Microbiology Commons^™

Identifying The Role Of Human Dna Polymerase Delta During Human Cytomegalovirus Genome Synthesis, Rachel Laree Dansereau

Dartmouth College Master’s Theses

Human cytomegalovirus (HCMV) is a large double stranded DNA virus and member of the Herpesviridae family. Like all herpesviruses, HCMV presents with characteristics of persistent, lifelong infections that transition between lytic and latent states. The replication of the viral genome is traditionally thought to occur by a rolling circle mechanism, but this model is supported by little evidence. It is known that HCMV actively recruits several host proteins to viral replication compartments (RCs), including proteins involved in the DNA damage response (DDR). The exact mechanisms of how these proteins are used and in what ways they help the virus remain …

Understanding The Role Of Toxs As A Bile Sensing Component Of The Toxrs Virulence Regulatory System In Vibrio Spp., Minje Kim

Dartmouth College Ph.D Dissertations

Pathogenic Vibrio bacteria, such as V. cholerae and V. parahaemolyticus, colonize in the human small intestine to cause severe gastrointestinal disease. During the infection, these bacteria encounter bile salts, antimicrobial cholesterol metabolites secreted into the intestine. Pathogenic Vibrio species have evolved to utilize bile salts as signals to regulate virulence. The signaling depends in part on ToxRS, a conserved co-component transmembrane transcription regulator. ToxRS consists of the transcription factor ToxR and its membrane-tethered binding partner ToxS. ToxS is required for ToxR stability and full transcriptional activity. Although bile salts are known to influence ToxRS-dependent virulence gene expression, the molecular …

Evolution Of Antibiotic Responses During Chronic P. Aeruginosa Infections In The Human Lung, David Frank Ritz

Dartmouth College Ph.D Dissertations

Typical antibiotic susceptibility testing (AST) of microbial samples is performed in homogeneous cultures in batch environments, which does not account for the highly heterogeneous and dynamic nature of antibiotic responses. The most common mutation found in P. aeruginosa lineages evolved during chronic cystic fibrosis infections in the human lung, a loss of function of repressor MexZ, increases basal levels of multidrug efflux MexXY, but does not increase resistance by traditional minimal inhibitory concentration (MIC) assays. Here, we use single cell microfluidics to show that P. aeruginosa’s response to aminoglycosides is highly heterogeneous, with only a subpopulation of cells surviving exposure. …

Biofilm Architecture Determines Proliferation Of Phages And Plasmids, James B. Winans

Dartmouth College Ph.D Dissertations

This thesis explores how biofilm architecture determines the spread and ecological consequences for the bacteria-dependent genetic elements phages and plasmids. Using Escherichia coli and Vibrio cholerae as model microbes, and the genetic elements T7 phages, λ phages, and conjugative plasmid pED208, we investigate how spatial structure influences phage infection dynamics, plasmid transfer, and interbacterial interactions within biofilms. We show that densely packed biofilms can protect sensitive bacteria from phage exposure and modulate competitive outcomes between obligately lytic and temperate phages. We find that entirely different outcomes can coöccur within microns of each other dependent on biofilm structure. We also demonstrate …

Investigating The Mechanisms Of Microbial Interactions Using A Model Cystic Fibrosis Polymicrobial Community, Bassam El Hafi

Dartmouth College Ph.D Dissertations

Cystic fibrosis (CF) airway microbial communities are complex ecosystems where interspecies interactions influence pathogen survival and disease progression. We leverage a CF-relevant in vitro four-species model composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, alongside a microbiome dataset with over 4,000 respiratory samples from publicly available sequencing data of CF patient samples, to explore the dynamics of microbial interactions in CF. On a broader scale, we performed microbial network analysis to examine how the latest modulator therapy, elexacaftor/tezacaftor/ivacaftor (ETI), might influence CF microbial communities. Our analysis revealed a reduction in node connections and …

The Impact Of Polymycovirus 1 On Aspergillus Fumigatus Pathogenesis During Lung Infection, Alexander W. Rapp

Dartmouth College Ph.D Dissertations

The genus Aspergillus comprises over 250 species of fungi. Within this genus, only a handful of species cause disease in humans. One species is Aspergillus fumigatus (A.f.), an environmentally ubiquitous, saprophytic fungal pathogen. Lung infections due to A.f. range from invasive infection to allergic forms of the disease. Allergic bronchopulmonary aspergillosis (ABPA) is one of the most severe forms and presents as a hypersensitivity that often results in off-target damage to the surrounding airways. Patients with ABPA are sensitized to fungal antigens and this is commonly seen in cystic fibrosis and genetic predispositions. Recent data from our lab shown how …

