibodies. Among the methods used for the production of human antibodies, the technology of Phage Display stands out. In this technique, the genes encoding the immunoglobulin variable regions are inserted into the genome of a bacteriophage, resulting in the production of hybrid virus particles which contain fragments of antibodies in fusion with one of the viral capsid proteins. In this work, we developed new vectors for the presentation of ScFv fragments in fusion with two proteins of viral capsid proteins, pIII and pVIII. The oligonucleotides used to amplify the immunoglobulin genes were redesigned and to minimize repertory loss during library production, we evaluated restriction enzymes in databases that lack restriction sites in the gene sequences. These restriction sites were used to construct the cloning regions of the Phagemid vector. Another critical step in the production of antibody libraries is the overlap PCR reaction, which may restrict the diversity of antibodies and result in the production of amplicons encoding truncated antibodies. Therefore, our vectors were designed to allow the direct cloning of human or murine Immunoglobulins variable regions without the need for overlap PCR. Our expectation is that these new reagents will be more effective for the production of new antibody libraries by the Phage Display system.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes. / Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions.

Noroviruses are the major cause of acute viral gastroenteritis worldwide. Thus, rapid and reliable pathogen detection and control are crucial to avoid epidemic outbreaks. Peptides which bind to these viruses with high specificity and affinity could serve as small and stable recognition elements in biosensing applications for a point-of-care diagnostic of noroviruses. They can be identified by screening large phage display libraries using the biopanning technique. In the present study, this method was applied to identify norovirus-binding peptide motifs. For this purpose, a biopanning based on column chromatography was established, and three rounds of selections were performed. After the second round, the cosmix-plexing recombination technique was implemented to enhance the chance of obtaining peptides with very high affinity. Biopanning data evaluation was based on next-generation sequencing (NGS), to show that this innovative method can enable a detailed analysis of the complete sequence spectrum obtained during and after biopanning. Highly enriched motifs could be characterized by their large proportion of the amino acids W, K, R, N, and F. Neighbourhood analysis was exemplarily performed for selected motifs, showing that the motifs FAT, RWN, and KWF possessed the fingerprints with the largest differences relative to the original library. This thesis thus presents next-generation sequencing-based analysis tools, which could now be transferred to any other biopanning project. The identified peptide motifs represent promising candidates for a future examination of their norovirus-specific binding. A new option for testing such phage-target interactions in the context of biopanning selections was studied in the second part of the thesis. For this purpose, a phage-based microarray was developed as a miniaturized binding assay. As a prerequisite, the different immobilization behaviour of phages on positively and negatively charged surfaces was studied, and a non-contact printing technique for bacteriophages was developed. Subsequently, the interaction of phages and antibodies directed against phage coat proteins was characterized in enzyme-linked immunosorbent assays, and the protocol was successfully transferred to the non-contact printed phage spots. At the proof-of-concept level, the phage array could finally be integrated into a microfluidic setup, showing a higher signal-to-background ratio relative to the static phage array. These results point the way towards a microfluidic phage array, allowing online monitoring, automation, and parallelisation of the phage array analysis. / Noroviren gelten als Hauptursache akuter viraler Magen-Darm-Erkrankungen. Nur eine zeitnahe und verlässliche Detektion und Kontrolle dieser Pathogene kann epidemische Ausbrüche vermeiden. Um dies zu ermöglichen, könnten Peptide, die an diese Viren mit hoher Spezifität und Affinität binden, als kleine und stabile Erkennungselemente in biosensorischen Anwendungen eingesetzt werden. Solche Peptide können mithilfe der Biopanning-Technik identifiziert werden, die auf dem Screening großer Phagen-Display-Bibliotheken beruht. In der vorliegenden Arbeit wurde diese Methode genutzt, um Norovirus-bindende Peptidmotive zu identifizieren. Dazu wurde ein auf Säulenchromatographie basierendes Biopanning entwickelt und drei Selektionsrunden durchgeführt. Die Cosmix-Plexing-Rekombinationstechnik wurde nach der zweiten Runde eingesetzt, um die Wahrscheinlichkeit der Gewinnung hochaffiner Binder zu erhöhen. Die Auswertung der Biopanningdaten erfolgte mittels Hochdurchsatzsequenzierung (Next-Generation Sequencing). Es konnte gezeigt werden, dass diese innovative