Cell Biology & Molecular Genetics Theses and Dissertations
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Item Roles of Female Sex Hormones in Regulating Neisseria gonorrhoeae Colonization of the Human Cervix(2024) Di Benigno, Sofia; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-exclusive pathogen that infects the genital tract. Gonococcal infection may present with or without symptoms and can lead to a variety of serious sequelae if left untreated, especially in female patients. Despite this, there are few models that can effectively mimic GC infection in the female reproductive tract (FRT); of these, even fewer consider the impact of the menstrual cycle, an important feature of the FRT, on GC infection. I used the human cervical tissue explant model previously developed in our lab, which can recapitulate GC infection in vivo. Tissue explants were treated with the sex hormones estradiol and progesterone to mimic various stages of the menstrual cycle and examine its impact on GC infectivity. Estradiol was used to mimic the late proliferative phase, and a combination of estradiol and progesterone was used to mimic the middle of the secretory phase. The effects of hormones on GC infectivity were examined after 72 total hours of hormone treatment and 24 hours of inoculation with GC of strain MS11. My results show that treatment with estradiol and with a combination of estradiol and progesterone both increase the level of GC colonization on the endocervix, but not on the ectocervix, compared to controls that were not treated with hormones. However, the hormone treatment did not affect GC penetration of the cervical epithelium. Both hormone treatments increased the number of GC colonies on the endocervical epithelium, and a combination of estradiol and progesterone produced an additional population of large GC colonies, leading to an increase in the average colony size. These increases in colony number and size were not associated with an increase in the expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are the host receptors for GC Opa proteins. In contrast, treatment with estradiol induced a redistribution of CEACAMs from the luminal surface to the inside of epithelial cells. Additionally, estradiol altered the morphology of endocervical epithelial cells from columnar to cuboidal, but the integrity of cell-cell junctions was unchanged. The increase in colonization under high estradiol conditions was correlated with a decrease in levels of certain pro-inflammatory cytokines and chemokines, but this decrease was not sufficient to fully explain the increase in colonization. Next, I investigated the impact of cervical mucus on GC infectivity and interactions, as gel-forming mucin MUC5B but not MUC5AC increases with estradiol at the proliferation phase. Under both hormone treatment conditions, GC were able to establish close interaction with the luminal surface of the endocervical epithelial cells, displacing membrane-spanning mucin MUC1 in the membrane. Furthermore, GC were able to diffuse through an artificial mucin hydrogel and diffused more efficiently through a MUC5AC-dominant than a MUC5B-dominant hydrogel. Gel-forming mucins collected from cervical tissue explants enhanced GC aggregation in vitro, even at very low concentrations. However, mucins collected from estradiol-treated tissues showed less impact on GC aggregation than those collected from untreated tissues or tissues treated with both estradiol and progesterone. MUC5B and MUC5AC purified from cows and pigs also increase GC aggregation in vitro with GC aggregating more in a MUC5AC- than a MUC5B-dominant mucin mixture. Taken together, my research reveals for the first time that female sex hormones regulate GC colonization at the human cervix by changing the composition of the cervical mucus, providing a mechanism of hormonal regulation underlying the varying susceptibility of female patients to mucosal GC.Item THE STANDALONE REGULATOR ROFA OF STREPTOCOCCUS PYOGENES EXHIBITS CHARACTERISTICS OF A PRD-CONTAINING VIRULENCE REGULATOR(2024) Hart, Meaghan Taylor; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Streptococcus pyogenes (Group A Streptococcus; GAS) is a human pathogen estimated to cause nearly 790 million cases of disease annually at diverse tissue sites. To successfully infect these sites, GAS must detect nutrient availability and adapt accordingly. One mechanism employed to detect and import carbohydrates is the phosphoenolpyruvate transferase system (PTS), which mediates both carbohydrate uptake and metabolic gene regulation. Gene regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several stand-alone regulators that contain PRDs, with corresponding regulons encoding both metabolic genes and important virulence factors. These regulators form a family called PRD-Containing Virulence Regulators (PCVRs). RofA is a putative member of this family and is known to regulate the expression of genes important for virulence. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate appropriate virulence gene expression. In this dissertation, the RofA regulon was determined in strain 5448, a representative strain of the globally disseminated M1T1 serotype. The pilus and capsule operons were consistently dysregulated across growth in the absence of RofA. This correlated with increased capsule production and decreased adherence to primary keratinocytes. Purified RofA-His was phosphorylated in vitro by the general PTS components EI and HPr, and phosphorylated species of RofA-FLAG were detected in vivo late in stationary phase in a glucose-dependent manner. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar detection and utilization with GAS virulence gene regulation. Additionally, a bioluminescent construct was generated for allelic exchange into any S. pyogenes strain. Allelic exchange of this construct into WT 5448 yielded strains that were highly bioluminescent, grew to a similar density as WT, and survived as well as WT when challenged with human neutrophils. This tool could be used to study the contribution of specific proteins on in vivo virulence in a non-invasive manner, including RofA and RofA phosphorylation.