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Browsing by Author "Hsu, Bryan B."

Now showing 1 - 10 of 10
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    A naturally derived biomaterial formulation for improved menstrual care
    Bataglioli, Rogerio Aparecido; Kaur, Harsimran; Muller, John; Geddes, Elizabeth; Champine, Carrie; Hsu, Bryan B. (Cell Press, 2024-07-10)
    Adequately managing menstruation is an important factor in the overall quality of life for women. With a growing discussion of the global need for its improvement, it is clear that better management of menstruation can positively influence social, educational, and professional outcomes. Herein, we describe a biopolymer-based formulation that gels blood in a mechanism alternative to coagulation. We first tested several biopolymer mixtures with blood and quantified increases in viscosity, finding that high-molecular-weight alginate in combination with glycerol could rapidly absorb and gel blood. We then demonstrated that this powder could be deployed both as a traditional menstrual pad filler and as an additive to menstrual cups to reduce leakage and spillage, respectively. Finally, we include an antimicrobial polymer to impair the growth of Staphylococcus aureus, a bacterium associated with toxic shock syndrome. Collectively, our work describes a biodegradable formulation derived from renewable resources that can improve menstrual care.
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    Bacteriophages in the honey bee gut and amphibian skin microbiomes: investigating the interactions between phages and their bacterial hosts
    Bueren, Emma Kathryn Rose (Virginia Tech, 2024-06-14)
    The bacteria in host-associated microbial communities influence host health through various mechanisms, such as immune stimulation or the release of metabolites. However, viruses that target bacteria, called bacteriophages (phages), may also shape the animal microbiome. Most phage lifecycles can be classified as either lytic or temperate. Lytic phages infect and directly kill bacterial hosts and can directly regulate bacterial population size. Temperate phages, in contrast, have the potential to undergo either a lytic cycle or integrate into the bacterial genome as a prophage. As a prophage, the phage may alter bacterial host phenotypes by carrying novel genes associated with auxiliary metabolic functions, virulence-enhancing toxins, or resistance to other phage infections. Lytic phages may also carry certain auxiliary metabolic genes, which are instead used to takeover bacterial host functions to better accommodate the lytic lifecycle. In either case, the ability to alter bacterial phenotypes may have important ramifications on host-associated communities. This dissertation focused on the genetic contributions that phages, and particularly prophages, provide to the bacterial members of two separate host-associated communities: the honey bee (Apis mellifera) gut microbiome and the amphibian skin microbiome. My second chapter surveyed publicly available whole genome sequences of common honey bee gut bacterial species for prophages. It revealed that prophage distribution varied by bacterial host, and that the most common auxiliary metabolic genes were associated with carbohydrate metabolism. In chapter three, this bioinformatic pipeline was applied to the amphibian skin microbiome. Prophages were identified in whole genome bacterial sequences of bacteria isolated from the skin of American bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), Spring peepers (Pseudacris crucifer) and American toads (Anaxyrus americanus). Prophages were additionally identified in publicly available genomes of non-amphibian isolates of Janthinobacterium lividum, a bacteria found both on amphibian skin and broadly in the environment. In addition to a diverse set of predicted prophages across amphibian bacterial isolates, several Janthinobacterium lividum prophages from both amphibian and environmental isolates appear to encode a chitinase-like gene undergoing strong purifying selection within the bacterial host. While identifying the specific function of this gene would require in vitro isolation and testing, its high homology to chitinase and endolysins suggest it may be involved in the breakdown of either fungal or bacterial cellular wall components. Finally, my fourth chapter revisits the honey bee gut system by investigating the role of geographic distance in bacteriophage community similarity. A total of 12 apiaries across a transect of the United States, from Virginia to Washington, were sampled and honey bee viromes were sequenced, focusing on the lytic and actively lysing temperate community of phages. Although each apiary possessed many unique bacteriophages, apiaries that were closer together did have more similar communities. Each bacteriophage community also carried auxiliary carbohydrate genes, especially those associated with sucrose degradation, and antimicrobial resistance genes. Combined, the results of these three studies suggest that bacteriophages, and particularly prophages, may be contributing to the genetic diversity of the bacterial community through nuanced relationships with their bacterial hosts.
