Tesi sul tema "Bactera/phage interactions"

Avec l'augmentation de la demande pour les produits fromagers, l'optimisation des procédés de production est devenue essentielle. L'une des premières étapes de la fabrication du fromage est l'acidification du lait, qui influence fortement les propriétés organoleptiques, la texture et la sécurité du produit final. Elle consiste à convertir le lactose, sucre du lait, en acide lactique par des bactéries lactiques. Cependant, ces bactéries sont sensibles aux attaques de virus appelés bactériophages. Ces attaques peuvent entraîner la lyse bactérienne, retardant ou arrêtant l'acidification, ce qui engendre des pertes économiques dues au rejet du lait et à la nécessité de nettoyer les installations. Cela souligne l'importance d'une meilleure compréhension des interactions phages-bactéries en fromagerie.Une approche novatrice pour étudier ces interactions est la modélisation mécaniste dynamique. Cette étude vise donc à contribuer à une meilleure compréhension des interactions phages-bactéries dans les processus de fermentation du lait en établissant un modèle dynamique.Pour ce faire, nous avons utilisé une méthode de mesure à haut débit du pH pour générer des données sur l'acidification dans différentes conditions initiales de bactéries et de phages. Cette approche nous a permis de distinguer trois résultats distincts en fonction de ces conditions : pour certaines conditions, l'acidification a été une réussite ; pour d'autres, elle a échoué ; et pour le reste, le résultat n'a été ni un échec complet ni une réussite complète.Le modèle mécaniste développé comprend cinq équations différentielles ordinaires (EDO) et prend en compte divers phénomènes, tels que l'inhibition par le produit, le temps de latence, l'adsorption des phages et la lyse cellulaire. Le modèle a donné des résultats satisfaisants, prédisant avec précision les données expérimentales et identifiant correctement le résultat de l'acidification. Nous avons également comparé différentes structures de modèle et effectué une analyse de sensibilité pour révéler les phénomènes dominants, ce qui a aussi aidé à concevoir de nouvelles expériences informatives.Une analyse théorique du modèle a révélé trois phases temporelles de l'attaque : d'abord, la phase de contamination, un court laps de temps où les phages s'adsorbent aux bactéries ; puis la phase de propagation, dominée par la propagation des phages et l'infection des bactéries sensibles ; et enfin, la phase de décharge, caractérisée par la lyse bactérienne et la libération de nouveaux phages. Le temps de transition entre les deux dernières phases, noté t*, a été relié aux conditions initiales. Nous avons également identifié une composante dynamique rapide qui peut être séparée des dynamiques lentes. En utilisant l'approximation de l'état quasi-stationnaire, nous avons établi une relation analytique entre les conditions initiales des bactéries et des phages et le pH final. Cela montre que l'acidification ne dépend pas uniquement du rapport des conditions initiales. Cette approximation a permis de réduire le modèle, économisant 83 % du temps de simulation.Enfin, nous avons développé un outil pour prédire le nombre d'acidifications réussies possibles avant qu'un nettoyage ne soit nécessaire. Les résultats sont basés sur des données faciles à obtenir, comme la quantité de bactéries utilisée et les résultats d'une acidification précédente. Cela représente une première étape vers la conception d'un outil d'aide à la décision pour les fromagers.Cette étude améliore notre compréhension des dynamiques d'attaque des phages lors de l'acidification du lait et permet des prédictions précises grâce à un système d'EDO et un modèle réduit
As the demand for diverse cheeses increases, there is a growing interest in optimizing production processes. One of the earliest steps in cheese-making is milk acidification, which highly influences the final product's organoleptic properties, texture, and safety. Milk acidification involves the conversion of lactose, the sugar in milk, into lactic acid by lactic acid bacteria. However, these bacteria are susceptible to attack by viruses known as bacteriophages. This attack can lead to bacterial lysis, resulting in delayed or halted acidification, which incurs significant economic losses as milk is discarded and production facilities require extensive cleaning. This highlights the need for a deeper understanding of phage-bacteria interactions in cheese-making. Research efforts in the dairy industry have primarily focused on characterizing the phages involved and finding new strategies to mitigate phage attacks.One novel approach to studying these interactions is through dynamic mechanistic modeling. Previous models have been developed but have never been applied to the dairy industry. This study aims to fill this gap by contributing to the broader understanding of phage-bacteria interactions in milk fermentation through the establishment of a dynamic model.To achieve this, we first employed a high-throughput pH measurement method to generate acidification data under different initial conditions of bacteria and phages. This methodology proved useful in distinguishing various dynamic behaviors depending on these conditions. It allowed us to delineate three distinct outcomes depending on these conditions: for some conditions the acidification was a success; for some others, it was a failure; and for the rest, the result was neither a complete failure nor a complete success.The mechanistic model we developed consists of five ordinary differential equations (ODEs) and accounts for various phenomena, including product inhibition, lag time, phage adsorption, and cell lysis. The model yielded satisfactory results, accurately predicting experimental data and correctly identifying the acidification outcome. We further investigated the model's structure by comparing various candidate structures and performing a sensitivity analysis to reveal the dominant phenomena throughout the process. The sensitivity analysis also contributed to the design of new informative experimental setups.A theoretical analysis of the model provided insights into the intrinsic dynamics of the system, revealing three time frames of the attack. First, the contamination phase, a short initial time where phages adsorb to the bacteria. Next, the spread phase, where the dominant dynamics involve the spread of phages and the infection of susceptible