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 A SYNTHETIC TMRNA PLATFORM FOR ELUCIDATION OF BACTERIAL PROTEOME REMODELING UNDER STRESS(2022) Turner, Randi; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Translational reprogramming is a key component of the bacterial stress response and is a function of mRNA stability, protein turnover and proteolysis. Total proteome measurements give a view of the stable proteome but can fail to capture dynamic changes under stress, including incomplete polypeptides that result from cleaved mRNAs or stalled translation events. Bacteria employ a nearly ubiquitous native ribosome rescue system, transfer-messenger RNA (tmRNA), that rapidly resolves stalled translational complexes and tags the incomplete polypeptides for degradation. Characterization of these tmRNA-tagged polypeptides could reveal previously unknown aspects of the bacterial stress response. To address this information gap, we have developed a synthetic tmRNA platform that reprograms the native system to allow for co-translational labeling of the incomplete polypeptides in live bacteria. A short tag reading frame (TRF) encoded on native tmRNA facilitates the addition of a natural peptidyl degradation tag to the polypeptides, and therefore offers an attractive modular domain to introduce synthetic peptide tag sequences and study the “degradome”. To study translational remodeling under stress, we modified the native tmRNA with an 6x-HIS isolation tag with the specific purpose of stabilizing, isolating, and characterizing the degradome in Escherichia coli. Using our inducible system, we have successfully isolated 6xHis-tagged proteins, verified dynamic controlled tagging, assessed broad-spectrum tag introduction with mass spectrometry. Our results capture known tmRNA substrates and excitingly show that tagged protein profiles are markedly different under stress. We investigated the shifting degradome in cells experiencing translational stress associated with serine starvation induced by serine hydroxamate. In cells lacking RelE, the mRNA interferease toxin that cleaves mRNA in the ribosome A site, we find a dramatic shift away from catalytic protein degradation and distinct, disparate enrichment of ribosomal proteins in the degradome under stress. These latter results suggest a new specific role for RelE in regulating ribosome protein abundance under translational stress conditionsItem THE IMPORTANCE OF NUTRIENT ADAPTATION AND UPTAKE BY GROUP A STREPTOCOCCUS FOR ITS GROWTH AND INFECTION(2021) Braza, Rezia Era; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A major human-specific pathogen, Streptococcus pyogenes (Group A Streptococcus, GAS) causes a wide range of infections, from superficial to life-threatening diseases throughout different host niches. Thus, it is necessary to gain a better understanding of how GAS is able to overcome environment-specific challenges. One critical strategy for successful infection is efficient nutrient acquisition; the phosphotransferase system (PTS), which couples the import of carbohydrates with their phosphorylation, has been linked to GAS pathogenesis. In a screen of an insertional mutant library of all 14 annotated PTS permease genes in MGAS5005, the ß-glucoside PTS permease (bglP) was found important for GAS growth and survival in human blood. This was validated in another clinically relevant strain, 5448. In 5448, bglP was shown to be in an operon with a phospho-ß-glucosidase (bglB) downstream and an antiterminator (licT) upstream, with the operon repressed by glucose and induced by the ß-glucoside salicin as the sole carbon source. Investigation of individual bglPB mutants determined they influence regulation of virulence-related genes that control biofilm formation, SLS-mediated hemolysis, and localized ulcerative lesions during murine subcutaneous infections. Another nutrient acquisition system was previously identified as critical for fitness during murine soft tissue infection, the subcutaneous fitness (scf) genes CDE. This previously uncharacterized locus was transcribed as an operon and predicted to encode an ATP-Binding Cassette (ABC) importer for nutrient uptake. Individual scfCDE deletion mutants exhibited attenuation in multiple infection-related environments such as in vivo murine soft tissue infection and ex vivo whole human blood, indicating their impact is not limited to superficial infections. This was evident when vaccination with the permease mutant, scfD, resulted in protection against severe GAS invasive infection. Additionally, growth defects of scfD in nutrient-limiting chemically-defined media (CDM) could be rescued by addition of excess peptides, suggesting ScfCDE is an amino acid or peptide importer. Metabolomics and RNA-seq experiments suggested losing scfD affects multiple amino acid pathways. Since scfCDE is conserved throughout Firmicutes, this work may contribute to the development of therapeutic strategies against GAS and other Gram-positive pathogens. Overall, results indicate that the ß-glucoside PTS and ScfCDE nutrient transporters can differentially influence GAS pathophysiology during infection.Item INVESTIGATING THE COMPETITION BETWEEN COMPONENTS OF DUAL-FUNCTION RNA(2021) Aoyama, Jordan James Masuo; Storz, Gisela T; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Non-coding RNAs (ncRNAs) and small proteins have both emerged as important regulators of gene expression. Dual-function RNAs encode a small protein and have a separate function as a regulatory RNA. Although first discovered in bacteria, dual-function RNAs have now been identified and characterized in eukaryotes as well. These RNAs allow two activities of a single gene to regulate targets at multiple levels. The work described here explores how two novel and one synthetic dual-function RNA act and how competition between the components of a dual-function RNA impacts their functions. AzuCR is a 164-nucleotide E. coli RNA that was previously shown to encode a 28 amino acid protein (AzuC). This work demonstrates that the AzuC small protein impacts glycerol metabolism, with the small protein increasing activity of GlpD, an essential enzyme in glycerol catabolism, while the RNA base pairs