Methode die detailierte Analyse des kompletten Sequenzspektrums während und nach dem Biopanning ermöglicht. Stark angereicherte Motive konnten durch ihren hohen Anteil an den Aminosäuren W, K, R, N und F charakterisiert werden. Eine Nachbarschaftsanalyse wurde exemplarisch für ausgewählte Motive durchgeführt. Dabei wurden die stärksten Unterschiede im Fingerprint im Vergleich zur Ausgangsbibliothek bei den Motiven FAT, RWN und KWF gefunden. Diese Dissertation stellt damit auf Next-Generation Sequencing basierende Analysetechniken vor, die für weitere Biopanningprojekte übernommen werden können. Die identifizierten Peptidmotive könnten als vielversprechende Kandidaten zukünftig auf ihre Norovirus-spezifische Bindung hin getestet werden. Eine neue Möglichkeit, solche Phagen-Analyt-Interaktionen zu untersuchen, wurde im zweiten Teil der Dissertation untersucht. Dafür wurde als miniaturisierter Bindungsassay ein Phagen-basiertes Mikroarray entwickelt. Als Voraussetzung wurde zunächst das unterschiedliche Immobilisierungsverhalten von Bakteriophagen auf positiv und negativ geladenen Oberflächen untersucht und eine kontaktfreie Drucktechnik für Bakteriophagen etabliert. Anschließend wurde die Interaktion von Phagen und gegen sie gerichteten Antikörpern in Enzym-gekoppelten Immunadsorptionstests charakterisiert und das Protokoll erfolgreich auf die kontaktfrei gedruckten Phagenspots übertragen. Schließlich wurde erstmals die grundsätzliche Möglichkeit gezeigt, das Array in ein mikrofluidisches Setup zu integrieren, was zu einem höheren Signal-zu-Hintergrund-Verhältnis im Vergleich zum statischen Array führte. Diese Ergebnisse zeigen damit den Weg zu einem mikrofluidischen Phagen-Array auf, das sowohl die Möglichkeit des Online-Monitorings als auch der Automatisierung und Parallelisierung der Phagen-Array-Analyse bietet.

La recherche de nouvelles molécules à visée thérapeutique ou diagnostic ciblant les récepteurs membranaires incluant les RCPGs, les récepteurs à tyrosine kinase et les canaux ioniques sont au coeur des recherches investies par les entreprises pharmaceutiques. Dans ce projet nous avons étudié et caractérisé des domaines variables de chaîne lourde d'anticorps de lamas (sdAbs) qui peuvent contourner certaines limites liées à l'utilisation des anticorps monoclonaux ou des petites molécules. En effet, de par leurs particularités structurales qui les rendent particulièrement intéressants en termes de stabilité, d'affinité et de reconnaissance d'antigène, les sdAbs représentent etre une alternative prométeuse. Dans ce manuscrit sont exposés les travaux effectués sur les récepteurs aux tyrosines kinases appartenant à la famille des ErbBs et les récepteurs au glutamate mGluRs (RCPG). En plus d'avoir sélectionné des sdAbs hautement spécifiques de ces différents antigènes, ces molécules ont également montré des caractéristiques très étonnantes et inattendues. En effet, la majeure partie des sdAbs sélectionnés présentent une spécificité pour une conformation du récepteur particulière (forme active ou inactive). Cette particularité très pertinente nous ouvre un spectre d'application très diversifié, car elle permet de cibler et d'analyser les récepteurs dans ces différents états d'activation. Ajouté à cela, certains sdAbs possèdent une activité de modulateur allostérique, voir même présentent un effet agoniste. Ces résultats très encourageants nous ouvrent de nouvelles perspectives, et ces molécules représentent une nouvelle approche pour la modulation et l'étude des ces récepteurs. / The research for new therapeutic or diagnostic molecules targeting membrane receptors, including GPCRs, tyrosine kinase receptors and ion channels are the heart of the research invested by pharmaceutical companies. In this project we used the variable domain of llama antibody heavy chain also called single domain antibodies (sdAbs) that can bypass some limitations to the use of monoclonal antibodies or small molecules. Indeed, because of their structural features that make them particularly interesting in terms of stability, affinity and antigen recognition, sdAbs represent a very promising candidates that can be used in various fiels of application: as fluorescent probes , screening tools , or therapeutic molecules. In this manuscript are exposed the work performed on the tyrosine kinases receptor belonging to the ErbBs family and metabotropic glutamate receptors, mGluRs (GPCRs).We selected very highly specific sdAbs directed against antigen of interest, but these molecules have also shown very surprising and unexpected particularities. Indeed, most of the selected sdAbs exhibit specificity for a particular conformation of the receptor (active or inactive form). This very relevant feature opens an highly diversified application spectrum, because it allows to identify and analyze these receptors in different states of activation. Added to this, some sdAbs present an allosteric modulator activity, or even present an agonist effect. These encouraging results open up new perspectives, and these molecules represent a new approach for modulation and study of these membrane receptors.