bacteria. Finally, the discharge phase, where the dominant dynamics are the lysis of bacteria and the release of new phages. The switch time between the last two phases was defined as t∗ and its dependency on the initial conditions was characterized.We also identified a faster dynamic component of the system that can be separated from slower ones. Utilizing the quasi-steady state approximation, we established an analytical relationship between the initial conditions of bacteria and phages and the resulting pH. This relationship indicates that the final outcome of acidification does not solely depend on the ratio of initial conditions but is more complex. The approximation resulted in a reduced model that saved 83% of the simulation time.Finally, we developed a tool to predict the number of potential successful acidifications that can be run before cleaning is required. The results are based on easily obtainable inputs. This represents a first step toward designing a decision aid tool to help cheese makers in their production.This study enhances our understanding of the dynamics of phage attack in milk acidification and facilitates accurate predictions of these dynamics through an ODE system and a reduced model

Bacteria and their viruses (bacteriophage, phage) are the most abundant and diverse taxonomic groups, but ecological and evolutionary research on bacteria-phage interactions has largely focused on studies of simplified communities using a few model organisms. The goal of the thesis is to understand how bacteria and phage interact within natural environments, and how these interactions impact the patterns of phage infectivity and bacterial resistance. Here I investigate the effects of natural environments on the coevolutionary patterns of bacteria (Pseudomonas fluorescens) and phage (SBW25Φ2). In chapter 3 I investigate the effects of nineteen different communities on the coevolutionary interactions of SBW25 and phage, and the degree to which the infectivity of phage to its host, SBW25, changes depending on their local microbial community. Chapter 4 aimed to understand the effects of varying diversities of communities on coevolutionary interactions. In Chapter 5, I looked at how coevolutionary interactions were affected by different communities in different abiotic conditions (pH, temperature and nutrient concentration) and the effect communities had on the ability of SBW25 to adapt to the abiotic conditions. Understanding how biological and physical factors affect coevolutionary interactions in natural environments allows predictions of how phage and bacteria coevolve in natural and unnatural settings.

Vibrio parahaemolyticus, a human pathogenic bacterium, is a naturally occurring member of the microbiome of the Eastern oyster. As the nature of this symbiosis in unknown, the oyster presents the opportunity to investigate how microbial communities interact with a host as part of the ecology of an emergent pathogen of importance. To define how members of the oyster bacterial microbiome correlate with V. parahaemolyticus, I performed marker-based metagenetic sequencing analyses to identify and quantify the bacterial community in individual oysters after culturally-quantifying V. parahaemolyticus abundance. I concluded that despite shared environmental exposures, individual oysters from the same collection site varied both in microbiome community and V. parahaemolyticus abundance, and there may be an interaction with V. parahaemolyticus and Bacillus species. In addition, to elucidate the ecological origins of pathogenic New England ST36 populations, I performed whole genome sequencing and phylogenetic analyses. I concluded ST36 strains formed distinct subpopulations that correlated both with geographic region and unique phage content that can be used as a biomarker for more refined strain traceback. Furthermore, these subpopulations indicated there may have been multiple invasions of this non-native pathogen into the Atlantic coast.

Bacteriophages cohabit with their bacterial hosts and shape microbial communities. To initiate infection, phages use bacterial components as receptors to recognize and attach to hosts. Flagellotropic phages utilize bacterial flagella as receptors. Studies focused on uncovering mechanistic details of flagellotropic phage infection are lacking. As the number of phage-based applications grows, it is important to understand these details to predict the potential outcomes of phage therapy. To this end, we studied two flagellotropic phages: Agrobacterium phage 7-7-1 and bacteriophage χ. Phage 7-7-1 infects Agrobacterium spp., while bacteriophage χ infects Salmonella and Escherichia coli. Chapter 1 consists of a literature review. Chapter 2 addresses factors underlying phage-bacteria coexistence. We document the emergence of a sector-shaped lysis pattern following co-inoculation of phage χ and one of its Salmonella hosts on swim plates. We propose that this pattern serves as a reporter for balanced phage-bacteria coexistence. Using a combined experimental and mathematical modelling approach, we discovered that variations to intrinsic factors (i.e., bacterial motility, phage adsorption) skews the pattern towards either bacterial or phage predominance. Thus, this computational model may be used to predict phage therapy application outcomes. Chapter 3 details the identification of cell surface receptors essential for phage 7-7-1 infection using a transposon mutagenesis approach. We identified three Agrobacterium sp. H13-3 genes involved in phage 7-7-1 infection. Using mass spectrometry and other analyses, we determined that the LPS profiles of strains lacking these genes varied compared to the wild type. Thus, LPS is a secondary cell surface receptor for phage 7-7-1. Chapter 4 focuses on the discovery of phage encoded receptor binding proteins (RBPs) in Agrobacterium phage 7-7-1. Using an RBP screen, we discovered three candidate RBPs. We learned that our top candidate, Gp4, inhibits the growth of Agrobacterium sp. H13-3 cells in a motility and glycan dependent manner. Because of its bacteriostatic activities, this protein is a promising candidate for therapeutic use. Overall, the described works contribute to a deepened understanding of flagellotropic phage infection and the factors influencing their coexistence with motile bacteria. These works will contribute towards the development of phage therapies using whole phage or their components.