with and represses galE mRNA, a gene essential for galactose metabolism. The second dual-function RNA studied in this work is Spot 42, a 109-nucleotide RNA known to base pair with and repress mRNAs encoding proteins involved in the metabolism of non-preferred carbon sources. Although Spot 42 is a well-characterized base pairing small RNA (sRNA) in E. coli, this work shows it also encodes a 15-amino acid protein, SpfP. SpfP was found to bind to cAMP receptor protein (CRP) and block activation of some target genes. For both AzuCR and Spot 42, the coding sequence for the small protein overlaps the base pairing region, and we have observed that translation interferes with base pairing activity suggesting competition between the sRNA and mRNA activities. Finally, a synthetic dual-function RNA was constructed from the Escherichia coli sRNA MgrR and the mRNA for the small protein MgtS. Various versions of this hybrid molecule are used to probe how the organization of components is important for the proper functioning of a dual-function RNA. These three studies highlight the complexities of regulation by dual-function RNAs and provide insights into how these molecules coordinate two different activities to carry out regulatory roles in the cell.Item Chromatin Control of Papillomavirus Infection(2020) Porter, Samuel Stephen; McBride, Alison A; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genomes of papillomaviruses are packaged into chromatin throughout the entire viral lifecycle. A peculiar feature of papillomaviruses genome organization is that the viral DNA is associated with host histones even inside the virion particle. However, little is known about the nature of the epigenome within papillomavirions, or its biological impact on early infection. Here, we use three approaches to study the epigenome of papillomavirions. Papillomaviruses can be assembled in packaging cells by expression of the capsid proteins in the presence of the viral genome. We have optimized and manipulated this process to generate viruses with replicated and genetically modified virion DNA and have used these “quasivirions” to evaluate early infection of primary human keratinocytes. We have also profiled the histone modifications on chromatin extracted from native virions isolated from human and bovine warts. We find that, compared to host cells, the viral chromatin is enriched in histone modifications associated with transcriptionally active chromatin (including histone acetylation), and depleted in those associated with transcriptional repression. To examine the biological role of histone acetylation in the early virus lifecycle, we produced HPV quasivirions with highly acetylated chromatin by assembling the virions in cells treated with histone deacetylase inhibitors. We show that acetylation of viral chromatin results in a reduction of early viral transcription in primary keratinocytes indicating that the histone modifications on virion chromatin do influence the early stages of infection. Collectively, these studies demonstrate that histone modifications on virion chromatin are important for the HPV infectious cycle.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.Item NEISSERIA GONORRHOEAE MODULATES INFECTIVITY BASED ON PROPERTIES OF HUMAN CERVICAL EPITHELIA AND PHASE VARIABLE BACTERIAL SURFACE STRUCTURES(2019) Yu, Qian; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) infection in the human female reproductive tract causes various clinical outcomes, from no symptom to severe complications. The major barrier to a better understanding of GC infection in women is the lack of experimental system closely mimicking in vivo infection. Here, I developed a human cervical tissue explant model, which maintains the heterogeneity of the cervical epithelium. Using this model, my thesis research examined the impact of the heterogeneity of the cervical epithelium and the phase variation of GC surface structures on GC infectivity. My research revealed that GC preferentially colonize the ectocervix and the transformation zone (TZ), but exclusively penetrate into the subepithelial tissues of the TZ and endocervix. Pili are essential for GC colonization in all regions of the cervix. Expression of Opa isoforms that bind to the host receptors CEACAM (OpaCEA) enhances GC colonization in the ecto/endocervix but inhibits GC penetration into the endocervix. However, GC infectivity in the TZ does not respond to Opa phase variation, due to the low expression level and intracellular location of CEACAMs in the TZ epithelial cells. OpaCEA enhances GC colonization in the ecto/endocervix by inhibiting epithelial exfoliation and suppresses GC penetration into the endocervical subepithelium by inhibiting GC-induced disassembly of the apical junction. Opa-mediated modulation of GC infectivity depends on the immune receptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and its downstream phosphatase SHP. The effect of epithelial cell polarity on GC invasion was studied using a cell line model. My results show that GC invade more efficiently into non-polarized than polarized epithelial cells without changing the adhesion efficiency. Opa (phase variable) expression enhances both adhesion and invasion in both non-polarized and polarized cells. In non-polarized cells, Opa expression induces F-actin accumulation and microvilli elongation underneath GC microcolonies, suggesting an actin-mediated uptake of GC. In contrast, GC expressing no Opa reduce F-actin and demolish microvilli underneath microcolonies in both polarized epithelial cell line and endocervical epithelial cells potentially by increasing calcium flux, NMII activation and the redistribution of actin nucleation factor Arp2/3 from the apical surface. Taking together, my research demonstrates that both the heterogeneity of the cervical epithelium and the phase variation of bacterial surface structures regulate GC infectivity in the human cervix, either dominated by colonization or penetration, consequently influencing the clinical outcomes of the infection.Item Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC(2019) Shang, Xiaoran; Nelson, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering.