Cell Biology & Molecular Genetics Theses and Dissertations
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Item INVESTIGATING MECHANISMS UNDERLYING MLO’S ROLE AS A HOST FACTOR ESSENTIAL FOR PATHOGENESIS OF POWDERY MILDEW FUNGI(2024) Bloodgood, David; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss-of-function mutations in Mildew Locus O (MLO) family genes confer broad-spectrum resistance to powdery mildew (PM) fungi in various plant species. mlo-mediated resistance is invariably coupled with increased defense responses and early leaf senescence; hence the molecular basis of mlo-mediated resistance remains unresolved. A saturated genetic screen in the background of an Arabidopsis triple mutant where three essential immune components, EDS1, PAD4 and SID2 are mutated, led to the identification of five allelic mutations in MLO2, each of which results in compromised immunity yet poor infection (cipi) to PM. Further CRISPR-targeted mutagenesis of two functional homologs, MLO6 and MLO12 in a cipi mutant background result in complete lack of infection from PM fungi. The sextuple mutant, eds1pad4sid2mlo2mlo6mlo12 (epsm3) showed no early leaf senescence, ROS accumulation or expression of defense genes, indicating that MLO2, MLO6 and MLO12 are bona fide host susceptibility factors for PM. Expression of MLO2-GFP as a transgene in epsm3 restores susceptibility to PM and MLO2-GFP focally accumulates at the fungal penetration site. Thus, restoration of susceptibility to PM in the epsm3 background can be used as a sensitive reporter to assess whether other MLO family members share a conserved molecular function when expressed in leaf epidermal cells. The Barley MLO and Arabidopsis MLO7 enabled PM pathogenesis whereas MLO1, MLO3 and MLO4 could not, suggesting the existence of two distinct classes of MLO family members. Sequence alignment identified three conserved amino acid residues in the C terminal calmodulin-binding domain of MLO2, and MLO7, which are absent in MLO1, MLO3 and MLO4. This observation suggests that the C-terminal domain of MLO proteins could contribute to their functional divergence. Creation and functional assays of chimeric MLO2/MLO1 proteins by swapping their C terminal domains revealed that the C terminus determines the localization pattern of MLO proteins. The Feronia (FER) receptor-like kinase is required for localization of MLO7 in synergid cells; however, CRISPR-targeted mutagenesis of FER did not disrupt the localization of MLO2 to the fungal penetration site. Based on the results described above, it can be inferred that MLO2 localization to and possible stabilization of the plasma membrane at the fungal penetration site is essential for allowing PM fungi to penetrate the host cell and subsequently differentiate the haustorium. Further multiplexed CRISPR mutagenesis of other gene families suggests that SYP121 and SYP122, two closely related SNARE genes play essential roles in focal accumulation of MLO2 at the fungal penetration site.Item Characterization of chromatin assembly dynamics mediated by the histone H3.3 chaperone HIRA and implications of innate immunity during Human Papillomavirus infection(2024) Della Fera, Ashley Nichole; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The circular double-stranded DNA genome of Human papillomavirus (HPV) is chromatinized throughout its viral lifecycle and relies on numerous host chromatin assembly processes, epigenetic modifications, and immune evasion to ensure genomic stability and productive infection. Despite its chromatinization, the HPV genome remains susceptible to innate immune pathways that sense and respond to foreign DNA. In this work next generation sequencing (RNAseq) was utilized to profile changes in the host transcriptome following cellular differentiation and HPV infection in keratinocyte cell lines. Global alterations in keratinocyte differentiation were observed upon HPV infection, and unexpectedly, upregulation of innate immune signaling upon differentiation. Recent findings indicate that packaged HPV genomes are enriched in histone H3.3. Notably, the replication-independent histone H3.3 chaperone HIRA has been implicated in several pro- and anti-viral responses, but its function during HPV infection has yet to be elucidated. Using in-situ approaches, the role of HIRA during the late phase of the HPV lifecycle was evaluated, which showed that HIRA and other chromatin assembly factors localize to sites of HPV replication. Here the requirements for this localization were further characterized, and the impacts of HIRA on HPV genome amplification and viral transcription during the late stage of the HPV life cycle were assessed. Moreover, histone H3.3 phosphorylated at serine 31 was shown to be highly associated with HPV replication factories. HIRA, in part through association with the PML nuclear body associated protein Sp100, has also been reported to promote innate immune responses following infection with other DNA viruses. Here, HIRA localization to PML-NBs was identified to