Bacteriophages (phages) are viruses that infect bacteria. The phages that are described by this dissertation encompass 2 classes, double-stranded DNA phages and single-stranded RNA phages. While both of these phages infect similar bacteria, they have adopted different mechanisms to lyse, or destroy, the cell in order to release phage progeny. dsDNA phages have large genomes (> 20 kb) and use multiple lysis proteins (holin, endolysin, and spanin complex) to lyse the cell. ssRNA phages, on the other hand, have small genomes (< 6 kb) and only encode one lysis protein. The two X-ray crystallography projects outlined here deal with the phage proteins involved in these lysis mechanisms. The project described in the first study deals with the holin (T) and the antiholin (RI) of the ds-DNA phage T4, the major players of the lysis inhibition (LIN) phenomenon. Crystal structures of the holin and of the holin-antiholin complex are presented. The structures provide new molecular level insights into the phenomenon of LIN in bacteriophage T4 and the T-even phages in general. The second investigation describes ongoing efforts at structural characterization of A2, the maturation protein of the ssRNA bacteriophage Qbeta that inhibits E. coli MurA. In addition, the structure of Bacillus subtilis MurA, which is not recognized by A2, is presented. The crystal structure of B. subtilis MurA, the first structure of MurA from a Gram-positive organism, allows for a direct comparison of Gram-positive and Gram-negative homologs and for identification of any significant structural differences. The more flexible catalytic loop of B. subtilis MurA protrudes farther out compared to the loop of E. coli MurA and creates enough hindrance to prevent A2 from establishing secure contact points.

No description available.

Plaques formed by DC1 on B. cepacia LMG 18821 and B. cenocepacia PC184 are surrounded by large and expanding halos when production of the exopolysaccharide (EPS) cepacian is induced. This plaque morphology indicates that DC1 putatively carries an EPS depolymerase enzyme. Plaque halos were absent when DC1 infected a PC184 cepacian knockout mutant and a non-mucoid LMG 18821 mutant, constructed using plasposon mutagenesis. The virulence of these mutants compared to wildtype PC184 and LMG 18821 was determined using the Galleria mellonella infection model. No major changes to virulence were observed for the LMG 18821 mutant. But, the PC184 cepacian knockout mutant was attenuated for virulence suggesting that this carbohydrate pathway may play a role in pathogenesis. The gene(s) involved in halo formation remain unknown although attempts were made to determine the gene(s) involved by cloning and expressing DC1 fragments in E. coli and assaying for EPS degradation. / Microbiology and Biotechnology

No description available.

Bacteriophages (phages) are bacterial viruses. Phages offer remarkable diversity and can be found in many shapes and sizes; however, what they all have in common is that they are made of protein nano-shells that encase their genome (DNA or RNA). In other words, phages are proteinous bionanoparticles. In this work, we use the filamentous phage M13. M13 is a simple virus with a high aspect ratio. It has 11 genes and only 5 structural proteins. The phage filament is almost entirely made of 2700 copies of pVIII, the major coat protein, and is capped off on one end by five copies each of the proteins pIII and pVI, while the opposite end displays five copies each of the proteins pVII and pIX. M13 phage can be genetically engineered to display certain peptides with affinity toward cancer cells, specific tissue, or even minerals and polymers. These filaments can further self-organize to form liquid crystals at high concentrations. All these properties make M13 a unique building block for the bottom up synthesis of advanced bioactive material. The objective of my proposed research is to develop hydrogels using M13 phage. Hydrogels can absorb large quantities of water without dissolving. They mark a breakthrough in the field of biomaterials, owing to their high water content, porosity and soft consistency. I crosslinked M13 at liquid crystalline concentrations using glutaraldehyde. The resulting hydrogels were characterized for swelling and mechanical properties. These hydrogels exhibited self-healing and autofluorescence properties. In addition, I demonstrated that M13 can from self-healing hydrogels at lower concentrations by adding the small globular protein, BSA. The developed M13 hydrogels mark the first step in the development of bioactive hydrogels that could be utilized to direct cell destiny and genuinely mimic the natural tissue. / Thesis / Master of Applied Science (MASc) / Filamentous phage are viruses that infect bacteria. These bio-filaments are ~1 𝜇𝑚 long, 6-8 nm in diameter and can propagate themselves by infecting bacteria. This means one bio-filament can make 300-1000 particles only by infecting a bacterial host, a characteristic that drastically increases their utility over synthetic filamentous nanomaterial. Filamentous phage can be readily genetically engineered to express foreign receptors on their surface. In this thesis, I demonstrate how these bio-filaments can self-organize at high concentrations and can be crosslinked to make hydrogels that can adsorb up to 12 times their weight in water. These hydrogels can also heal themselves if broken or cut and exhibit autofluorescence, which are very useful properties for hydrogels used for biomedical applications. We further demonstrate that adding small proteins to the bio-filaments can expand the range of hydrogel formation, to the extent that even low concentrations of bio-filament can form hydrogels.

