Browsing by Author "Matthews, Kathleen"

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    A Synthetic Biology Approach to Engineering Bacterial Two- Component Systems for Sensor Development and Discovery of Anti-Virulence Agents
    (2019-12-06) Ekness, Felix; Tabor, Jeffrey J; Matthews, Kathleen
    Bacterial two-component systems (TCSs) are the largest family of signal transduction pathways that enable bacteria to sense a diversity of stimuli including small peptides, environmental pollutants, and light. Canonical TCSs are composed of a transmembrane sensor histidine kinase (SK) that converts stimulus detection into phosphorylation of a cognate response regulator (RR). Upon phosphorylation, the cytoplasmic RR binds target output promoters, hereby modulating gene expression. TCSs are valuable sensors for synthetic biology due to their diverse sensing capabilities and straightforward transduction of detected stimulus into transcriptional regulation. TCSs are also emerging targets for novel therapeutic development due to their extensive role in regulating bacterial virulence and antibiotic resistance. Although TCSs are exciting sensors for synthetic biology and targets for therapeutic applications, most TCSs remain difficult to harness for applications and study due to output promoters that are unknown, subject to cross- regulation, or silent in heterologous hosts. In the first portion of my work, I develop a method to overcome the hurdles in characterizing and utilizing TCSs as biosensors. Through the framework of synthetic biology, I demonstrate that the two largest families of RR DNA binding domains (DBDs) can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. In collaboration with Kristina Daeffler, we exploit this plasticity to eliminate cross-regulation and in collaboration with Brian Landry, we un-silence a gram-negative TCS in a gram-positive host and engineer a sensor with over 1,300-fold activation. In collaboration with Kathryn Brink, we also apply DBD swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a novel pH sensor. In the second portion of my work, I demonstrate a method for identifying inhibitors of a virulence regulating TCS. This work focuses on the methicillin-resistant Staphylococcus aureus (MRSA) virulence regulating TCS SaeRS. I first heterologously express saeRS in Bacillus subtilis to remove the native, confounding regulation of the TCS and its output promoter. I then screen heterologous SaeRS against a diverse 1,593 small molecule library to identify inhibitors of its signaling. A lead compound emerged from the screen as an effective inhibitor of SaeRS signaling, leading to decreased exoprotein secretion and virulence from treated MRSA similar to MRSA lacking saeRS. My work described herein should accelerate 1) fundamental TCS studies and the engineering of a large family of biosensors with diverse applications and 2) the discovery of new anti-virulence compounds.
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    Antiangiogenic Factor Receptor PlexinD1 is Required for Proper Formation of the Periocular Vasculature and Establishment of Corneal Avascularity
    (2015-08-03) Kwiatkowski, Sam C; Matthews, Kathleen; McNew, James; Stewart, Charles; Qutub, Amina
    The cornea is an avascular component of the visual system located in the anterior eye. Avascularity of the cornea is critical for proper vision since the cornea functions by transmitting light into the eye. Impaired vision from loss of avascularity may occur as a result of tissue damage which induces corneal neovascularization from the highly vascularized tissues of the anterior eye. Neovascularization in adult corneas is regulated by secreted pro- and anti-angiogenic factors. These factors function by maintaining corneal avascularity under healthy conditions while permitting neovascularization in damaged corneas. Several pro- and anti-angiogenic factors that function to maintain corneal avascularity during adult life have been identified, however little is known about how pro- and anti-angiogenic factors function to establish avascularity during corneal development. The purpose of this work was to study the role of pro- and anti-angiogenic factors during corneal development. First, the spatial and temporal expression patterns of numerous secreted pro- and anti-angiogenic factors were determined in the anterior eye during avian corneal development using semi-quantitative RT-PCR and RNA in situ hybridization. These techniques were also used to show that known receptors for secreted pro- and anti-angiogenic factors were simultaneously expressed in angioblasts and blood vessels located in the developing anterior eye. These experiments suggested that pro- and anti-angiogenic factor signaling mechanisms may contribute to the patterning of periocular vasculature and establishment of corneal avascularity. Next, I exemplified the role of pro- and anti-angiogenic factors during avian corneal development by using shRNA to knock down the expression of PlexinD1, an antiangiogenic factor receptor expressed in periocular angioblasts and blood vessels. Knockdown of PlexinD1 resulted in multiple patterning defects of the developing periocular vasculature including corneal neovascularization. These phenotypes implicated PlexinD1 as a critical component of the genetic mechanisms that establish corneal avascularity and were suggestive of the role that other pro- and anti-angiogenic factors may play during anterior eye development. These results demonstrate how pro- and antiangiogenic factors are used to simultaneously promote vascularization of the anterior eye and corneal avascularity during development. This information may lead to the creation of novel therapeutic treatments for vascular patterning abnormalities in the anterior eye and corneal neovascularization.
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    Functionalized protein-based materials and their uses
    (2021-03-16) Bondos, Sarah; Bayless, Kayla; Matthews, Kathleen; Patterson, Jan; Abbey, Colette; Howell, David; Hsiao, Hao-ching; Churion, Kelly; Tsai, Shang-pu; Ramasamy, Sandhya; Porterpan, Dustin; Northern, Keira; Rice University; The Texas A&M University System, College Station, TX; United States Patent and Trademark Office
    Embodiments of the invention are directed to Ubx-fusion molecules that maintain their mechanical strength and properties even after being fused with Ubx. Ubx fusions with VEGF and other growth factors, cell signaling proteins, and cell binding proteins can be used to induce angiogenesis. Ubx fibers and mesh, embedded within a tissue engineering scaffold, induce formation of vasculature within the scaffold. The presence of vasculature is necessary to provide oxygen and nutrients to other cells growing within the scaffold.