Doctor of Philosophy
Bacteriophages, or phages for short, are the natural killers of bacteria. Like antibiotics, they can also be used as medicines to treat bacterial infections. Their attack on bacteria begins by recognizing specific parts of the bacterial cell and attaching to them. These parts are called receptors. To use phages as medicines it is important to understand how they recognize and kill bacteria. This information is helpful when deciding which phage should be given to treat a bacterial infection and to predict the outcomes of these treatments. In this work, we focused on two phages to answer different questions. Both phages use long helical thread-like structures, called flagella, as receptors. Flagella help the bacteria to move through the environment and reach new areas with more nutrients. One of these flagella-dependent phages, called phage 7-7-1, infects plant pathogens that cause tumor-like growth in plants. We found that this phage uses two very different host cell components during infection and identified one of the phage proteins that interacts with these receptors. This protein prevents the growth of the plant pathogen, which makes it a promising candidate for therapeutic use. We also investigated how another bacterial virus, bacteriophage χ, is spread throughout the environment and co-exists with its motile bacterial host. We built a computational model that can predict how altering different variables affects phage-bacteria coexistence. With additional research, this model will be a useful tool for predicting the outcomes following phage treatment.

Abstract The scope of this study includes aspects of phage evolution and antagonistic/mutualistic coevolution between a phage and its host. As a basic study it may provide tools for developing phage resistant starters and offer regulatory elements and factors for biotechnological applications. The LL-H anti-receptor was characterized by isolation of spontaneous LL-H host range mutants and subsequent sequencing of candidate genes. All LL-H host range mutants carried a single point mutation at the 3' end of a minor tail protein encoding gene g71. The genomic location of g71 is congruent with the other verified anti-receptor genes found in the λ supergroup. The C-terminus of Gp71 determines the adsorption specificity of phage LL-H similarly for the number of phages infecting Gram-positive and Gram-negative bacteria. A Gp71 homolog of phage JCL1032 showed 62% identity to LL-H Gp71 within the last 300 amino acids at the C-terminus. Lactobacillus delbrueckii phage receptors were investigated by the purification of different cell surface structures. Certain Lb. delbrueckii phages from homology groups a and c including LL-H, LL-H host range mutants and JCL1032, were specifically inactivated by the LTAs. In structural analyses LTAs showed differences in the degree of α-glucosyl and ᴅ-alanyl substitution. α-glucose is necessary for LL-H adsorption. A high level of ᴅ-alanine esters in LTA backbones inhibited Lb. delbrueckii phage inactivation in general. Lysogenization of strain ATCC 15808 with the temperate phage JCL1032 revealed a rarely described coexistence of phage adsorption resistance and phage immunity, which could not be explained by lysogenic conversion. In this case the role of spontaneously induced JCL1032 may be significant. The LL-H early gene region was localized between the dysfunctional lysogeny module and the terminase encoding genes. The function of five ORFs could be connected to phage DNA replication and/or homologous recombination. Transcription of LL-H genes could be divided into two, possibly three, phases in which large gene clusters were sequentially transcribed. The intensity of the late transcripts exceeded the intensity of the early transcripts by several times. Two candidate genes for transcription regulators were found. One of the two candidates is the first ORF in the LL-H early gene region.

Bacteriophages (viruses that infect bacteria) are the most abundant biological life-forms on Earth. However, very little is known regarding the structure of phage-bacteria infections. In a recent study we showed that phage-bacteria infection assay datasets are statistically nested in small scale communities while modularity is not statistically present. We predicted that at large macroevolutionary scales, phage-bacteria infection assay datasets should be typified by a modular structure, even if there is nested structure at smaller scales. We evaluate and confirm this hypothesis using the largest study of the kind to date. The study in question represents a phage-bacteria infection assay dataset in the Atlantic Ocean region between the European continental shelf and the Sargasso Sea. We present here a digitized version of this study that consist of a bipartite network with 286 bacteria and 215 phages including 1332 positive interactions, together with an exhaustive structural analysis of this network. We evaluated the modularity and nestedness of the network and its communities using a variety of algorithms including BRIM (Bipartite, Recursively Induced Modules), NTC (Nestedness Temperature Calculator) and NODF (Nestedness Metric based on Overlap and Decreasing Filling). We also developed extensions of these standard methods to identify multi-scale structure in large phage-bacteria interaction datasets. In addition, we performed an analysis of the degree of geographical diversity and specialization among all the hosts and phages. We find that the largest-scale ocean dataset study, as anticipated by Flores et al. 2013, is highly modular and not significantly nested (computed in comparison to null models). More importantly is the fact that some of the communities extracted from Moebus and Nattkemper dataset were found to be nested. We examine the role of geography in driving these modular patterns and find evidence that phage-bacteria interactions can exhibit strong similarity despite large distances between sites. We discuss how models can help determine how coevolutionary dynamics between strains, within a site and across sites, drives the emergence of nested, modular and other complex phage-bacteria interaction networks. Finally, we releases a computational library (BiMAT)to help to help the ecology research community to perform bipartite network analysis of the same nature I did during my PhD.

