Les interactions entre les membres d'une communauté microbienne peuvent être un moyen d'adaptation dans l'environnement. Parmi les nombreuses interactions qui ont lieu dans un écosystème et qui joue un rôle majeur sur la diversité et la dynamique des populations microbiennes est celui des virus procaryotes et leurs hôtes. Les virus peuvent également arbitrer le transfert de matériel génétique entre les procaryotes (transduction), qui pourrait être un mécanisme d'adaptation rapide. Afin de déterminer l'impact potentiel des virus et la transduction, nous avons besoin d'une meilleure compréhension de la dynamique des interactions entre virus et leurs hôtes dans l'environnement. Les données sur les virus de l'environnement sont rares, et les méthodes pour le suivi de leurs interactions avec les procaryotes sont nécessaires. Clustered regularly interspaced short palindromic repeats (CRISPRs), qui contiennent des séquences virales dans les génomes bactériens, pourraient aider à documenter l'histoire des interactions virus-hôte dans l'environnement. Ainsi, cette thèse vise à explorer les interactions virus-hôte dans un environnement donné à travers du séquences CRISPR.Les virus de la cryosphère sont considérés comme abondantes, très actif et avec de larges gammes d'hôtes. Ces caractéristiques pourraient faire de la transduction virale, un facteur clé pour l’adaptation microbienne dans ces environnements. Des métagénomes publics créés à partir des environnements avec une gamme de températures différents ont été examinés. De cette manière, certaines dynamiques d'interactions virus-hôte se sont révélées comme ayant une corrélation avec la température. Un flux de travail a ensuite été développé pour créer un réseau reliant les virus et leurs hôtes en utilisant des séquences CRISPR obtenus à partir de données métagénomiques de la glace des glaciers et du sol de l'Arctique. La création de réseaux d'infection à traves du CRISPRs a fourni une nouvelle perspective sur les interactions virus-hôte. En outre, nous avons cherché des événements de transduction dans les données métagénomiques par la recherche de séquences virales contenant de l'ADN microbien. L’analyse indiquée que les bactériophages du Ralstonia pourraient être des agents de transduction dans la glace des glaciers de l'Arctique. / Interactions between the members of a microbial community can be a means of adaptation in the environment. Among the many interactions that take place in an ecosystem and have been seen to play a major role on microbial diversity and population dynamics is that of prokaryotic viruses and their hosts. Viruses can also mediate the transfer of genetic material between prokaryotes (transduction), which could be a mechanism for rapid adaptation. In order to determine the potential impact of viruses and transduction, we need a better understanding of the dynamics of interactions between viruses and their hosts in the environment. Data on environmental viruses are scarce, and methods for tracking their interactions with prokaryotes are needed. Clustered regularly interspaced short palindromic repeats (CRISPRs), which contain viral sequences in bacterial genomes, might help document the history of virus-host interactions in the environment. Thus, this thesis aimed to explore virus-host interactions in a given environment through CRISPRs. Viruses in the cryosphere have been seen to be abundant, highly active and with broad host ranges. These characteristics could make viral transduction a key driver of adaptation in these environments. Public metagenomes created from environments over a range of temperatures were examined through sequence and CRISPR analysis. In this fashion, certain virus-host interaction dynamics were found to have a correlation with temperature. A workflow was then developed to create a network linking viruses and their hosts using CRISPR sequences obtained from metagenomic data from Arctic glacial ice and soil. The creation of CRISPR-based infection networks provided a new perspective on virus-host interactions in glacial ice. Moreover, we searched for transduction events in metagenomic data by looking for viral sequences containing microbial DNA. Further analysis of the viral sequences in the CRISPRs indicated that Ralstonia phages might be agents of transduction in Arctic glacial ice.

Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC. EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber. Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC. After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells. Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.