Le but de ce travail est de découvrir des nouvelles protéines suceptibles d'interférer avec le cycle vital du virus HIV. De par leur repliement, les protéines à motifs ankyrines peuvent constituer une ossature protéique trés bien adaptée à cet objectif. Plusieurs interacteurs spécifiques de la protéine MA-CA du HIV ont été sélectionnées par exposition sur phage à partir d'une bibliothèque de variants d'ankyrines. Trois protéines isolées ont été produites à partir de clones ayant une forte activité de liaison. Le meilleur interacteur protéique (1D4) interagit avec un épitope situié sur le domaine CA. La constante de dissociation entre 1D4 et la protéine HACA a été déterminée par Calorimétrie de Titrage Isotherme (ou ITC) et est égale à 0,45M. La protéine 1D4 n'a pas d'effet détectable sur la maturation virale suivie par une technique ELISA de dosage de la Protease du HIV. En revanche, cette protéine interfere avec l'assemblage viral dans des cellules supT1 qui exprime de façon stable la protéine 1D4 sous forme myristoylée. Ce resultat ouvre une perspective d'appoche pour interferer avec le cycle vital du HIV. / Presently, the standard regimen for antiretroviral treatment is highly active antiretroviral therapy (HAART). However, this strategy inherits the well-known side effects and is prone to promote the HIV drug-resistant strains. As a consequence, gene therapy has been introduced as an alternative approach. In this study, we aimed to discover the novel protein-based agents for intervening viral replication by gene targeting procedure. Regarding the efficient folding dynamic in cytoplasm, ankyrin repeat protein was considered to be a candidate scaffold. Several engineered ankyrin binders specific to HIV MA-CA domain were successfully retrieved from the ankyrin-displayed phage library. Three positive clones with high binding activity by ELISA were selected for further analyzing their binding property in soluble form. The best binder, 1D4, recognized its epitope located on CA domain as shown by Western immunobloting and ELISA. The affinity of 1D4 against H6MA-CA was 0.45 μM with one to two moles of target molecule determined by isothermal titration calorimetry (ITC). Although 1D4 exhibited no effect on viral maturation as verified by an ELISA based HIV protease assay technique, it disturbed the viral assembly process in Sup-T1 cells which stably expressed the myristoylated 1D4. This finding has provided a concrete prospect for HIV life cycle interruption by stem cell gene therapy in the future.

<div> <div> <div> <p>Phages, viruses that infect bacteria, have been used for many studies in understanding fundamentals of molecular biology and taking advantage of their natural antimicrobial properties (Harper 2021). They are often noted for their overwhelming abundance and are recognized as the most abundant biological entities in the world (Harper 2021). The field has grown since the early 20th century, and now, there are several classes of phages that have been observed and characterized (Ackermann 2009). Within this abundant class of biological organisms, the order called Caudovirales, is the most populated group of phages to date (Harper 2021). In this order of viruses, the dsDNA genome phages have 2 main components, the icosahedral capsid, and a tail (Harper 2021). Though many tailed phages have been studied for many decades, new information about phages is still being found. Important findings such as the CRISPR gene editing tool adapted from phages in 2007 (Barrangou, Fremaux et al. 2007) have contributed to new biotechnology that impacts human health. For this reason, studies on phages have proven to be valuable in understanding fundamental biological questions and advancing basic research. </p><p><br></p> <p>In this dissertation, we investigated phage G, which has the largest capsid and genome of propagated phage studied to date (Donelli 1968, Sun and Serwer 1997, Pope 2011, Hua, Huet et al. 2017). By studying phage G, we may add to the knowledge of this relatively unexplored group of Jumbo phages with remarkably larger genomes (>200kbp) (Yuan and Gao 2017)to understand how their structure and function may be similar or different to the commonly studied, smaller bacteriophages, such as T4 and λ. For a majority of these studies, we outline how our structural biology insights of phage G using cryo-EM (cryo-Electron Microscopy) have shown it’s icosahedral capsid of ~ 180 nm in diameter at the 5-fold icosahedral vertex is composed of hexamer and pentamer proteins similar to what’s been discovered in other, smaller tailed phages (González, Monroe et al. 2020). Our observations from microscopy data also show unique mechanistic properties in phage G’s tail that are inconsistent with current model of tail contraction within the Myoviridae family of tailed phages. Data suggest phage G’s structure and organization of its helical tail are still similar to contractile phages such as T4 (Amos and Klug 1975, Abuladze, Gingery et al. 1994) and phi812 (Nováček, Šiborová et al. 2016), however, the mechanism of the tail sheath movement is inconsistent with the existing ideas of myophage function (Harper 2021).</p></div></div></div>