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    Genetic suppressors reveal varying methods for improving peroxisome function in Arabidopsis peroxin mutants
    (2021-12-02) Llinas, Roxanna J.; Bartel, Bonnie; Matthews, Kathleen
    Peroxisomes are eukaryotic organelles that support several metabolic pathways critical to survival and development. These pathways include diverse oxidative reactions with harmful reactive byproducts that are neutralized with enzymes accumulated in peroxisomes. New peroxisomes can be generated via de novo formation from the endoplasmic reticulum or growth and fission of mature peroxisomes with the assistance of proteins called peroxins (PEX proteins). Once formed, peroxisomes acquire necessary enzymes by using other peroxins to import lumenal proteins. Although we have a general framework for understanding peroxisome biogenesis, we lack a complete molecular understanding of the activity and specific sequence of events through which these peroxins function. Unlike in mammals, -oxidation is exclusively peroxisomal in plants, making plants an ideal multicellular system in which to study peroxisomes and peroxisomal metabolism. Germinating Arabidopsis seedlings rely on peroxisomes to catabolize fatty acids stored in lipid droplets, and mutations in peroxin genes confer a range of easily assayed developmental defects that report peroxisome function. I elucidated molecular mechanisms underlying Arabidopsis peroxisomal biogenesis and function by characterizing peroxin mutants and peroxin mutant suppressors. These suppressors carry secondary mutations that restore peroxisomal function to Arabidopsis peroxin mutants. I examined suppressors of pex10-2 and pex12-1, mutants defective in ubiquitin-protein ligases that aid in peroxisome receptor recycling, and pex14-1 and pex14-6, mutants defective in a docking complex peroxin that aids in peroxisomal protein import. I clarified details of how these peroxins function and uncovered novel associations between peroxins by assessing the steps in peroxisomal function that are restored in the suppressors. One pex14-6 suppressor corrects the splicing defect of the original lesion, and I used it to investigate how splicing factors maintain splicing fidelity. One pex12-1 suppressor is defective in a peroxin implicated in peroxisomal membrane protein insertion and pre-peroxisome budding from the endoplasmic reticulum, and I used it to investigate a potential role in protein import. Finally, I examined the role of PEX16, an early-acting peroxisome biogenesis factor, in plants. Together, these studies revealed varying methods for modulating peroxisome function and suggest additional obscure factors in peroxisome competence.
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    Modulating the Lysosome-Autophagy System to Restore Homeostasis in in vitro Model Systems of Lysosomal Storage Disorders
    (2014-11-14) Song, Wensi; Segatori, Laura; Matthews, Kathleen; Gonzalez, Ramon
    The protein quality control system is a complex network that promotes the folding and trafficking of newly synthesized proteins and regulates the degradation of misfolded proteins and protein aggregates. Failure of the quality control system to maintain protein homeostasis (or proteostasis) characterizes the cellular pathogenesis of a number of human diseases. In particular, this study focuses on lysosomal storage disorders, a group of inherited metabolic diseases characterized by deficiencies in specific lysosomal hydrolytic activities that result from mutations in genes encoding for lysosomal proteins and consequent buildup of lysosomal storage material. The ultimate goal of this work is to develop cell engineering strategies to modulate cellular quality control machineries that control protein folding, processing, and degradation to restore cellular homeostasis under conditions of proteotoxic stress. Specifically, this study aims to manipulate the lysosome-autophagy system to enhance folding and processing of lysosomal enzymes as well as to enhance the cellular clearance capacity. To achieve this goal, I investigated the role of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in regulating lysosomal proteostasis and autophagic clearance. Specifically, chemical and genetic modulation of TFEB was found to enhance folding, trafficking and activity of unstable, degradation-prone lysosomal enzymes in in vitro models of lysosomal storage disorders. Moreover, pharmacologic activation of autophagy achieved by treating cells with 2-hydroxypropyl-β-cyclodextrin was found to enhance autophagic clearance of storage material specifically by activating TFEB. To further investigate the molecular mechanism of autophagy induction and activation of autophagic clearance, I tested the impact of polystyrene nanoparticles of different size and surface charge on the lysosome-autophagy system with the ultimate goal to link the physicochemical properties of nanomaterials with the specific nature of the autophagic response activated upon nanomaterial uptake into cells. Efficient autophagic clearance was found to depend highly on the surface charge. Specifically, cell exposure to polystyrene nanoparticles presenting neutral or negative surface charge results in activation of autophagic clearance, whereas cell exposure to polystyrene nanoparticles presenting cationic surface charge results in impairment of lysosomal function and blockage of autophagic flux. Ceria nanoparticles (or nanoceria) are widely used in a variety of applications including as UV blockers and catalysts in industrial processes. Recent studies also revealed that ceria nanoparticles present antioxidant properties, suggesting a potential role of nanoceria in a variety of biomedical applications. In this study, I investigated the impact of ceria nanoparticles stabilized by organic surface coatings on the lysosome-autophagy system, Ceria nanoparticles were found to activate the lysosome-autophagy system and enhance autophagic clearance. In summary, this work provides proof-of-principle demonstration of chemical and biological strategies to activate the lysosome-autophagy system for restoring lysosomal proteostasis and enhancing autophagic clearance in model systems of diseases characterized by deficiencies in lysosomal enzymes activities and aberrant accumulation of undegraded lysosomal substrates. These findings lay the foundation for the development of nanotherapeutics for the treatment of diseases associated with inefficient autophagic clearance.