Colonization-Persistence Trade-Offs In The Human Microbiome, Liam F. Nokes

Environmental Studies Senior Theses

Understanding how diverse microbial taxa coexist in the human body without competitively excluding one another remains a key challenge in microbiome ecology. One proposed explanation is the colonization-persistence trade-off, where species with high colonization ability are poor persisters, and vice versa. We test for this trade-off across human associated microbial communities among many individuals and in multiple body sites using a large-scale participant-level meta-analysis of longitudinal microbiome datasets curated from the MGnify database. We applied island biogeography-based models to calculate effective colonization and persistence rates for microbial families and genera across 606 individuals over twenty-six studies. Regression of the log-transformed …

Belowground Biomass Shifts Along A Temperate Forest Edge, Lara Holbrook Roelofs

Dartmouth College Undergraduate Theses

Anthropogenic land use change and development has caused widespread global forest fragmentation, resulting in a greater proportion of forests at or near a forest edge. Temperate forests are the most fragmented forest biome, and forests in the Northeastern U.S. are particularly fragmented due to a history of intense land use and development pressure. Nearly a quarter of these forests are now within 30 meters of a forest edge. These edge ecosystems have distinct abiotic and biotic gradients that differentiate them from forest interiors, including increased light, temperature, and exposure to wind and other disturbances, as well as decreases in humidity …

Emergence Of Heterogeneity In Bacteria During Antibiotic Exposures, Mirjana Stevanovic

Dartmouth College Ph.D Dissertations

Bacterial populations exposed to antibiotics often exhibit phenotypic heterogeneity, with individual cells displaying distinct responses with fates ranging from survival to arrest or death. The emergence of this heterogeneity is largely driven by feedback mechanisms mediated by the interactions between antibiotic action, gene regulation, and cellular metabolism. These responses unfold in dynamic environments where conditions such as nutrient availability and drug concentration fluctuate over time and space. Yet, these complex interactions and their role in shaping population-level outcomes during drug responses remain poorly understood. Using tetracycline resistance in E. coli as a model system, this thesis focuses on how these …

Metabolite Analysis To Understand Factors Limiting Microbial Fermentation At High Substrate Concentrations, Bishal D. Sharma

Dartmouth College Ph.D Dissertations

Clostridium thermocellum, a thermophilic anaerobic bacterium, holds promise as a biocatalyst for conversion of lignocellulosic biomass into biofuels. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes encoding secondary fermentation products such as H₂, formate, acetate, and lactate, as well as the expression of heterologous genes to redirect carbon flux toward ethanol production. Despite these efforts, achieving economically viable ethanol titers remains a challenge. We hypothesized that studying how intracellular metabolite concentrations change as fermentation stops could reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at …

Biochemical And Structural Insights Into The Yersinia Effector Yopm And Its Negative Regulation Of The Pyrin Inflammasome, Bethany Wairimu Mwaura

Dartmouth College Ph.D Dissertations

Bacteria utilize sophisticated secretion systems to inject effectors into host cells in order to facilitate their survival and replication in the host. Some pathogenic species of Gram-negative bacteria use a conserved contact-dependent T3SS to inject a wide array of effectors. While the effectors diverge in their structures, functions and cell-localization, the T3SS is well conserved. Several effectors discussed in this thesis target key host inflammasome responses. The focus of this work is how the Yersinia effector YopM inhibits the pyrin inflammasome. During Yersinia infection, two effectors, YopE and YopT inadvertently activate the pyrin inflammasome while targeting RhoA to avoid phagocytosis. …

Modeling The Cystic Fibrosis Intestinal Environment To Study Microbial Interactions, Kaitlyn Emma Barrack

Dartmouth College Ph.D Dissertations

The microbiome of the cystic fibrosis (CF) intestine is altered significantly in structure and function beginning in early life. Key taxa associated with health, digestion and immune-training are reduced in abundance, including but not limited to Bacteroides, Faecalibacterium, Akkermansia, Ruminococcus and other short-chain fatty acid producers. Conversely, there is an expansion of microbes with pathogenic potential, such as Escherichia, Veillonella, Pseudomonas and Staphylococcus. The shifts in microbiota composition direct community-wide changes with differences in beta diversity by genotype, whereby children with CF (cwCF) exhibit significantly different gut microbial compositions compared to nonCF children. Additionally, shifts in microbiota structure often …