Le travail de thèse présente une approche innovante pour la construction d’une bibliothèque de protéines basées sur l’ossature protéique. L’objectif est de générer une source de biodiversité artificielle permettant la création de nouvelles capacités d’interaction avec des cibles d’intérêts. Une banque, basée sur une ossature protéique bactérienne avait déjà été construite dans l’équipe, mais elle nécessitait d’être optimisée. L’étape initiale a été d’explorer les raisons de l’instabilité des protéines de la banque de première génération, ceci par des approches d’étude de la structure in silico suivie d’une stratégie de mutagenèse dirigée. Des positions déstabilisantes existant dans la première banque ont donc été remplacées dans la banque de deuxième génération. La deuxième étape a eu pour objectif de diminuer le nombre de positions diversifiées et de simplifier le schéma de diversification des variants de la banque. Puis un procédé de filtration et de shuffling de ces variants a été mis au point pour augmenter la proportion de séquences codantes correctes. Une nouvelle stratégie de filtration basée sur la technique d’exposition sur phage a été élaborée, en exploitant le fait que la protéine matrice de la banque, l'ossature protéique a un partenaire biologique capable d’interagir sur la zone « constante », non modifiée par le schéma de diversification. Ainsi les variants de la banque exposés dans une conformation correcte à la surface des phages ont pu être capturés par ce partenaire. Ensuite, les séquences correspondant à ces variants ont été recombinées entre elles pour recréer une plus grande diversité utile. Une bibliothèque optimisée composée de 2.8 x 108 protéines indépendantes a ainsi été obtenue. Cette nouvelle banque optimisée a permis de sélectionner par Phage display, des interacteurs contre plusieurs cibles de structures différentes. Ces nouveaux interrupteurs sont spécifiques de leurs cibles et présentent des affinités de l’ordre du μM. Une approche de séquençage à haut débit a également été entreprise pour réaliser une analyse plus approfondie des séquences de cette bibliothèque et de notre processus de sélection. Cette approche nous a apporté une nouvelle dimension pour la caractérisation des banques construites au laboratoire notamment concernant la diversité réelle de ces banques. Pour le suivi de sélections, nous avons appréhendé le séquençage haut débit comme un moyen d’identifier les interacteurs spécifiques d’une cible par l’analyse exhaustive des séquences issues des sélections. L’objectif est ici de mettre au point un protocole utilisant l’approche NGS pour identifier les interacteurs spécifiques isolées par Phage Display. / Here new methods to build a library of artificial proteins based on a new protein framework have been developed. The objective is to generate a source of artificial biodiversity allowing the creation of new interaction capacities with various specific targets. A first-generation library was previously built with this scaffold, but it needed to be optimized. The first step was to explore the reasons of the instability of the first generation proteins library through an in silico approaches followed by a site-directed mutagenesis strategy. The second step was to reduce the number of diversified positions and simplify the randomization scheme of the variants of the library. Then a method of filtering and shuffling of the variants of the bank was elaborated. To increase the proportion of correct coding sequences, a new filtration strategy based on the phage display technique has been developed, exploiting the fact that the scaffold of the librarie. This scaffold has a particularly interesting biological partner able to interact on the "constant" zone. This filtration made it possible to recover a set of well-folded clones. Then, DNA sequences corresponding to these clones were recombined with each other to recreate a greater useful diversity. An optimized library of 2.8 x 108 independent proteins was obtained. This new optimized library has enabled us to select, by a phage display approach, binders against several targets of different structures. These new binders are specific for their targets and have affinities in the μM range. A high throughput sequencing (NGS) approach was also undertaken to further analyse the library sequences and the selection process. This approach offers a new dimension for the characterization of the library built in the laboratory, especially concerning it actual diversity. To follow the selections, we have considered the NGS as a way to identify the target-specific binders through exhaustive analysis of the sequences obtained from selections. The objective here is to develop a general protocol using the NGS approach to identify specific binders isolated by Phage Display.