L'excision de la méthionine N-terminale (NME) est la première modification se produisant au N-terminal des protéines (NPM). Les peptides déformylases (PDF) sont les enzymes impliquées dans ce processus co-traductionnel essentiel et conservé. Les PDFs suppriment le groupe formyle lié à la méthionine initiatrice (iMet) présente au début de toutes les chaînes procaryotes naissantes. Les PDFs agissent au niveau du tunnel de sortie des ribosomes, plaque tournante de nombreux facteurs de biogénèse des protéines associées aux ribosomes (PRB), impliqués non seulement sur les MNP, mais également dans le repliement et la translocation des protéines. La déformylation N-terminale implique 95% du protéome bactérien et contribue directement à la stabilité des protéines. Le récent séquençage à haut débit de milliers de génomes a révolutionné notre perception de la distribution des PDFs dans les différents règnes, révélant des PDFs putatives dans tous les organismes, y compris les virus. En particulier, les études concernant les virus présents dans les échantillons microbiens océaniques ont permis d’identifier des gènes inhabituels de PDF chez les phages, constituant la famille la plus abondante de protéines auxiliaires conservées de ces génomes. La comparaison des séquences identifiées révèle que les PDF virales présentent une forte conservation des trois motifs constituant le site catalytique. Cependant, ces PDFs virales ne présentent pas d'extension C-terminale, région réputée importante des PDFs des autres organismes. Sachant que cette extension est impliquée dans la liaison de la PDF d’E. coli au ribosome et est requise pour son activité déformylase in vivo, il était incertain que les PDFs de phage découvertes avaient une activité déformylase classique. Ainsi, la découverte de ces PDFs virales soulève un certain nombre de questions parmi lesquelles: a) Ces PDFs virales présentent-elles une activité déformylase classique? b) Ces PDFs sont-elles capables de se lier aux ribosomes ? c) Pourquoi autant de virus portent-ils une ou plusieurs déformylases ? Dans ce contexte, l’objectif de ma thèse a été d’entreprendre la caractérisation de ces PDFs de phages marins et en particulier la PDF de Vp16 provenant de bactériophages isolés à l’origine de la souche 16 de Vibrio parahaemolyticus. Nos études révèlent que ces PDFs de phages présentent une activité déformylase à la fois in vitro et in vivo, avec une spécificité de substrat similaire à celle des autres PDFs bactériennes. D'autre part, nous avons montré par des études biochimiques et structurales, combinées à des analyses par mutagenèse dirigée, que les propriétés de la PDF de Vp16 diffèrent significativement de celle des autres PDFs caractérisées précédemment. Il faut aussi noter que l'expression de la PDF Vp16 dans les souches d'E. Coli, même à de faibles concentrations, montre un effet bactéricide marqué à une température inférieure à 37 °C. L’effet bactéricide de la PDF Vp16 est indépendant de la présence de la PDF endogène bactérienne et repose strictement sur son activité déformylase. La caractérisation de ce phénotype a révélé que la létalité induite par Vp16 PDF montrait un lien génétique fort avec des gènes codants pour des facteurs cellulaires impliqués dans le ciblage et le repliement précoce des protéines (Trigger Factor et Sec). Contrairement à ce qui a été montré pour les PDFs bactériennes, Vp16 PDF a une forte affinité pour le ribosome bactérien d’E. coli en cours de traduction, interagissant avec une région ribosomale chevauchant celles des facteurs impliqués dans le transit des protéines vers les voies de sécrétion. Une