Philosophiae Doctor - PhD / There are approximately 10³¹ bacteriophages in the biosphere, outnumbering bacteria 10:1, hence, the dynamic and specific nature of phage-host interactions exerts significant influence on microbial communities. Bacteriophages also represent the reservoir of the highest known genetic diversity making them a potential source of novel biotechnological products. However, the isolation of novel bacteriophages is limited by the observation that less than 1% of bacterial hosts have been cultured. This study aimed to bypass this problem by developing novel culture independent approaches to improve our ability to isolate novel phage-host pairs. Samples were collected from an abandoned copper prospecting site near the Gobabeb Desert Research and Training Station and a Salt lake located in the Swakopmund region of the Namibian desert. Two approaches were explored in this study namely viral tagging and reverse metaviromics. For viral tagging, fluorescently labelling the environmental phage fraction before challenging the environmental bacterial fraction with tagged phages proved difficult. This was most likely due to the complex interaction of the labelling agent with phages and requires further studies. For the reverse metaviromics approach, total DNA from the environmental phage fractions was extracted, sequenced and analyzed for novel phages. Analysis of the phage diversity showed that the copper site was dominated by tailed viruses as has been shown for other extreme arid environments. However, the saline site was atypical of marine environments, with tailed viruses being the most abundant, suggesting that the diversity present is not only driven by salinity. Using the metaviromic sequence data to guide the selection of potential bacterial hosts, two strategies were employed. In the first, putative hosts were predicted based on similarity of phage sequences to those identified in databases. Media targeting these specific genera were employed, 8 bacterial species were isolated and based on 16S rRNA similarity to the closest known species were identified as Halomonas caseinilytica, Halomonas eurihalina, Halomonas sinaiensis, Idiomarina loihiensis, Marinobacter xestospongiae, Virgibacillus salarius and two Salinivibrio species. The 16S rRNA analysis also suggested that H. sinaiensis, V. salarius and both Salinivibrio species are novel. All 8 isolates were challenged with the environmental phage fraction. A novel phage, SMHB1, was isolated on one of the Salinivibrio spp. and is only the second characterized phage ever described for this genus. SMHB1 is a 32 kb myovirus, with a head diameter of 56 nm, and a tail length of 106 nm. The second approach involved the design of fluorescently labelled probes targeting phages identified from the metaviromic sequence data. In a control E. coli system to detect cloned phage DNA fragments, 87% of the interrogated cells showed significant hybridization of the phage specific probe to the target. The optimized method was applied to a simulated environmental bacterial fraction and a detection limit of 1:100 was observed for the bacteria containing the phage DNA fragment of interest. This study demonstrates the possibility of improving the specificity of isolating phage-host pairs in a culture-independent manner by incorporating sequence data in the experimental design; and contributes to our knowledge of the phage diversity of an understudied extreme environment.

Virtually all organisms exhibit circadian rhythms, this includes mosquitoes. Many aspects of their biology are under the control of their circadian clocks like their vision, olfaction, host-seeking, mating, oviposition, metabolism, locomotion, and more. However, how the circadian clock regulates mosquito-host interactions and adapts to specific environmental conditions remains largely unknown, despite its importance to vector disease control. Here, we relied on a multidisciplinary, integrative, and comparative approach to shed light on the mechanisms underlying mosquitoes adaptations to various temporal niches. We use CRISPR/Cas9 to knockout timeless in Aedes aegypti and show this mutation causes an increase in their free running periods under continuous darkness conditions. External factors can also influence a vector's activity pattern like climate, light pollution, as well as host preference and availability. To investigate the influence these factors have on activity patterns, we compare the activity patterns of multiple lab rear strains of Aedes albopictus as well as two field collected populations of Aedes japonicus. Our results suggest host availability and light pollution could cause the differences in activity profiles that we observed. With vision playing an important role in both circadian rhythms, host seeking, and threat detection, we compared a nocturnal mosquito (Aedes japonicus) and a diurnal mosquito (Aedes aegypti). We introduced a looming visual stimulus in an LED arena and found Aedes aegypti to be more responsive to the looming stimulus than Aedes japonicus. Finally, the underlying mechanisms that mediate this threat detection and escape behavior were examined, revealing a possible candidate for a giant fiber neuron in Aedes aegypti. Overall these results provide novel insights into mosquito biology that have the potential to be applied towards vector control methods. / Doctor of Philosophy / Most living things, including mosquitoes, follow a daily pattern called a circadian rhythm. This rhythm affects many parts of their lives including their vision, smell, and activity. Understanding how their circadian clock functions can help us control populations of mosquitoes and prevent the transmission of diseases they may carry. By studying different species and populations of mosquitoes in the lab and from the wild, we have found that factors like light pollution and host availability could affect when mosquitoes are active. In addition to these results we have also demonstrated that mosquitoes that are active during the day and mosquitoes that are active during the night will respond differently to a looming visual stimulus. We examined the brain of the mosquitoes that were most responsive to the looming stimulus and found a system of large neurons that could potentially be responsible for eliciting their escape behaviors. Overall these results provide new and important information about mosquito biology and can be applied to mosquito control.