Neutrophil Inflammatory Death Regulation By Bacterial Type Iii Secreted Effectors, Arianna D. Reuven

Dartmouth College Ph.D Dissertations

Yersinia and Pseudomonas use a T3SS to secrete effectors into host cells that inhibit phagocytosis and modulate immune responses, such as inflammasome activation. Inflammasomes promote the activation of the proinflammatory protease caspase-1. The activation of an inflammasome sensor in response to a signal or ligand initiates the assembly of the inflammasome leading to activation of caspase-1 which cleaves GSDMD and pro-inflammatory cytokines IL-1β and IL-18 into their mature forms. As neutrophils are recruited in large quantities to sites of infection, they are excellent candidates to drive inflammasome dependent responses. Neutrophils have been shown to produce multiple inflammasomes, like pyrin and …

Of Mice And Men: The Regulation Of Herpes Simplex Virus 1 Latency In Mouse And Human Neurons, Paige N. Canova

Dartmouth College Ph.D Dissertations

Herpes simplex virus 1 (HSV-1) establishes latent infections in sensory neurons, from which HSV sporadically reactivates, often due to external stress and other stimuli. Latency and reactivation are commonly studied using in vivo and ex vivo models in a variety of hosts, as well as in vitro models including primary mouse neurons and neurons derived from human pluripotent stem cells (iPSCs). The criteria for authentically modeling HSV latency include the ability to easily manipulate host genetics and biological pathways, as well as mimicking the immune response and viral pathogenesis in human infections. Presently, there remains a pressing need for …

Engineering Extracellular Vesicles (Evs) To Combat Infection And Inflammation In Cystic Fibrosis, Sharanya Sarkar

Dartmouth College Ph.D Dissertations

Cystic Fibrosis (CF) is a multisystemic genetic disorder caused by mutations in the CFTR gene, resulting in chronic pulmonary infections, hyperinflammation, and progressive lung damage. Among the pathogens that colonize CF lungs, Pseudomonas aeruginosa (P. aeruginosa) is predominant, infecting over 50% of adults with CF and developing antibiotic resistance over time. Current modulator therapies fail to address persistent bacterial infections, chronic inflammation, or irreversible lung damage, underscoring the need for novel therapeutic strategies. In our previous in vitro studies, we demonstrated that extracellular vesicles (EVs) secreted by primary Human Bronchial Epithelial Cells (HBECs) inhibit P. aeruginosa growth by reducing the …

Biochemical And Molecular Analysis Of Anr-Dependent Microoxic Fitness Factors In Pseudomonas Aeruginosa, Stacie E. Balsam

Dartmouth College Ph.D Dissertations

Pseudomonas aeruginosa is a prominent opportunistic pathogen that is often found in microoxic environments like sites of infection and colony biofilms. P. aeruginosa can grow at oxygen (O2) at concentrations as little as 3 μM and also grows anaerobically by generating energy using nitrate as an alternate electron acceptor. An important way P. aeruginosa adapts to low O2 is through Anr, a transcription factor that upregulates many genes important for microoxic and anoxic fitness.

Specifically, Anr regulates mhr which encodes a hemerythrin protein (Mhr) that binds O2 with affinities relevant to microoxia and is also necessary for full fitness of …

Targeting Aspergillus Fumigatus Hypoxia Response Pathways For Novel Antifungal Drug Development., Cecilia Gutierrez Perez

Dartmouth College Ph.D Dissertations

Invasive fungal disease present are difficult to diagnose and treat and present a high mortality rate across the world with approximately 1.5 million people a year succumbing to fungal infections worldwide. Currently, there are limited antifungal classes used therapeutically, and antifungal drug resistance on the rise further exacerbates the dire need for novel therapeutics with innovative mode of actions. The filamentous fungi, Aspergillus fumigatus is the causative agent of invasive pulmonary aspergillosis (IPA). It is estimated that contemporary antifungals fail in about 50% of patients with invasive filamentous fungal infections, likely due to the biofilm mode of growth exhibited in …

Fleq And Gac/Rsm-Mediated Regulation Of Biofilm Formation And Swarming Motility In Pseudomonas Fluorescens, Alexander Benito Pastora

Dartmouth College Ph.D Dissertations

Pseudomonas fluorescens is a gram-negative Gammaproteobacterium that survives and persists in diverse environments. This survivability is dependent on the bacterium’s ability to form a biofilm. Within Pseudomonas fluorescens, biofilm formation is a complex and dynamic process involving multiple protein- and polysaccharide-based components which each individually serve a specific purpose. Of these components, the repeats-in-toxin (RTX) adhesins LapA and MapA are critical for biofilm formation. Specifically, LapA is important for initial establishment of a biofilm and MapA is important in forming a fully mature biofilm. Deployment of the RTX adhesins during biofilm formation is a highly coordinated process involving multiple …