L'intestin des mammifères est peuplé de nombreux et divers microbes comprenant des bactéries et leurs prédateurs viraux, des bactériophages (phages). Les interactions entre les phages et les bactéries intestinales sont encore mal comprises. Des expériences indépendantes ont montré que les phages virulents n’avaient aucun effet majeur sur l’abondance des bactéries intestinales ciblées, en dépit de leur amplification durable. Cela suggère que des facteurs encore inconnus de l'environnement intestinal modulent ces interactions. À l’aide d’une analyse transcriptomique comparative de la bactérie Escherichia coli cultivée in vitro et in vivo (dans l’intestin de mammifères), nous avons constaté que, dans l’intestin, les bactéries réduisent l’expression de gènes liés aux récepteurs du phage. Ceci permet d’expliquer l’absence de sélection des bactéries mutées devenues résistantes aux phages lors d'expériences in vivo. D’autre part, nous avons montré que l'acquisition d'un îlot de pathogénicité, souvent associé aux souches intestinales humaines d'E. coli, affecte la susceptibilité aux phages en régulant négativement un mécanisme de défense contre l'ADN étranger. Enfin, nous avons examiné la répartition des phages et des bactéries dans les parties mucosales et luminales de l’intestin et avons observé une distribution spatiale hétérogène de ces deux populations antagonistes, corroborant l'hypothèse d'une dynamique « source-sink ». Globalement, nos données démontrent que de multiples facteurs incluant la distribution spatiale, la physiologie bactérienne et les défenses contre l’ADN étranger modulent les interactions entre bactéries et phages dans l’intestin des mammifères
The mammalian gut is a heterogeneous environment inhabited by a large and diverse microbial community, including bacteria and their viral predators, bacteriophages (phages). Dynamic interactions between virulent phages and bacteria in the gut are still poorly understood, which is also an obstacle for the design of successful therapeutic interventions based on phages. Independent experiments have shown that virulent phages were found to have no major effects on their targeted bacteria in the gut, in spite of sustainable phage amplification. This suggests that there are unknown factors in the gut environment that modulate these interactions. Using comparative transcriptomics analysis of E. coli grown in vitro and in vivo (within the mammalian gut) we found that in the gut, bacteria downregulate the expression of genes related to phage receptors, which provides an explanation for the lack of selection of phage-resistant bacteria during in vivo experiments. We also found that the acquisition of a pathogenicity island commonly found in human E. coli isolates affects phage susceptibility possibility by downregulating a defense mechanism against invading DNA. Finally, we examined the repartition of phages and bacteria through mucosal and luminal gut sections and observed a heterogeneous spatial distribution of these two antagonist populations, supporting the hypothesis of source-sink dynamics. Altogether our data demonstrates that multiple factors encompassing, spatial distribution, bacterial physiology and defenses against foreign DNA modulate the interactions between bacteria and phages within the gut

The rise of antibiotic resistance has rekindled interest in the development of alternative antimicrobial agents. Bacteriophages, bacteria's obligate parasites, have drawn a lot of interest from the scientific community and from the industry due to their many advantages. However, there are various challenges hindering the use of bacteriophages as antimicrobial agents. In this dissertation, a number of these challenges have been addressed. After a brief introduction on the advantages and drawbacks of using phage in chapter one, the inherent limitation of using immobilized phage for antimicrobial surfaces is presented in chapter two. In the subsequent three chapters, one of the main issues of challenging bacterial communities with phage was addressed, namely emergence of phage-resistant bacteria variants. The effect of phage on biofilm formation was investigated and it was observed that in some cases they can lead to increase in biofilm formation. Furthermore the colonies of phage-resistant bacteria emerged in contact with phage were studied. It was reported that their phenotype, their virulence traits as well as their in vitro virulence towards mammalian cells had been significantly affected. This investigation highlights the importance of awareness of the effect of phage on bacterial communities for effective utilization of their potential.
L'augmentation de la résistance aux antibiotiques a ravivé l'intérêt dans le développement d'agents antimicrobiens alternatifs. Les bactériophages, parasites bactériens obligatoires, ont suscité beaucoup d'intérêt de la part de la communauté scientifique et de l'industrie à cause de leurs nombreux avantages. Cependant, plusieurs défis restent à relever pour résoudre les problèmes qui empêchent l'utilisation des bactériophages comme agents antimicrobiens. Un certain nombre de ces défis sont adressés dans cette dissertation. Après une brève introduction sur les avantages et désavantages de l'utilisation des bactériophages dans le premier chapitre, nous présentons dans le second chapitre les limitations intrinsèques de l'utilisation de bactériophages immobilisés pour créer des surfaces antimicrobiennes. Dans les trois chapitres suivants, nous traitons de l'une des principales questions concernant l'infection de cultures bactériennes par les bactériophages, à savoir l'émergence de variants bactériens résistants. L'effet des bactériophages sur la formation de biofilms a été étudié et nous avons observé que dans certains cas, les bactériophages peuvent augmenter la formation de biofilms. En outre, nous avons étudié les colonies bactériennes résistantes qui émergent après l'infection par des bactériophages. Nous avons trouvé que leur phénotypes, leurs caractères de virulence ainsi que leurs virulences in vitro envers les cellules mammifères avaient été affectés de manière significative. Cette investigation souligne l'importance des effets des bactériophages sur les cultures bactériennes pour une utilisation efficace de leur potentiel antimicrobien.