increase following stimulation with IFN in an Sp100-dependent manner. However, while Sp100 is required for localization of HIRA at PML-NBs, it was not required for HIRA localization at sites of HPV replication. In summary, this work highlights the broad changes in the host transcriptome following cellular differentiation and HPV infection, elucidates a previously undescribed role for histone H3.3 chaperone HIRA during the late phase of the HPV life cycle, and further characterizes the relationship between HIRA and Sp100 at PML-NBs.Item AN INTERSECTING NETWORK OF REGULATORS IS REQUIRED FOR RNA SILENCING AND NUCLEAR INTEGRITY IN C. ELEGANS(2024) Knudsen-Palmer, Daphne R; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Regulation of gene expression is required for an organism to develop, maintain homeostasis, and respond to environmental stimuli. While each cell in a multicellular organism contains the same genetic information, the epigenetic control of the expression of genes at different times is crucial for processes such as cell differentiation, division, and for allowing for cells to carry out different functions from one another. One type of such epigenetic regulation is mediated by small non-coding RNAs. Introduction of double-stranded RNA (dsRNA) and subsequent production of small interfering RNAs can result in sequence-specific mRNA silencing, creating the potential for highly specific therapeutics and pesticides. However, some targets are more easily silenced than others, and the mechanisms of silencing are not fully understood. Here we investigate regulators of small RNA-mediated silencing in the nematode C. elegans and find that they function in an intersecting network, allowing the potential for regulators to contribute to the silencing of any target. Quantitative modeling suggests that the production and turnover rates of a target at steady-state can affect the ease with which a target can be knocked down, and experimentally we found that changing the cis-regulatory sequences of a target can make it more susceptible to silencing. We found restricted production of RNA silencing intermediates, allowing for the recovery of a target in response to dsRNA, which we observed experimentally in non-dividing cells. In addition to roles in response to dsRNA, we report that disruption of small RNA-based regulation can result in germline nuclear defects. In the absence of the intrinsically disordered and perinuclear granule-forming protein MUT-16, some of the nuclei in the syncytial germline appear enlarged, suggesting that small RNA-based regulation may be playing an active role in maintaining nuclear size. Taken together, these findings suggest that (1) regulators of small RNA silencing can contribute to the silencing of all targets as part of an intersecting network, as opposed to operating in specialized pathways and (2) small RNA-based regulation is required for nuclear integrity, providing a paradigm for studying control of nuclear size, where enlarged nuclei can be compared with wildtype nuclei in a shared syncytium. We speculate that these findings will improve understanding of RNA silencing across species and provide insight into understanding how nuclear size is controlled, a fundamental ability of all eukaryotes.Item INVESTIGATING GENE REGULATORY ARCHITECTURES THAT DICTATE TRANSGENERATIONAL EPIGENETIC EFFECTS IN C. ELEGANS(2023) Chey , Mary Somontha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The form and function of an organism rely on the recreation of similar gene expression patterns in every generation. The information for these expression patterns is stored in a single cell (e.g., zygote) in two forms – the genome sequence and the spatial arrangements of gene regulators. The interactions of regulators and the genome form intricate networks with different regulatory architectures. However, a change in the environment can impact gene expression by disrupting the physical and/or chemical properties of regulators or interactions without mutations in DNA sequence. Such epigenetic information can be transmitted across generations, but how long these effects can last is unclear. Here, we investigate the regulatory elements that promote transient or permanent epigenetic effects by analyzing the properties of a recombinant two-gene operon that expresses the fluorescent proteins mCherry and GFP and is susceptible to long-term RNA silencing in the nematode C. elegans. We reveal that 1) multiple mechanisms regulate transgenerational gene silencing and 2) the presence of the mCherry sequence can perturb RNA regulation within the germline to facilitate heritable epigenetic changes. Previous studies showed that the Argonaute protein HRDE-1 is required for the maintenance of silencing in the germline initiated by double-stranded (ds)RNAs and that poly-UG (pUG)-RNAs are key intermediates generated from the target mRNA. We found that loss of HRDE-1 can selectively rescue the expression of one cistron in a two-gene operon, suggesting that the two cistrons are not regulated by the same silencing pathway, but rather by a chromatin-independent mechanism that requires an unknown regulator. Surprisingly, we detected distinct populations of pUG-RNAs associated with expressed and silenced genes, suggesting that pUG-RNAs could potentially prime expressed genes for long-term silencing. Consistently, total RNA sequencing revealed trace amounts of anti-sense RNAs against mCherry and gfp that could trigger the production of pUG-RNAs. Examining the endogenous genes perturbed by the presence of mCherry suggests that long-term RNA silencing relies on the synergy between the sensing and processing of dsRNAs. Together, our results provide insights into the regulatory architectures and mechanisms of heritable gene silencing that occur without genetic mutations.Item Characterizing the full complement of antimicrobial resistance genes and linking the resistance genes and plasmid to source bacteria(2023) sarria, saul; Song, Jiuzhou; Tadesse, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antimicrobial resistance (AMR) is a global public health threat. Selective pressure exerted by antimicrobial use has been the major driving force, more than 2.8 million AMR infections occur yearly in the United States. The intestinal microbiome is an important reservoir of antimicrobial resistance genes. Yet, little is known about the true diversity of the antimicrobial resistance genes in food animal intestinal microbiomes. We employed deep shotgun metagenomic sequencing and proximity ligation (Hi-C) library sequencing to characterize the resistome, assemble genomes from metagenomic samples, and accurately attribute antibiotic resistance genes and plasmids to host bacteria cells. We randomly selected 21 cecal samples from food animal sources (cattle n= 6, swine n= 6, chicken n= 3, and turkey n= 6). We generated more than 75 million reads/sample for Hi-C and more than 100 million reads/sample for shotgun metagenomic sequence reads. Bioinformatics analysis revealed over 200 bins containing metagenome assembled genomes (MAGs) with different levels of completeness, novelty scores, and contamination based on CheckM. A total of 245 previously uncharacterized genomes were reconstructed with high level of confidence (>90% Completeness, >90% Novelty, < 5% contamination). Of the 245 newly reconstructed MAGs, 24 were at bacteria taxonomic rank level, 5 at phyla (Actinobacteria; 11 genomes. Firmicutes; 3 genomes. Bacteroidetes; 17 genomes. Euryarchaeota; 2 genome), 4 at class (Bacilli; 1 genome. Clostridia; 9 genomes. Deltaproteobacteria; 3 genomes. Gammaproteobacteria; 1 genome), 5 at order level (Actinomycetales; 2 genomes. Bacteroidales; 25 genomes. Clostridiales; 114 genomes. Lactobacillales; 2 genomes. Selenomonadales; 2 genomes), and 3 at family level (Lachnospiraceae, 24 genomes. Spirochaetaceae; 1 genome. Spirochaetaceae; 2 genomes). We identified over 400 antimicrobial resistance genes representing 22 antimicrobial classes including: aminoglycoside (40 gene variants), beta-lactams (37 gene variants), bleomycin (2 gene variants), colistin (3 gene variants), fosfomycin (4 gene variants), glycopeptide (6 gene variants), lincosamide (9 gene variants), lincosamide/streptogramin (2 gene variants), macrolide (16 gene variants), macrolide/lincosamide/streptogramin (4 gene variants), nitroimidazole (1 gene variant), phenicol (9 gene variants), phenicol/oxazolidinone (1 gene variant), phenicol/quinolone (2 gene variants), pleuromutilin ( 1 gene variant), quinolone (5 gene variants), streptogramin (1 gene variant), streptothricin (3 gene variants), sulfonamide (3 gene variants), tetracycline (25 gene variants), and trimethoprim (8 gene variants). Plasmid characterization using Hi-C proximity ligation and shotgun metagenomics allowed the identification of 146 plasmids (>= 85% completeness, >= 90% reference sequence similarity), and over 13000 plasmid-contigs (<85% completeness, < 90% reference sequence similarity). Shotgun metagenomics provide valuable insights into the diversity and identity of the resistome present in a microbiome, while Hi-C generates millions of paired-end reads linking DNA fragments in close proximity. When shotgun metagenomics is coupled with the Hi-C proximity ligation approach it shows a great capability in genome binning and simultaneous retrieval of high-quality MAGs from a single sample, thusly enabling the link of resistance genes and plasmids to host bacterial cells and facilitating the public health management decisions aimed at reducing the source and exposure routes of AMR to humans.Item ANTIBACTERIAL MECHANISM OF PLANT-DERIVED PHENOLICS AGAINST SALMONELLA ENTERICA SEROVAR TYPHIMURIUM(2023) Alvarado-Martinez, Zabdiel; Biswas, Debabrata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Salmonella enterica serovar Typhimurium (ST) remain one of the main bacterial pathogens responsible for illnesses, hospitalizations, and deaths in the USA. Its ubiquitous prevalence in nature, invasive pattern and increasing antibiotic resistance make it a public health threat, warranting the discovery of novel antimicrobials that can be implemented as either treatments or as forms of control. Plant-derived compounds have been proposed as potential antimicrobials that can be used against gram-negative pathogens, with phenolic acids being of interest for their prevalence in nature and bioactivity. This research studied the effects of gallic acid (GA), protocatechuic acid (PA) and vanillic acid (VA) against ST. Findings showed these compounds to be able to inhibit bacterial growth in vitro, while also showing a reduction in the expression of key virulence genes, without inducing resistance over multiple passages. Further studies using a human epithelial cell line for studying host-pathogen interactions, showed their capability to reduce the number of ST that were able to invade the host cells. Further studies were performed in cecal fluid to test their potency in more complex environments and assess their effects on the microbiome. When in cecal fluid, compounds showed a reduced inhibitory potency compared to in vitro, but still exerted antimicrobial pressure against ST. When analyzing relative abundance of other bacteria through 16S-rRNA gene sequencing, there was an overall decrease in the Protobacteria phylum, while no significant negative effect was seen for other phyla like that of the Firmicutes and Actinobacteria. Experiments to determine the mechanism of action against ST showed these phenolic acids to permeabilize the cell plasma membrane, in addition to reducing cell wall synthesis. Scanning electron microscopy showed treated bacteria to have dents at the polar ends of the cell, while others were found in a duplet formation, suggesting further disruption of specific bacterial functions associated to cell division and structure. These findings suggest that despite their similarities, these compounds are capable of exerting different types of antimicrobial pressure against ST that could better inform their future use as control measures against ST, and their potential use case based on the desired outcome.Item INVESTIGATION OF ACCELERATED SKIN AGING AND PEROXISOMAL ABNORMALITIES IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2022) Mao, Xiaojing; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. In this dissertation, I study the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, I investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. Chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to early-stage epithelial development markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS. One important feature in both HGPS and normal aging is the elevated levels of Reactive Oxygen Species (ROS), which are generated from metabolic pathways to cause oxidative damage to macromolecules within the cells. Although peroxisomal bioreactions can generate free radicals as their byproducts, many metabolic enzymes within the peroxisomes play critical roles as ROS scavengers, particularly catalase. In this dissertation, I observed impaired peroxisomes-targeting protein trafficking, which suggested that the poorly assembled peroxisomes might cause high oxidative stress, contributing to the premature senescent phenotype in HGPS. I also investigated the ROS clearance efficiency by peroxisomal enzymes and found a significantly decreased catalase expression in HGPS. Furthermore, I evaluated the effects of two promising HGPS-treatment drugs Methylene Blue and RAD001 (Everolimus, a rapamycin analog), on catalase in HGPS fibroblasts. I found that both drugs effectively reduced cellular ROS levels. As a well-known antioxidant, MB did not affect catalase expression or activity. Interestingly, the RAD001 treatment significantly upregulated catalase activity in HGPS cells. This is the first characterization of peroxisomal function in HGPS and provides new insights into the cellular aspects of HGPS and the ongoing clinical trial.Item Intercellular transport of RNA can limit heritable epigenetic changes(2021) Shugarts, Nathan Maxwell; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs in circulation carry sequence-specific regulatory information between cells in animal, plant, and host-pathogen systems. The accumulation of specific RNA species in circulation during human disease states therefore implicates such RNAs in disease-related gene regulation. However, mechanisms of RNA secretion, accumulation and import into cells are not well understood and yet are directly taken advantage of in the delivery of recently approved RNA-based therapeutics. In the tractable animal Caenorhabditis elegans, double-stranded RNA (dsRNA) can be delivered into circulation, accumulate within the germline and reach progeny, raising the potential for intergenerational effects from endogenous RNAs released into parental circulation. Here we provide evidence for spatial, temporal, and substrate specificity in the transport of dsRNA in C. elegans from parental circulation to progeny. Temporary loss of dsRNA transport resulted in the persistent