Establishment Of Bacteroides In The Gut And Its Role In The Cystic Fibrosis Gut-Lung Axis, Rebecca A. Valls

Dartmouth College Ph.D Dissertations

In the quest to understand the complex interplay between the gut microbiota and cystic fibrosis (CF), a disease characterized by chronic upper respiratory complications and an exacerbated proinflammatory response, our lab engaged in a collaborative study with Dartmouth-Hitchcock Medical Center (DHMC) to characterize the gut microbiota of pediatric CF patients. Initial analyses established a connection between gut microbial composition and respiratory health, particularly highlighting a scarcity of Bacteroides species compared to healthy controls. In following investigations, we utilized random forest models to understand how stool microbiota may influence clinical outcomes of these pediatric CF patients, including: age, upper respiratory infections, …

Understanding The Non-Canonical Regulation Of Srebp In Aspergillus Fumigatus, Muhammad Abubakar Khan

Dartmouth College Ph.D Dissertations

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive pulmonary aspergillosis (IPA), with high mortality rates in immunocompromised individuals. Adaptation to the hypoxic microenvironment of IPA is crucial for fungal virulence. The transcription factor SrbA, a Sterol Regulatory Element-Binding Protein (SREBP) homolog, plays a key role in this hypoxic response and regulates genes involved in hypoxic growth, sterol biosynthesis, and azole resistance, making it an attractive therapeutic target.

However, SrbA activation in A. fumigatus lacks critical components of the canonical SREBP pathway, such as SCAP, Site-1 protease (S1P), and Site-2 protease (S2P). Instead, the rhomboid protease RbdB, signal peptide peptidase …

Mechanistic Insights On Positive Regulation Of The Pyrin Inflammasome In Macrophages, Haleema Sadia Malik

Dartmouth College Ph.D Dissertations

The mammalian innate immune defense employs sophisticated mechanisms, such as effector triggered immunity (ETI), to discern between pathogenic and non-pathogenic bacteria and initiate a protective response. ETI is involved in immune detection of virulent activities of bacterial effector proteins in the host cytosol. Inflammasome sensors, such as pyrin, detect cellular infection or stress as part of ETI, leading to the assembly of multiprotein complexes, caspase-1 activation and proinflammatory cytokine release. Pyrin uniquely initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors such as Yersinia's YopE and YopT, and C. difficile’s toxins TcdA/B. Understanding pyrin regulation is crucial due to its …

Investigating The Mechanisms Of Surface Sensing Using Motility Appendages By Pseudomonas Aeruginosa Pa14, Christopher James Geiger

Dartmouth College Ph.D Dissertations

Biofilms are surfaced attached communities of cells encased in an extracellular matrix. The transition from free-swimming planktonic cells to a surface attached biofilm begins with cellular changes that occur after surface contact. This process is known as "surface sensing" and the opportunistic pathogen Pseudomonas aeruginosa PA14 uses its two motility appendages, type IV pili (T4P) and a single, polar flagellum to sense and traverse surfaces. The first cellular changes to occur within this organism upon surface contact is an increase in the second messengers cAMP and cdi- GMP. While the genes involved in surface sensing by P. aeruginosa are known, …

Dpr-Mediated H2o2 Resistance Contributes To Streptococci Survival In A Cystic Fibrosis Airway Model System, Rendi R. Rogers

Dartmouth College Master’s Theses

The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). Streptococcus spp. is highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown Streptococcus spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of Streptococcus spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more …

Social Interactions And Spatial Structure Drive Community Assembly Of Bacterial Biofilm, Matthew C. Bond

Dartmouth College Ph.D Dissertations

Despite the critical nature which microbial communities play in the natural world and human civilization, the breadth of understanding remains shallow. Challenged by scale, high variability between environments, and extensive diversity, microbial ecologists strive to understand connections between a community’s structure and function, as well as the ecological and evolutionary mechanisms underlying observed natural patterning of communities. Biofilms are the predominate mode of growth for microbial communities—characterized by cellular attachment to a surface via a self-produced matrix and heterogeneous structure, often resulting in a primary growth front along the biofilm surface due to differential access to bulk nutrients (cite). This …