compétition au niveau du ribosome entre Vp16 PDF et ces RPBs pourrait contribuer à la lyse cellulaire de l’hôte. Mon travail suggère un nouveau mécanisme utilisé par les bactériophages permettant de contrôler la viabilité de l'hôte. / N-terminal Methionine Excision (NME) is the first occurring N-terminal Protein Modification (NPMs). Peptide deformylases (PDFs) are the enzymes involved in this essential and conserved co-translational process. PDFs remove the formyl group bound to the iMet present at the beginning of all prokaryotic nascent chains. PDFs act on the nascent chain at the level of the ribosome exit tunnel, a central hub for a number of Ribosome-associated Protein Biogenesis factors (RPBs) involved not only on NPMs but also in protein folding and translocation. Deformylation involves 95% of bacterial proteome and it is suggested to directly contribute to protein stability. Recent high-throughput sequencing of thousands of genomes has strongly contributed to revolutionizing our perception of the distribution of PDFs among kingdoms, revealing putative PDFs in all organisms, including viruses. In particular, studies of viruses within oceanic microbial samples retrieved unusual PDFs genes as the most abundant family in most of phage genomes. Sequence comparisons reveal that viral PDFs show high conservation in the three motifs that build the catalytic site; however, viral PDFs do not display a C-terminal extension when compared to the different active PDFs from other organisms. Since this C-terminal extension was shown to be important for PDF-ribosome binding and is required for the in vivo deformylase activity of E. coli PDF, it was unclear whether the discovered phage PDFs might support a classical deformylase activity. Thus, the discovery of these viral PDFs raises a number of questions among which: a) Have these viral PDFs a classical deformylase activity? b) Are these PDFs able to still bind to the ribosomes? c) Why so many viruses carry a peptide deformylase? In this context, the objective of my thesis was to undertake the characterization of these marine phage PDFs and particularly Vp16 PDF derived from the bacteriophages originally isolated from Vibrio Parahaemolyticus strain 16. Our studies reveal that phage PDFs display deformylase activity both in vitro and in vivo with a substrate specificity similar to that of other bacterial PDFs. On the other hand, we showed by biochemical and structural data, combined with site-directed mutagenesis analyses, that Vp16 PDF significantly differs from previously characterized PDFs in terms of their properties, which can be related to its few uncommon peculiarities. Interestingly, expression of Vp16 PDF in E. coli strains, even at low concentrations, exhibited a severe bactericidal effect at temperature lower than 37 °C. This bactericidal effect of Vp16 PDF was independent of the presence of the bacterial endogenous PDF and strictly relied on its PDF activity. Characterization of this phenotype revealed that Vp16 PDF-induced lethality showed a strong genetic link with genes encoding cellular factors involved in nascent pre-secretory protein targeting and folding (Trigger Factor and Sec). Differently from bacterial PDF, I could show that Vp16 PDF has strong affinity for ribosomes with a specific nascent chain, interacting with a ribosomal region overlapping that of factors involved in pre-secretory protein targeting. A competition between Vp16 PDF and these RPBs at the level of the ribosome may contribute to the host lysis, revealing a possible new unrecognized mechanism developed by viruses to control host viability.