accumulation of mRNA from a germline gene. The expression of this gene varied among siblings and even between gonad arms within one animal. Perturbing RNA regulation of the gene created new epigenetic states that lasted for many generations. Thus, one role for the transport of dsRNA into the C. elegans germline in every generation is to limit heritable changes in gene expression. We speculate that transport of extracellular RNA into germ cells in other systems could similarly buffer against heritable change across generations.Item Investigation of the molecular mechanisms of vascular endothelial dysfunction in Hutchinson-Gilford progeria syndrome through in vitro 2D and 3D models(2021) Gete, Yantenew; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. Predominantly, HGPS is caused by a de novo point mutation in the LMNA gene (c.1824C > T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes endothelial function in HGPS. In this study, I used human iPSC-derived endothelial cell (iPSC-EC) models that cultured under both static and fluidic culture conditions. HGPS iPSC-ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared to healthy controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary-like microvascular network formation. In addition, expression of matrix metalloproteinase 9 (MMP-9) was reduced in HGPS iPSC-ECs while expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) were upregulated relative to healthy controls. Moreover, I used an adenine base editor (ABE7.10max-VRQR) to correct the pathogenic c.1824C > T allele in HGPS iPSC-ECs. Remarkably, ABE7.10max-VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized TIMPs , and restored angiogenic competence in HGPS iPSC-ECs. Furthermore, to elucidate the effects of progerin on endothelial cells and vascular remodeling, in collaboration with Dr. Truskey’s lab at Duke university, we developed tissue-engineered blood vessels (TEBVs) using iPSC-ECs and smooth muscle cells (iPSC-SMCs) from normal and HGPS patients. Relative to normal TEBVs, HGPS TEBVs showed reduced function and exhibited markers of cardiovascular disease associated with endothelium, including a reduction in both vasoconstriction and vasodilation with increased inflammation markers, VCAM-1 and E-selectin protein. Hence, the TEBV model has identified a role of the endothelium in HGPS. Together, the results of the study provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE7.10max-VRQR could be a promising therapeutic approach for correcting HGPS-related cardiovascular phenotypes.Item INVESTIGATION OF CYCLIC DINUCLEOTIDE HOMEOSTASIS AND THE HYDROLYSIS OF THEIR LINEAR INTERMEDIATES IN BACTERIA(2019) Weiss, Cordelia Anne; Winkler, Wade C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The synthesis of cyclic dinucleotides as signals is one strategy bacteria use to sense and adjust to environmental changes. Cyclases synthesize the cyclic dinucleotide, while phosphodiesterases cleave it to yield a linear diribonucleotide, which is recycled into monoribonucleotides by other enzymes. For many bacteria, cyclic di-GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile community. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. Bacillus subtilis is a model for the study of bacterial differentiation, yet how c-di-GMP functions in this organism is not fully understood. This work began with construction of a fluorescent reporter to measure c-di-GMP abundance in B. subtilis, which showed that c-di-GMP levels are strikingly different among differentiated subpopulations. These data highlight how single-cell approaches can be used to analyze metabolic trends within bacterial populations and demonstrate that for some bacteria, c-di-GMP levels are adjusted heterogeneously across bulk populations. The enzymes Orn, NrnA, NrnB, and NrnC have been proposed to act as general 3’-5’ exoribonucleases that preferentially process ‘short’ oligoribonucleotides. Intriguingly, Orn also performs a crucial role in c-di-GMP homeostasis by processing the pGpG generated from c-di-GMP production. To discover the molecular basis for Orn’s ability to ‘select’ short RNAs, and to elucidate the relationship between Orn and the diribonucleotide pGpG, we combined structural, biochemical, and in vivo analyses of RNA cleavage. These data reveal that Orn is not a general exoribonuclease of short RNA oligoribonucleotides, as previously believed, but instead acts as a dedicated ‘diribonucleotidase’. Our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of diribonucleotides, which affect cellular physiology and viability. Examination of the roles of NrnA and NrnB is underway. We conducted an initial study to determine if NrnA and NrnB are redundant proteins, as has been proposed, and if they might also act as ‘diribonucleotidases’. These data show that they exhibit different substrate preferences and that they may have unique cellular functions. Therefore this work changes the perception of the role(s) Orn plays and that a re-evaluation of ‘short’ RNases is needed.