<div>Viruses are a group of contagious microbes that have compact structures, containing a nucleic acid core and a protein shell. The replication of viruses requires assistance from hosts which can be almost any cellular organism. Viral infections are often associated with diseases and have been a major threat to the human race. To cope with viral diseases, we need to understand viruses, including their structures, life cycle, pathogenesis and interactions with their hosts. The first structure of a human virus was determined by the Rossmann lab in 1985 using X-ray crystallography.</div><div>Thanks to the recent advances in both hardware and software, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool to study virus structures. Cryo-EM allows structural determination for a wide range of specimens to high resolution comparable to what can be achieved by X-ray crystallography. Currently two techniques of cryo-EM are commonly used in structural virology: single particles analysis (SPA) and electron tomography (ET). </div><div>Single particle analysis has been used to determine the structures of viruses complexed with host factors in three studies that are to be discussed with more details in chapters 2-4. </div><div>The structure of B19 parvovirus complexed with Fabs of a neutralizing human antibody was determined to 3.2 Å resolution. This structure showed that amino acids from three neighboring VP2 proteins form a quaternary structure epitope. In addition, the structure of human rhinovirus-C (RV-C) complexed with its cellular receptor, CDHR3, was determined to 3.9 Å resolution. Despite the low occupancy of the receptors, a “powerful” localized 3D classification procedure helped to select viral particles that had more bound receptors. Furthermore, structures were determined to 10 Å resolution of bacteriophage ΦX174 bound to lipopolysaccharide (LPS) bilayers, before and after genome ejection. These structures showed a series of conformational changes that occurred when a phage penetrated the bacterial membranes. These studies are good examples of applying cryo-EM to investigate virus-host interactions.</div><div>However, single particle analysis requires samples to be isolated, homogenous and monodispersed. On the contrary, tomography allows in situ studies and is applicable to samples with more flexibility and more heterogeneity. In the case of ΦX174, the structural changes that are involved in the assembly of the H-tube during infection remains a huge mystery. To provide an environment that is more similar to the surface of a bacterial cell, LPS-containing liposomes were mixed with ΦX174 viruses. It was then observed that the ΦX174 particles bound to these liposomes in a very compact manner which was impossible interpret with single particle analysis. Using cryo-ET, 3D volumes of liposome-ΦX174 complexes were reconstructed and structural details were visualized by sub-tomogram classification and averaging.</div><div>The emergence of cryo-EM has not only made high-resolution structural studies possible but also broadened the scope of samples with which virologists could work. Moreover, studies on flexible and heterogeneous complexes between viruses and host factors are now possible using either single particle analysis or electron tomography. These techniques will help us to understand virus-host relationships and finally, to develop effective anti-viral therapies.</div>

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.

The development of a new technique capable of analyzing compounds crystallographically without first needing to crystallize them has been recently described. The present thesis aims to demonstrate the potential of such a technique, which utilizes crystalline sponges, in order to regularly order guest compounds in a porous media. The structural stability of the molecular sponges, which are highly porous metal-organic frameworks (MOFs), is first investigated, revealing that the Co-based MOF, 1, undergoes two remarkable transformations. This thesis also demonstrates how the technique can be employed to visualize the motion and occupancy of gaseous guests in a MOF. The Zn-based MOF, 4, was found to physisorb and chemisorb molecular iodine, leading to the formation of a variety of polyiodide species. The flexible nature of the host was determined to be an essential component in the exceptionally large iodine uptake capacity of the MOF. These results illustrate that the crystalline sponge method can be an effective strategy for directly visualizing guest molecules and obtaining vital information on the interactions formed between the host and guest.

Cyclodextrins are well known for their ability to encapsulate molecules and have captured the attention of scientists for many years. This ability alone makes cyclodextrins attractive for study, research, and applications in many fields including food, cosmetics, textiles, and the pharmaceutical industry. In this thesis, we specifically look at the ability of the three native cyclodextrins, alpha, beta, and gamma cyclodextrin (α-CD, β-CD, and γ-CD, respectively), to encapsulate the drug molecule, quinine, a small hydrophobic, lipophilic molecule used to treat malaria, leg cramps, and other similar conditions. This encapsulation process is driven by the molecular interactions, which have been studied by NMR techniques at different temperatures (288 K, 293 K, 298 K, 303 K, 308 K) and pH values (7.4, 11.5). These factors (temperature and pH) influence these molecular interactions, which in turn significantly affects the entire encapsulation process. Detailed studies of the influences of temperature and pH on the interactions that drive the encapsulation may suggest some new directions into designing controlled drug release processes. Results obtained throughout the course of this work indicate that β-CD is the best native cyclodextrin to bind quinine, and that binding is best at pH = 11.5. It was found that temperature does not significantly affect the binding affinity of quinine to either α-CD, β-CD, or γ-CD.