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    The Cell Membrane Proteome of the SKBR3/HER2+ Cells and Implications for Cancer Targeted Therapies
    Karcini, Arba (Virginia Tech, 2023-06-02)
    Breast cancer is the second most common type of cancer among women in the US and the second leading cause of cancer death. HER2+ breast cancers represent ~20% of all cancer types, are highly invasive, and can be treated by using targeted therapies against the HER2 receptor. However, these therapies are challenged by the development of drug resistance, often induced by the presence of mutations in the cell-membrane proteins and receptors and/or by alternative signaling pathways that cross-talk with- or transactivate HER2+ triggered signaling. This study was aimed at investigating the cell membrane proteome of SKBR3 cells, representative of HER2+ breast cancers, and the signaling landscape and cellular responses elicited by the cell membrane receptors when the cells are stimulated with either growth factors or therapeutic drugs. It was hypothesized that the identification of a broad range of cell membrane proteins with roles in cancer progression and signaling crosstalk will lead to a more comprehensive understanding of the biological processes that sustain the proliferation of cancer cells, and will guide the selection of more efficient drug targets. The project was conceptualized in three stages: (1) profiling the cell membrane proteins of SKBR3 cells, (2) determining the functional role of the detected cell membrane proteins in the context of cancer hallmarks and exploring their mutational profile, and (3) analyzing the cellular events that occur in response to treatment with a single therapeutic agent or a combination of drugs. Mass spectrometry technologies were used for performing proteomic and phosphoproteomic profiling of SKBR3 cells, detecting changes in the abundance of the detected proteins, and identifying the presence of mutations in the cell membrane proteins. Orthogonal enrichment methods were developed for profiling the low-abundance cell membrane proteins, for generating a rich landscape of cell membrane receptors with various functional roles and relevance to the cancer hallmarks, and for enabling the detection of potentially new drivers of aberrant proliferation. The analysis of serum-starved, stimulated (with growth factors), or inhibited (with kinase inhibitors) cells revealed alternative protein players and crosstalk activities that determine the fate of cells, and that may fuel the development of resistance to treatment with drugs. The proteome profiles that were generated in this project expand the opportunities for targeting cancer-relevant processes beyond proliferation, which is commonly attempted, broadening the landscape to also include apoptosis, invasion, and metastasis. Altogether, the findings that emerged from this work will lay the ground for future studies that aim at developing more complex and effective targeted cancer treatment approaches.
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    Decoding novel virulence strategies in Fusobacterium invasion and survival
    Nguyen, Tam (Virginia Tech, 2022-06-08)
    Fusobacterium nucleatum is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacterium species exhibit genetic recalcitrance, which has led to hindrance in the understanding of their biology and molecular pathogenesis. Though the association of Fusobacterium in diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum ATCC 23726 because it is genetically tractable. Here, in this dissertation, we show that we are able to enhance our existing molecular tools for genome editing to introduce the first mutants in a clinically relevant strain, F. nucleatum ATCC 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cellular invasion and survival. In this work, we identified a novel small adhesin, FadA2, that played a significant role in the invasive ability of F. nucleatum ATCC 25586 to colorectal cancer cells. This dissertation also sheds the first insight into the roles of the type 5a autotransporters. Using a deletion library of genes encoding for the type 5a autotransporter proteins in F. nucleatum ATCC 23726, we systemically characterized altogether 12 type 5a proteins with a focus on the invasion of colorectal cancer cells. Most notably, we found that a wide assortment of type 5a proteins contributing to binding and invasion of F. nucleatum to HCT116 cancer cells. Furthermore, we identified that RadD was not directly involved in inducing secretions of the cytokines IL-8 and CXCL1 while confirmed the specific association of Fap2 in bacterial-induced cytokine secretion. Thus, our findings provided the first comparative and functional analysis of Fusobacterium type 5a autotransporter proteins in colorectal cancer cells which will be crucial to the understanding of Fusobacterium involvement in cancer progression. Finally, this dissertation reported on the first ever observation on the survival strategy of Fusobacterium inside the host cells. We uncovered a novel protein that contributed to enhanced survival of Fusobacterium residing in colorectal cancer cells. This work undoubtedly helps expand the current Fusobacterium genetic toolkit to study proteins and mechanisms relevant to Fusobacterium-accelerated diseases. By identifying and characterizing novel virulence strategies that Fusobacterium can take advantage of, we can increase our comprehension on this opportunistic microbe while devising innovative therapeutic treatments.