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The bacterial flagellum is a rotary motor that propels motile bacteria through their surroundings via swimming motility, or on surfaces via swarming motility. The flagellum is a key virulence factor for motile pathogenic bacteria. Viruses that infect bacteria via this appendage are known as flagellotropic or flagellum-dependent bacteriophages. Much like other phages, flagellotropic phages are of interest for clinical applications as antibacterial agents, particularly against multidrug resistant (MDR) bacteria. Bacteriophage χ is a flagellotropic phage that infects multiple species of motile pathogens. In the projects described below, we characterized several aspects of the complex interactions between χ and two of its hosts: Salmonella enterica and Serratia marcescens. In Chapter I, we describe in detail the existing knowledge on flagellum-dependent bacteriophages, pathogenic bacteria, and the flagellar motility system. We also expand significantly on flagellotropic phage χ. In Chapter II, we describe our discovery of S. enterica cellular components other than motility that are crucial for bacteriophage χ infection, making the key discovery that the AcrABZ-TolC multi-drug efflux system is required for infection to proceed. We additionally found that the host molecular chaperone trigger factor is important for the χ phage lifecycle. In Chapter III, we outline our characterization of the initial binding interaction between χ and the flagellum, determining that of flagellin's seven domains, C-terminal domain D2 is the most important for χ adsorption. In Chapter IV, we expand on this by discussing our work that determined that the χ tail fiber protein is encoded by the gene CHI_31, purification of this recombinantly-expressed protein, and demonstration of its direct interaction with the flagellar filament. Lastly, in Chapter V, our findings indicate that S. marcescens is able to detect χ infection and lysis in the surroundings and alter gene expression, resulting in an increase in the production of the red pigment prodigiosin. Overall, our hypothetical model for χ infection is as follows: χ binds to the flagellum of its host using its single tail fiber, composed of monomers of the CHI_31 gene product gp31. This tail fiber interacts with CTD2 of flagellin, and the rotation of the flagellum brings the phage to the cell surface, where it interacts with AcrABZ-TolC to inject its genetic material into the host cytoplasm. At some point during the process of production of phage particles and subsequent cell lysis, the host molecular chaperone trigger factor likely assists with proper folding of χ proteins. After cell lysis, cells in the surroundings are capable of detecting lysis and responding accordingly, at least in the case of S. marcescens. This research is clinically relevant for a number of reasons. Phage therapy, the use of bacteriophages as antibacterial agents, requires knowledge of phage infection pathways for optimal implementation. The fact that the flagellum and a complex mediating MDR are both essential for χ infection leads to particular interest in χ for this application. Knowledge of the host-determining factors between χ and Salmonella may lead to the ability to alter the χ phage genome to target specific pathogenic Salmonella or Escherichia coli strains while avoiding disruption of beneficial bacterial communities. / Doctor of Philosophy / Bacteriophages (phages) are viruses that only infect bacteria. They do not harm animal cells or the human body, despite being highly effective predators of bacteria. As such, they have applications in the medical field as antibacterial agents, similar to antibiotics. Phages that infect pathogenic bacteria like Salmonella are of particular interest for scientific research. Bacteriophage χ (Chi) infects bacteria by binding to their flagella, propeller-like appendages that a bacterial cell uses to swim through its surroundings. In many bacterial species, flagella and the ability to swim are closely involved in human infection. Due to this, flagellotropic (flagellum-dependent) phages like χ may be particularly useful as antibiotics. Throughout this project, we characterized the χ phage infection process, including exploring how it attaches to flagella, interactions it has on the surface of and inside Salmonella cells, and the largely unexplored relationship with Serratia marcescens, another bacterial species that causes illness in humans and is highly antibiotic resistant. Overall, our research contributes to the medical field, and indicates that χ may serve as a highly effective antibacterial treatment.