Viruses infecting bacteria (phages) are the most abundant and ubiquitous entities on Earth and likely critical to any ecosystem, as they influence nutrient cycling, mortality and evolution. Ultimately, their impact depends on whether phage—host interactions lead to intracellular phage coexistence (temperate phage) or cell death (lytic phage). Temperate phages in the lysogenic cycle replicate their genome (either integrated into the host chromosome or extrachromosomally), until induced to become lytic, when they create and release progeny via cell lysis. While knowledge on lytic versus lysogenic outcomes is vast, it largely derives from few model systems that underrepresent natural diversity. Further, less is known about the efficiency of phage—host interactions and the regulation of optimal versus sub-optimal lytic infections, which are predicted as relevant under environmental (nutrients, temperature) and host (availability, density) conditions that are common in the ocean. In this dissertation I characterize the phage—host interactions in a new marine model system, phage ϕ38:1 and its Cellulophaga baltica bacterial host, member of the ubiquitous Bacteroidetes phylum. First, I show ϕ38:1’s ability to infect numerous, genetically similar strains of the C. baltica species, two of which display contrasting infection outcomes–lytic versus sub-optimally lytic or lysogenic on the original versus alternative hosts, respectively. Second, I collaboratively apply new gene marker-based approaches (phageFISH and geneELISA) to study ϕ38:1’s infection at the single-cell level and show that it is sub-optimal on the alternative host, rather than lysogenic. Third, I collaboratively develop whole-genome transcriptome datasets for ϕ38:1 infecting both, the optimal and sub-optimal hosts, to characterize the cellular response to infection and hypothesize potential transcriptional and post-transcriptional regulation of the sub-optimal infection. Together, these findings advance our knowledge of naturally-occurring phage—host interactions with a focus on nearly-unstudied sub-optimal infections.

A prevalent morphology in the microscopic world of artificial microswimmers, bacteria and viruses is that of a helix. The intriguingly different physics at play at the small scale level make it necessary for bacteria to employ swimming strategies different from our everyday experience, such as the rotation of a helical filament. Bio-inspired microswimmers that mimic bacterial locomotion achieve propulsion at the microscale level using magnetically actuated, rotating helical filaments. A promising application of these artificial microswimmers is in non-invasive medicine, for drug delivery to tumours or microsurgery. Two crucial features need to be addressed in the design of microswimmers. First, the ability to selectively control large ensembles and second, the adaptivity to move through complex conduit geometries, such as the constrictions and curves of the tortuous tumour microvasculature. In this dissertation, a mechanics-based selective control mechanism for magnetic microswimmers is proposed, and a model and simulation of an elastic helix passing through a constricted microchannel are developed. Thereafter, a theoretical framework is developed for the propulsion by stiff elastic filaments in viscous fluids. In order to address this fluid-structure problem, a pertubative, asymptotic, elastohydrodynamic approach is used to characterise the deformation that arises from and in turn affects the motion. This framework is applied to the helical filaments of bacteria and magnetically actuated microswimmers. The dissertation then turns to the sub-bacterial scale of bacteriophage viruses, 'phages' for short, that infect bacteria by ejecting their genetic material and replicating inside their host. The valuable insight that phages can offer in our fight against pathogenic bacteria and the possibility of phage therapy as an alternative to antibiotics, are of paramount importance to tackle antibiotics resistance. In contrast to typical phages, flagellotropic phages first attach to bacterial flagella, and have the striking ability to reach the cell body for infection, despite their lack of independent motion. The last part of the dissertation develops the first theoretical model for the nut-and-bolt mechanism (proposed by Berg and Anderson in 1973). A nut being rotated will move along a bolt. Similarly, a phage wraps itself around a flagellum possessing helical grooves, and exploits the rotation of the flagellum in order to passively travel along and towards the cell body, according to this mechanism. The predictions from the model agree with experimental observations with respect to directionality, speed and the requirements for succesful translocation.