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    In situ reprogramming of gut bacteria by oral delivery
    Hsu, Bryan B.; Plant, Isaac N.; Lyon, Lorena; Anastassacos, Frances M.; Way, Jeffrey C.; Silver, Pamela A. (2020-10-06)
    Abundant links between the gut microbiota and human health indicate that modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We demonstrate that an engineered temperate phage lambda expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose. It is difficult to precisely target bacterial populations in the mammalian gut. Here the authors use encapsulated phages to deliver dCas9 to E. coli in the mouse gut to modulate RFP expression.
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    The interplay between pathogenic bacteria and bacteriophage Chi: New directions in motility and phage-host interactions in Enterobacterales
    Esteves, Nathaniel Carlos (Virginia Tech, 2024-04-15)
    The bacterial flagellum is a rotary motor that propels motile bacteria through their surroundings via swimming motility, or on surfaces via swarming motility. The flagellum is a key virulence factor for motile pathogenic bacteria. Viruses that infect bacteria via this appendage are known as flagellotropic or flagellum-dependent bacteriophages. Much like other phages, flagellotropic phages are of interest for clinical applications as antibacterial agents, particularly against multidrug resistant (MDR) bacteria. Bacteriophage χ is a flagellotropic phage that infects multiple species of motile pathogens. In the projects described below, we characterized several aspects of the complex interactions between χ and two of its hosts: Salmonella enterica and Serratia marcescens. In Chapter I, we describe in detail the existing knowledge on flagellum-dependent bacteriophages, pathogenic bacteria, and the flagellar motility system. We also expand significantly on flagellotropic phage χ. In Chapter II, we describe our discovery of S. enterica cellular components other than motility that are crucial for bacteriophage χ infection, making the key discovery that the AcrABZ-TolC multi-drug efflux system is required for infection to proceed. We additionally found that the host molecular chaperone trigger factor is important for the χ phage lifecycle. In Chapter III, we outline our characterization of the initial binding interaction between χ and the flagellum, determining that of flagellin's seven domains, C-terminal domain D2 is the most important for χ adsorption. In Chapter IV, we expand on this by discussing our work that determined that the χ tail fiber protein is encoded by the gene CHI_31, purification of this recombinantly-expressed protein, and demonstration of its direct interaction with the flagellar filament. Lastly, in Chapter V, our findings indicate that S. marcescens is able to detect χ infection and lysis in the surroundings and alter gene expression, resulting in an increase in the production of the red pigment prodigiosin. Overall, our hypothetical model for χ infection is as follows: χ binds to the flagellum of its host using its single tail fiber, composed of monomers of the CHI_31 gene product gp31. This tail fiber interacts with CTD2 of flagellin, and the rotation of the flagellum brings the phage to the cell surface, where it interacts with AcrABZ-TolC to inject its genetic material into the host cytoplasm. At some point during the process of production of phage particles and subsequent cell lysis, the host molecular chaperone trigger factor likely assists with proper folding of χ proteins. After cell lysis, cells in the surroundings are capable of detecting lysis and responding accordingly, at least in the case of S. marcescens. This research is clinically relevant for a number of reasons. Phage therapy, the use of bacteriophages as antibacterial agents, requires knowledge of phage infection pathways for optimal implementation. The fact that the flagellum and a complex mediating MDR are both essential for χ infection leads to particular interest in χ for this application. Knowledge of the host-determining factors between χ and Salmonella may lead to the ability to alter the χ phage genome to target specific pathogenic Salmonella or Escherichia coli strains while avoiding disruption of beneficial bacterial communities.