The interactions between parasites, hosts, and the ecosystems they live in are complex and diverse. This can make predicting how they will respond to pressures like nutrient enrichment difficult. However, this endeavor is important, due to the far-reaching effects it has on ecosystems functioning, industries like aquaculture, and even human health. Even with the significance of this topic, there are key parts of the picture that have received little research attention. For this reason, I chose to study the effects of nutrient enrichment on parasite-host interactions at two different scales, first investigating the interaction directly between parasites and their host, and then manipulating experimental ponds to look at trophic effects. Using the theory of ecological stoichiometry as a conceptual framework allowed for comparison across the two investigations. In both studies, I used largemouth bass and the macro parasites of their digestive tract as my study system, due to the importance of largemouth bass as sport fish, their high trophic status, and diverse parasite assemblages. My first study examined the effects of eutrophication on parasite-host interactions occurring within the host. It centered on four hypotheses: 1) the nutrient content of different host tissues (infection sites) varies within and across hosts, 2) the nutrient contents of parasite genera differ from that of their host tissue(s), 3) the nutrient contents of parasite genera differ from one another, and 4) nutrient availability within specific host tissues is related to the nutrient content and abundance of parasite genera within tissues. I quantified the nutrient content (carbon (C), nitrogen (N), and phosphorous (P) and their ratios) of parasitic infracommunities in the tissues of the liver, stomach, pyloric caeca, intestines, and visceral mesentery of 30 largemouth bass (Micropterus salmoides). I found stoichiometric variation across host tissues and that fish collection site explained variation in pyloric caeca N:P ratios. Parasitic genera differed in their nutrient content with actively feeding parasite forms having higher %N and lower C:N ratios than encysted/non-reproductive forms. In addition, the %C of both actively feeding parasites varied across organs, and, for one genus, this variation reflected differences in host tissue %C. Finally, I found that the total number of actively feeding parasites in the pyloric caeca increased with that tissue’s N:P ratio. My results suggest that parasites encounter significant variation in nutrient availability within and across hosts, and that this variation can influence the nutrient content and abundance of some actively feeding parasites within specific tissues. To determine if the effects from my first study were a result of nutrient changes to host tissues or if they were an artifact of changes occurring at other trophic levels caused by nutrient addition, I conducted an experimental manipulation of a short trophic system. I applied two treatments, a fertilized treatment and a control, to 14 ponds stocked with largemouth bass. I hypothesized that adding a low N:P fertilizer to the ponds would result in fewer parasites in the pyloric caeca of the bass in accordance with findings from my previous work. Alternatively, I hypothesized that indirect changes to intermediate hosts would be responsible for changes in parasite abundance. To measure the effects of nutrient additions on multiple trophic levels, samples of fine particulate organic matter (FPOM), benthic invertebrates, and vertebrate prey were taken from each pond. Stocked bass were retrieved and dissected to obtain samples for nutrient analysis from their pyloric caeca, and to quantify parasite abundances. My treatment was able to affect the %P, C:N, C:P, and N:P of the FPOM in the ponds. Benthic invertebrate biomass was negatively correlated with FPOM %P. I did not find a direct effect of benthic invertebrates on vertebrate prey species, but fertilized ponds had a smaller decrease in the number of vertebrate prey over the course of the experiment. Counterintuitively, the %N of the pyloric caeca of fish in the fertilized ponds decreased, and the %C and C:N increased in relation to the number of vertebrate prey, but %P did not change. The treatment status of the pond and the nutrients of the pyloric caeca did not affect the number of parasites that were found, but parasite numbers were positively associated with prey fish. This result would suggest the number of intermediate hosts, in this case, prey fish, is the determining factor for parasitic loads in the bass I collected. The indirect effects of trophic interaction and the direct effects of changes to host and parasite dynamics both play important roles in predicting the outcome of eutrophication on disease. The conflicting results of these two studies highlight the need for in-depth knowledge of the systems being affected by nutrient additions.

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