Kohlenhydrate stellen aufgrund der strukturellen Vielfalt und ihrer oft exponierten Lage auf Zelloberflächen wichtige Erkennungsstrukturen dar. Die Wechselwirkungen von Proteinen mit diesen Kohlenhydraten vermitteln einen spezifischen Informationsaustausch. Protein-Kohlenhydrat-Interaktionen und ihre Triebkräfte sind bislang nur teilweise verstanden, da nur wenig strukturelle Daten von Proteinen im Komplex mit vorwiegend kleinen Kohlenhydraten erhältlich sind. Mit der vorliegenden Promotionsarbeit soll ein Beitrag zum Verständnis von Protein-Kohlenhydrat-Wechselwirkungen durch Analysen struktureller Thermodynamik geleistet werden, um zukünftig Vorhersagen mit zuverlässigen Algorithmen zu erlauben. Als Modellsystem zur Erkennung komplexer Kohlenhydrate diente dabei das Tailspike Protein (TSP) aus dem Bakteriophagen HK620. Dieser Phage erkennt spezifisch seinen E. coli-Wirt anhand der Oberflächenzucker, der sogenannten O-Antigene. Dabei binden die TSP des Phagen das O-Antigen des Lipopolysaccharids (LPS) und weisen zudem eine hydrolytische Aktivität gegenüber dem Polysaccharid (PS) auf. Anhand von isolierten Oligosacchariden des Antigens (Typ O18A1) wurde die Bindung an HK620TSP und verschiedener Varianten davon systematisch analysiert. Die Bindung der komplexen Kohlenhydrate durch HK620TSP zeichnet sich durch große Interaktionsflächen aus. Durch einzelne Aminosäureaustausche im aktiven Zentrum wurden Varianten generiert, die eine tausendfach erhöhte Affinität (KD ~ 100 nM) im Vergleich zum Wildtyp-Protein (KD ~ 130 μM) aufweisen. Dabei zeichnet sich das System dadurch aus, dass die Bindung bei Raumtemperatur nicht nur enthalpisch, sondern auch entropisch getrieben wird. Ursache für den günstigen Entropiebeitrag ist die große Anzahl an Wassermolekülen, die bei der Bindung des Hexasaccharids verdrängt werden. Röntgenstrukturanalysen zeigten für alle TSP-Komplexe außer für Variante D339N unabhängig von der Hexasaccharid-Affinität analoge Protein- und Kohlenhydrat-Konformationen. Dabei kann die Bindestelle in zwei Regionen unterteilt werden: Zum einen befindet sich am reduzierenden Ende eine hydrophobe Tasche mit geringen Beiträgen zur Affinitätsgenerierung. Der Zugang zu dieser Tasche kann ohne große Affinitätseinbuße durch einen einzelnen Aminosäureaustausch (D339N) blockiert werden. In der zweiten Region kann durch den Austausch eines Glutamats durch ein Glutamin (E372Q) eine Bindestelle für ein zusätzliches Wassermolekül generiert werden. Die Rotation einiger Aminosäuren bei Kohlenhydratbindung führt zur Desolvatisierung und zur Ausbildung von zusätzlichen Wasserstoffbrücken, wodurch ein starker Affinitätsgewinn erzielt wird. HK620TSP ist nicht nur spezifisch für das O18A1-Antigen, sondern erkennt zudem das um eine Glucose verkürzte Oligosaccharid des Typs O18A und hydrolysiert polymere Strukturen davon. Studien zur Bindung von O18A-Pentasaccharid zeigten, dass sich die Triebkräfte der Bindung im Vergleich zu dem zuvor beschriebenen O18A1-Hexasaccharid verschoben haben. Durch Fehlen der Seitenkettenglucose ist die Bindung im Vergleich zu dem O18A1-Hexasaccharid weniger stark entropisch getrieben (Δ(-TΔS) ~ 10 kJ/mol), während der Enthalpiebeitrag zu der Bindung günstiger ist (ΔΔH ~ -10 kJ/mol). Insgesamt gleichen sich diese Effekte aus, wodurch sehr ähnliche Affinitäten der TSP-Varianten zu O18A1-Hexasaccharid und O18A-Pentasaccharid gemessen wurden. Durch die Bindung der Glucose werden aus einer hydrophoben Tasche vier Wassermoleküle verdrängt, was entropisch stark begünstigt ist. Unter enthalpischen Aspekten ist dies ebenso wie einige Kontakte zwischen der Glucose und einigen Resten in der Tasche eher ungünstig. Die Bindung der Glucose in die hydrophobe Tasche an HK620TSP trägt somit nicht zur Affinitätsgenerierung bei und es bleibt zu vermuten, dass sich das O18A1-Antigen-bindende HK620TSP aus einem O18A-Antigen-bindenden TSP evolutionär herleitet. In dem dritten Teilprojekt der Dissertation wurde der Infektionsmechanismus des Phagen HK620 untersucht. Es konnte gezeigt werden, dass analog zu dem verwandten Phagen P22 die Ejektion der DNA aus HK620 allein durch das Lipopolysaccharid (LPS) des Wirts in vitro induziert werden kann. Die Morphologie und Kettenlänge des LPS sowie die Aktivität von HK620TSP gegenüber dem LPS erwiesen sich dabei als essentiell. So konnte die DNA-Ejektion in vitro auch durch LPS aus Bakterien der Serogruppe O18A induziert werden, welches ebenfalls von dem TSP des Phagen gebunden und hydrolysiert wird. Diese Ergebnisse betonen die Rolle von TSP für die Erkennung der LPS-Rezeptoren als wichtigen Schritt für die Infektion durch die Podoviren HK620 und P22.
Carbohydrates are important for recognition events because of their diverse structure and their exposition on cell surfaces. Interactions between proteins and carbohydrates mediate a specific exchange of information crucial for manifold biological functions. The energetics of protein-carbohydrate-interactions are not very well understood so far due to the lack of structural data of proteins in complex with extensive oligosaccharides consisting of more than two building blocks. This dissertation improves the understanding of how proteins recognize complex carbohydrates by analysis of structural thermodynamics, which might lead to reliable algorithms for predictions of protein-carbohydrate-interactions. As model system for this work the tailspike protein (TSP) from coliphage HK620 was used. This phage recognizes specifically the surface O-antigen of its E. coli host by its TSP. HK620TSP does not only bind the O-antigen of host lipopolysaccharide (LPS), but also cleaves the polysaccharide (PS) by its endo-N-acetylglusaminidase activity. HK620TSP binds hexasaccharide fragments of this PS with low affinity (KD ~ 130 μM). However, single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants (KD ~ 100 nM). Strikingly, at room temperature association is driven by enthalpic and entropic contributions emphasizing major solvent rearrangements upon complex formation. Regardless of their affinity towards hexasaccharide the TSP complexes showed only minor conformational differences in crystal structure analysis accept of mutant D339N. The extended sugar binding site can be subdivided into two regions: Firstly, there is a hydrophobic pocket at the reducing end with minor affinity contributions. Surprisingly, access to this site is blocked by a single exchange of aspartate to asparagine (D339N) without major loss in hexasaccharide affinity. Secondly, there is a region where specific exchange of glutamate for glutamine (E372Q) creates a site for an additional water molecule. Upon sugar binding side chain rearrangements lead to displacement of this water molecule and additional hydrogen bonding. Thereby this region of the binding site is defined as the high affinity scaffold. HK620TSP is not only specific for the O18A1-antigen, but also the lacking of the branching glucose in the O18A1-antigen can be tolerated so that the accordant O18A PS can be bound and cleaved by HK620TSP as well. Surprisingly, in binding studies with the smallest O-antigen units of these PS the O18A pentasaccharide was bound by TSP variants with nearly the same affinity or even a slightly increased one compared to the O18A1 hexasaccharide. However, there is a change in thermodynamic contributions to binding: the lack of the glucose moiety leads to a less entropically favored binding compared to binding of O18A1-hexasaccharide (Δ (-TΔS) ~ 10 kJ/mol). In contrast the enthalpic contribution to the binding is more favorable (ΔΔH ~ -10 kJ/mol) for the binding of O18A pentasaccharide. The side-chain glucose contributes to entropy by the release of four water molecules out of a hydrophobic pocket. The binding of this branching glucose is paid by an enthalpic penalty because of the breakup of hydrogen bonding of displaced water molecules and destabilizing contacts between sugar and protein in this hydrophobic pocket. Therefore the binding of the glucose in this pocket does not account for generating affinity and an evolutionary relation of HK620TSP to an O18A-antigen binding protein is presumed. Finally, the infection mechanism of phage HK620 was studied as well. In analogy to the related phage P22 the DNA-ejection could be triggered by incubation of HK620 with the host LPS in vitro. The morphology and chain length of the LPS as well as the activity of HK620TSP towards the LPS are crucial for this in vitro DNA-ejection. Thus, the DNA-ejection could also be induced by LPS from bacteria of serogroup O18A which can be bound and hydrolyzed by HK620TSP. These results stress the role of TSP for the recognition of host LPS-receptors as a crucial step of infection by podoviruses P22 and HK620.

In this study, process development for glucose isomerase (GI) was aimed. In this context, firstly, thermostable xyl genes, PCR amplified from Thermus thermophilus and Pyrococcus furiosus cells, were recombined to the E.coli BL21 (DE3) and P.pastoris strains, respectively. But significant increase in the term of GI activity compared with wild type cells only detected in recombinant E.coli strain so this strain was selected for further experiments. Then, the effect of different natural and artificial inducers on the production of rGI under control of LacUV5 promoter was investigated in laboratory-scale bioreactors. Lactose was shown to be more efficient in the term of operon induction for long time bioprocesses. Thereafter, in order to increase thermostable rGI production rate, to achieve high cell density culture of E.coli BL21 (DE3) pLysS pRSETA::xylT as well as to evade acetate accumulation, the effect of exponential feeding strategy of carbon source on the production of thermostable GI enzyme, cell concentration and acetate formation by recombinant E.coli BL21 (DE3) pLysS was investigated at four sets of fed-batch bioreactor experiments at three different predetermined specific growth rates 0.1 h-1 (M-0.1), 0.15 h-1 (M-0.15), 0.2 h-1 (M-0.2) and a glucose based exponential feeding at specific growth rate of 0.15 h-1(G-0.15) were performed by recombinant E.coli BL21 (DE3) pLysS cells. The highest biomass was obtained in M-0.15 condition as 9.6 kg m&minus
3 at t=32 h and the highest rGI activity was achieved in M-0.1 operation as A=16399 U L-1 at t=32 h of bioprocess. Moreover, peptide ligand with specific affinity toward histidin-tag peptide was selected by phage display technology. Isothermal titration calorimetry and surface plasmon resonance analyses were carried out to determine peptide-peptide interaction properties.