The Paenibacillaceae family of bacteria includes two species known to infect the hives of honeybees, Paenibacillus larvae and Brevibacillus laterosporus. P. larvae, the causative agent of American Foulbrood (AFB) causes a lethal infection of honeybee larvae, while B. laterosporus is a secondary invader following European Foulbrood (EFB) infection. Increasing antibiotic resistance of P. larvae bacteria has prompted a search for alternative treatment methods for this disease. Bacteriophages are the most diverse life forms on earth and can provide important insights about the bacterial hosts they infect. However, few Paenibacillaceae phages have been isolated or characterized. In this study, the first B. laterosporus phages are characterized with respect to host range, structural morphology, and sequence similarity. The isolation and characterization of many P. larvae field isolates together with 38 novel P. larvae phages made possible the first broad phage typing study of P. larvae. Phage typing data indicated that P. larvae strains tested could be categorized into one of two groups. Comparative genomics of bacteriophages was made easier by modifying Phamerator to make it broadly accessible and usable to phage researchers throughout the world. Additionally, raw sequencing data can now be used to identify phage DNA packaging strategies that are indicative of a phage’s physical ends. Using these data, phage genomes can be published in an orientation and complementarity that reflects the physical structure of the phage chromosome, providing order and consistency that will benefit all future phage researchers.

Plasmid DNA (pDNA) vectors are the current conventional technology driving therapeutic gene transfer, whether for use toward mal/nonfunctional gene replacement, DNA vaccination, or production of therapeutic proteins in mammalian cells. However, the conventional pDNA vector suffers from several safety and efficiency limitations: 1) it imparts adverse immune responses to bacterial sequences required for maintenance and amplification in prokaryotes; 2) its bioavailability can be compromised due to size; and 3) it may be genotoxic due to its potential to integrate into the host chromosome and yield an oncogenic event. In this study we have constructed a robust in vivo bacterial platform for the production of bacterial sequence-free linear covalently closed (LCC) DNA vectors, termed DNA Ministrings, through the manipulation and application of bacteriophage-encoded recombination systems. Phage N15 and PY54 lysogenize their bacterial hosts as a linear plasmid with covalently closed ends (LCC plasmid). LCC morphology is conferred by the phage-encoded telomerase via a single cleaving-joining reaction of the perfect palindrome target site. This system was exploited to generate DNA Ministring vectors, encoding only the gene(s) of interest and necessary complementary eukaryotic expression/enhancement genetic elements that are devoid of unwanted bacterial sequences and are linearized through a single in vivo enzymatic reaction. The tel and telN prokaryotic telomerase (protelomerase) genes were amplified from PY54 and N15 lysates, respectively, and cloned into a bacterial vector that expresses the gene under control of the temperature sensitive bacteriophage ?? CI857 repressor that confers conditional expression from ?? pL/pR promoters. This regulatory circuit was integrated into a RecA+ lacZ+ E. coli K-12 strain via homologous recombination, where successful recombinants were disrupted for the lacZ gene. Recombinant cells are capable of conditional expression of the phage-derived telomerase enzymes by shifting the temperature to >37 ??C. Phage P1-derived Cre recombinase was applied as a positive control, since its functionality in generating DNA minicircle vectors has been previously shown. A multi-purpose 342 bp target site termed Super Sequence (SS) that possesses the Cre, Flp, Tel, and TelN target sites in addition to two flanking SV40 enhancer sequences was cloned into two different sites of a GFP expression eukaryotic pDNA vector. The amplification of this DNA vector through telN / tel or cre expressing Recombinant E. coli cells (R-cells) generated bacterial sequence-depleted (LCC) DNA Ministring and (CCC) Minicircle vectors, respectively, as evidenced by digestion patterns of the purified vector. Transfection efficiency of these vectors was assessed in rapidly dividing human ovarian cancer and in relatively slowly dividing human embryonic kidney cell lines. In vitro experiments with DNA Ministrings in human cells lines resulted in significantly higher transfection efficiency, bioavailability, and cytoplasmic diffusion levels compared to the parental plasmid precursor and isogenic DNA Minicircle counterparts. The safety of the LCC DNA vector conformation, with respect to insertional genotoxicity, was assessed by forcing LCC pDNA vectors into bacterial and human genomic DNA. The integration of LCC DNA into bacterial and human host genomic DNA resulted in chromosomal DNA disruptions at site of integration, loss of genome stability, and subsequent cell death. LCC integration-induced apoptotic cell death and natural elimination of the integrant from human cell population improves the safety profile of DNA Ministrings by eliminating integrants following the potential genotoxic side effects of undesired vector integration into the host genome.