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    Metagenomic approaches for examining the diversity of large DNA viruses in the biosphere
    Farzad, Roxanna (Virginia Tech, 2023-07-28)
    The discovery of large DNA viruses has challenged the traditional perception of viral complexity due to their enormous genome size and physical dimensions. Previously, viruses were considered small, filterable agents until the discovery of large DNA viruses. Among large DNA viruses, the phylum Nucleocytoviricota and its members, which are often called "giant viruses" have large genome sizes (up to 2.5 Mbp) and virion sizes (up to 1.5 um). Due to having large virion and genome sizes, these viruses were often excluded from viral surveys and remained understudied for years. Luckily, the advancement of metagenomic analysis has facilitated the study of large DNA viruses by analyzing them directly from their environment without cultivating them in the lab, which could be challenging for viruses. In the first chapter of the thesis, I investigated 11 metagenome-assembled genomes (MAGs) of giant viruses previously surveyed from Station ALOHA in the Pacific Ocean. St. ALOHA is located near Hawaii and represents oligotrophic gyres which the majority of the ocean is made of them. I focused on 11 MAGs of giant viruses to get insight into their phylogenetic characteristics, genomic repertoire, and global distribution patterns. Despite the fact that metagenomic analysis has facilitated the study of genetic materials of microbes and viruses on a huge scale, it is essential to benchmark the performance of metagenomic tools and understand the associated biases, particularly in viral metagenomics. In the second chapter, I evaluated the performance of metagenomic tools (contigs assembler and binning tool) in recovering viral genomes using annotated dataset. We used a metagenome simulator (CAMISIM) to generate simulated short reads with known composition to assess these processes. Moreover, I emphasized the importance of binning contigs for viral genomes to fully recover the genomes of viruses along with discussing how diversity metrics were differed for contigs, bins populations.
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    Rehabilitation of a misbehaving microbiome: phages for the remodeling of bacterial composition and function
    Baaziz, Hiba; Baker, Zachary Robert; Franklin, Hollyn Claire; Hsu, Bryan B. (Cell Press, 2022-03-23)
    The human gut microbiota is considered an adjunct metabolic organ owing to its health impact. Recent studies have shown correlations between gut phage composition and host health. Whereas phage therapy has popularized virulent phages as antimicrobials, both virulent and temperate phages have a natural ecological relationship with their cognate bacteria. Characterization of this evolutionary coadaptation has led to other emergent therapeutic phage applications that do not necessarily rely on bacterial eradication or target pathogens. Here, we present an overview of the tripartite relationship between phages, bacteria, and the mammalian host, and highlight applications of the wildtype and genetically engineered phage for gut microbiome remodeling. In light of new and varied strategies, we propose to categorize phage applications aiming to modulate bacterial composition or function as “phage rehabilitation.” By delineating phage rehab from phage therapy, we believe it will enable greater nuance and understanding of these new phage-based technologies.
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    Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut
    Hsu, Bryan B.; Way, Jeffrey C.; Silver, Pamela A. (2020-01-28)
    Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria. Here, we deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered lambda phage to transcriptionally repress Shiga toxin by using genetic hybrids between lambda and other lambdoid phages to overcome resistance encoded by the virulence-expressing prophage. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces Shiga toxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. Our work reveals a new framework for transferring functions to bacteria within their native environment. IMPORTANCE With the increasing frequency of antibiotic resistance, it is critical to explore new therapeutic strategies for treating bacterial infections. Here, we use a temperate phage, i.e., one that integrates itself into the bacterial genome, to neutralize the expression of a virulence factor by modifying bacterial function at the genetic level. We show that Shiga toxin production can be significantly reduced in vitro and in the mammalian gut. Alternative to traditional applications of phage therapy that rely on killing bacteria, our genetics-based antivirulence approach introduces a new framework for treating bacterial infections.
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    Towards the characterization and engineering of bacteriophages in the gut microbiome
    Hsu, Bryan B. (American Society for Microbiology, 2021-07-01)
    The gut microbiome and its importance to human health are a rapidly evolving area of study. Bacteria often take center stage. However, the composition is much more complex with other microbial members of the gut also playing key roles. Bacteriophages (phages), the viruses that infect bacteria, are an integral component of gut microbiomes and can often be found cocolonizing with their commensal bacterial hosts. Recent studies have shown associations between the composition of resident phage communities and human health and disease, but the mechanisms of these associations remain elusive. My research laboratory is focused on understanding the role of phages in the gut microbiome and exploring their possible therapeutic applications.