Bacteriophages (phages) have broad applications in diverse areas including phage therapy, agriculture, food safety, and environmental protection. In order to fully realize the potential for phage applications, it is critical to understand phage-bacteria interactions and characterize bacterial responses/targets to phage infection. Previous studies have largely focused on other classes of phages other than mycobacteriophages. This research provides the first global proteome investigation of the dynamic relationship between a mycobacteriophage and a mycobacterial host. Mycobacteriophages are viruses that infect mycobacteria. They have been reported to have vital potential uses in various fields, especially as an alternative in the prevention and treatment of mycobacterial diseases such as tuberculosis. Despite their potential, not much is known about the molecular interaction with mycobacteria during a mycobacteriophage infection, especially at the translational level. To better understand this, a novel mycobacteriophage, Ochi17 was first isolated and characterized based on the genome and structure. I then applied label-free quantitative proteomics using the model host, Mycobacteria smegmatis,which was infected with Ochi17 at different infection time points. Phage Ochi17 was found to be a temperate phage and classified as a Siphoviridae. The proteome changes occurring at the mid-lytic stage of Phage Ochi17 infection was first examined followed by a temporal study of the global changes. More than 2,000 M. smegmatis proteins and at least 50 Ochi17 proteins were identified across all time points. Homologous recombination and host macromolecular synthetic processes were significantly upregulated, while lipid metabolism was significantly downregulated. The results suggested that Ochi17 suppressed the growth of Mycobacterium smegmatis not just by utilizing the macromolecular synthesis of the host, but also by suppressing host transcription, and fatty acid biosynthesis, in addition to the degradation of fatty acids irrespective of infection time. The two-component system was a target at only 24 h post infection. I also showed that phage Ochi17 proteome expression is time-dependent and the proteins typically cluster based on functional relatedness. The results presented here may contribute in the development of mycobacteriophages as antimicrobial therapies that can overcome various defense strategies employed by host mycobacteria.

Dissertação de mestrado em Biotechnology
Pseudomonas aeruginosa and Acinetobacter baumannii are two Gram-negative pathogens frequently involved in hospital infections, including pneumonia or cystic fibrosis. Most infections caused by these bacteria are difficult to treat because of their low susceptibility to antibiotics, which is often associated with their ability to adhere to surfaces and form biofilms. Therefore, the World Health Organization has classified these microorganisms as top priority bacteria that urgently require the development of novel therapeutic approaches. Bacteriophages (phages) are bacterial viruses that have emerged as a possible novel treatment against antibiotic-resistant infections. The aim of this work was to study phage interaction with biofilms of P. aeruginosa PAO1 and A. baumannii RUH 134. To achieve this goal, single- and dual-species bacterial biofilms were characterized, revealing that both bacteria were able to form thick biofilms although depending on the culture media used. Interestingly, growth of these bacteria in dual-species biofilms led to the complete eradication of A. baumannii by P. aeruginosa in just 48h. After biofilm characterization, the efficacy of phages against single species biofilms with 24h, 72h and 7 days was evaluated. Although a significant reduction in viable cells and total biomass was observed in the first 3h and 6h post-infection of 24h- and 72h-old biofilms for both bacteria, after 24h of treatment the emergence of phage-resistance variants was observed. It was further observed that older biofilms (7 days) were in general less susceptible to phage treatment unless biofilms were cultured on DMEM:F12. The efficacy of phages was also evaluated against bacteria colonizing the normal human airway epithelial cell line NuLi-1. Phage treatment of NuLi-1 epithelial cells colonized with each bacterial species resulted in a significantly reduced mammalian cell death, with an effect that lasted longer than the activity of the phage on the bacterial cells. In conclusion, both phages revealed to be non-toxic for mammalian cells and a valuable approach for both early biofilm treatment and control of bacteria adhered to human epithelium.
Pseudomonas aeruginosa e Acinetobacter baumannii são duas bactérias Gram-negativas patogénicas que estão frequentemente associadas a infeções hospitalares, incluindo pneumonia e fibrose cística. A maioria das infeções causadas por estas bactérias são difíceis de tratar devido à sua baixa suscetibilidade aos antibióticos, que está frequentemente associada com a sua capacidade de aderir a superfícies e formar biofilmes. Assim, a Organização Mundial de Saúde classificou estes microrganismos como bactérias de prioridade máxima que requerem o desenvolvimento urgente de novas abordagens terapêuticas. Bacteriófagos (fagos) são vírus bacterianos que têm emergido como uma potencial estratégia no tratamento de infeções resistentes a antibióticos. O objetivo deste trabalho foi o estudo da interação entre fagos e biofilmes de P. aeruginosa PAO1 e A. baumannii RUH 134. Para isso, foi avaliada a formação de biofilme das duas bactérias isoladamente e em conjunto, demostrando que ambas foram capazes de formar um biofilme espesso, embora dependendo do meio de cultura usado. Curiosamente, quando as bactérias cresceram em conjunto, em apenas 48h verificou-se uma erradicação completa de A. baumannii causada pel P. aeruginosa. Após a caracterização dos biofilmes, foi avaliada a eficácia dos fagos no controlo de biofilmes de 24h, 72h e 7 dias. Embora se tenha observado uma redução significativa no número de células viáveis e biomassa total nas primeiras 3h e 6h de infeção dos biofilmes formados durante 24h e 72h para ambas as bactérias, após 24h de tratamento verificou-se uma proliferação de variantes resistentes aos fagos. Foi também observado que em biofilmes mais maduros (7 dias), geralmente houve uma menor suscetibilidade ao tratamento com os fagos, exceto quando cultivados em meio DMEM:F12. A eficácia dos fagos foi também avaliada em células epiteliais das vias respiratórias humanas, NuLi-1. O tratamento com fagos nas células epiteliais NuLi-1 colonizadas com cada uma das espécies bacterianas resultou numa redução significativa da morte celular, com um efeito mais prolongado do que a atividade dos fagos nas células bacterianas. Em conclusão, ambos os fagos revelaram ser não ser tóxicos para as células animais e uma abordagem promissora para o tratamento inicial de biofilmes e para o controlo de bactérias aderidas ao epitélio.