Dust-Driven Mechanism For Marine Particle Aggregation: Interactions Between Clay Minerals, Heterotrophic Bacteria And Organic Matter Influences Aggregation In Marine Systems, Vignesh Gokuladas Menon

Dartmouth College Master’s Theses

Atmospheric dust deposition plays a crucial role in marine biogeochemical cycles, yet mechanisms underlying its impact on particle dynamics remain unclear. This study investigates the synergistic interactions between marine heterotrophic bacteria and clay minerals in seawater, focusing on their effect on Transparent Exopolymer Particles (TEP) production and organoclay aggregation. Field experiments in the Gulf of Maine and laboratory roller tank studies were conducted using natural microbial communities and model heterotrophic bacterial strains (Alteromonas sp. EZ55, Marinobacter adhaerens HP15, and Sulfitobacter sp. NAS-14.1). Results demonstrate that clay exposure significantly enhances TEP production by bloom-associated heterotrophic bacteria, with increases up to …

Tracing Evolution Of Gene Transfer Agents Using Comparative Genomics, Roman Kogay

Dartmouth College Ph.D Dissertations

The accumulating evidence suggest that viruses and their components can be domesticated by their hosts, equipping them with convenient molecular toolkits for various functions. One of such domesticated system is Gene Transfer Agents (GTAs) that are produced by some bacteria and archaea. GTAs morphologically resemble small phage-like particles and contain random fragments of their host genome. They are produced only by a small fraction of the microbial population and are released through a lysis of the host cell. Bioinformatic analyses suggest that GTAs are especially abundant in the taxonomic class of Alphaproteobacteria, where they are vertically inherited and evolve …

Causes And Consequences Of Lasr Mutant Selection In Pseudomonas Aeruginosa Populations, Dallas L. Mould

Dartmouth College Ph.D Dissertations

Change is the only constant in life, and these changes, though random in nature, can have consequences. Quorum sensing is heterogeneous in phenotype and prone to negative selection. In P. aeruginosa, the regulator LasR is frequently non-functional in phylogenetically diverse isolates. Through repeated experimental evolution and mathematical modeling, we show that differences in growth enable lasR mutant evolutionary success and this requires a system enabling metabolic choices, known as carbon catabolite repression (or catabolite repression). The differences in catabolite repression between wild type and lasR mutants enable altered metabolite preferences, and the resulting differences in metabolic states enable intraspecies …

The Immune Modulation On Innate Immunity, From Pathogen Recognition To Fungal Clearance., Ko-Wei Liu

Dartmouth College Ph.D Dissertations

The human lung is not sterile but a complex environment with various microorganisms. Besides commensals in the lung, hundreds to thousands of individual microbiomes enter the lung every day but without causing the symptom. Host innate immunity plays an important role in maintaining homeostasis of the lung environment and as the first defense line against pathogens. Aspergillus fumigatus (A. fumigatus) is a saprophytic filamentous fungus that can cause human disease in immune compromised patients. However, with functional innate immunity, immune cells can quickly recognize pathogen associated molecular patterns (PAMPs) from A. fumigatus through pattern recognition receptors (PRRs). The activation of …

Consequences Of Biofilm Architecture On Vibrio Cholerae Ecology And Life History, Benjamin Ray Wucher

Dartmouth College Ph.D Dissertations

The diversity of microbes and the environments they inhabit are staggering. In many of these environments, bacteria have evolved to form sessile surface attached communities called biofilms. These biofilms have wide reaching impacts from importance in global carbon cycling, to persistent catheter infections, to biofouling and wastewater treatment. While many species of microbes form biofilms to survive in their environment, the architectures of these structures vary widely between organisms. Even though a great deal of work has been done to understand bacterial communities and their functions, little work has examined how the spatial aspects of biofilm architecture can affect the …

The Role Of L-Alanine Signaling In Aspergillus Fumigatus Biofilm Adherence, Carbon Catabolism, And Echinocandin Susceptibility., Joshua D. Kerkaert

Dartmouth College Ph.D Dissertations

Aspergillus fumigatus is a saprophytic filamentous fungus that participates in environmental carbon and nitrogen cycles through the degradation of complex organic substrates. In addition to its ecological role, A. fumigatus is the primary causative agent of a spectrum of diseases depending on the immune status of the individual, the most lethal of which is invasive aspergillosis (IA). Treatment strategies for IA are limited and far too frequently fail. Despite the high rates of treatment failure, antifungal resistance remains relatively low, albeit rising at a concerning rate. Insights into this discrepancy between the rate of treatment failure and the rate of …