UMD Theses and Dissertations
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Item Dual Quorum Quenching Capsules: Disrupting two bacterial communication pathways that lead to virulence(2016) Rhoads, Melissa Katherine; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Healthcare Associated Infections (HAIs) in the United States, are estimated to cost nearly $10 billion annually. And, while device-related infections have decreased, the 60% attributed to pneumonia, gastrointestinal pathogens and surgical site infections (SSIs) remain prevalent. Furthermore, these are often complicated by antibacterial resistance that ultimately cause 2 million illnesses and 23,000 deaths in the US annually. Antibacterial resistance is an issue increasing in severity as existing antibiotics are losing effectiveness, and fewer new antibiotics are being developed. As a result, new methods of combating bacterial virulence are required. Modulating communications of bacteria can alter phenotype, such as biofilm formation and toxin production. Disrupting these communications provides a means of controlling virulence without directly interacting with the bacteria of interest, a strategy contrary to traditional antibiotics. Inter- and intra-species bacterial communication is commonly called quorum sensing because the communication molecules have been linked to phenotypic changes based on bacterial population dynamics. By disrupting the communication, a method called ‘quorum quenching’, bacterial phenotype can be altered. Virulence of bacteria is both population and species dependent; each species will secrete different toxic molecules, and total population will affect bacterial phenotype9. Here, the kinase LsrK and lactonase SsoPox were combined to simultaneously disrupt two different communication pathways with direct ties to virulence leading to SSIs, gastrointestinal infection and pneumonia. To deliver these enzymes for site-specific virulence prevention, two naturally occurring polymers were used, chitosan and alginate. Chitosan, from crustacean shells, and alginate, from seaweed, are frequently studied due to their biocompatibility, availability, self-assembly and biodegrading properties and have already been verified in vivo for wound-dressing. In this work, a novel functionalized capsule of quorum quenching enzymes and biocompatible polymers was constructed and demonstrated to have dual-quenching capability. This combination of immobilized enzymes has the potential for preventing biofilm formation and reducing bacterial toxicity in a wide variety of medical and non-medical applications.Item Silencing bacteria with small molecules(2014) Guo, Min; Sintim, Herman O; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quorum sensing (QS) is a phenomenon in bacteria where the accumulation of extracellular signaling molecules (autoinducers, AIs), which enable bacterial cells to sense neighboring cells (population density), reaches certain threshold and triggers group behaviors of bacteria including virulence production and biofilm formation. The inhibition of QS and hence toxin production or biofilm formation by pathogenic bacteria has been suggested as an alternative strategy to deal with the problem of bacterial resistance to traditional antibiotics. Inhibiting QS will not kill bacteria, however the expectation is that resistance to a QS antagonist will not be as widespread as it is for traditional cytotoxic antibiotics. In Chapters 2 and 3 of this dissertation, we report the syntheses and biological evaluations of various analogs (C1 substituted, ester protected and 3,3-dihalogenated) of a universal QS signaling molecule, AI-2, which is found in both Gram-positive and Gram-negative bacteria. We report that modifications to the native AI-2 molecule affords analogs that can potently inhibit QS processes in E. coli and Salmonella. In Chapter 4, we explore the development of small molecule modulators of species-specific acylhomoserine lactone autoinducers, called AI-1. In the past three decades, intensive efforts have been dedicated to the development of modulators of AI-1-based QS signaling. The majority of modulators, reported to date, have kept the lactone head group and modified the acyl tail. These synthetic modulators, although effective, are not drug-like because lactones are susceptible to chemical and enzymatic hydrolysis. We demonstrate that 3-aminooxazolidinone based AI-1 analogs, which are hydrolytically more stable than homoserine lactone-based compounds, can also modulate AI-1-based QS.Item Structural Variants of AI-2 Analogs to Probe Quorum Sensing in Diverse Bacteria(2011) Gamby, Sonja Josette; Sintim, Herman O.; Master of Life Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial infections which were once easily managed with antibiotics are now reemerging as a serious threat to human health. The difficulty in managing infectious diseases is arising out of bacterial resistance to front line antibiotics. A new paradigm for fighting bacterial infection via the inhibition of quorum sensing has emerged. Quorum sensing is the process by which small diffusible molecules (autoinducers) are used to sense population density and upregulate genes. Notably, genes for virulence production and biofilm formation have been found to be controlled by this process. Thus, quorum sensing, offers an alternative target for the treatment of bacterial infections. One autoinducer which has been identified across many bacterial species is AI-2. The goals of this thesis were to make more hydrolytically stable analogs of AI-2 as potent inhibitors of quorum sensing, as well as, exploring the effects of AI-2 analogs on QS in P. aeruginosa. In this study, the processing of bis ester protected AI-2 analogs was examined. Also, two long chain AI-2 analogs were synthesized and tested for their ability to inhibit QS in P.aeruginosa. It was found that bis protected analogs are processed different across bacterial species. Also, long chain AI-2 analogs were found to be inhibitors of QS in P. aeruginosa, specifically, by inhibiting a LasR receptor which typically responds to a different class of autoinducer.Item ALTERING THE AI-2 MEDIATED QUORUM SENSING CIRCUITRY TO QUENCH BACTERIAL COMMUNICATION NETWORKS(2011) Roy, Varnika; Bentley, William E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The emergence of antibiotic resistant bacteria poses a global threat to human health and has been classified as a clinical super-challenge of the 21st century. This has necessitated research on new antimicrobials that inhibit bacterial virulence by mechanisms other than those that target bacterial growth or viability. Such approaches have been reported to pose less evolutionary pressure on bacteria to evolve and become resistant to antibiotics. Bacterial cell-cell communication, termed quorum sensing (QS), is mediated by signatures of small molecules. QS via these small molecules has been linked to numerous undesirable bacterial phenotypes such as biofilm formation, onset of pathogenicity, triggering of virulence genes etc. The small signaling molecules represent targets for intercepting bacterial communication (and their resultant undesirable phenotypes). We have devised two strategies that interrupt bacterial communication in multispecies bacterial cultures by targeting the interspecies signaling molecule autoinducer-2 (AI-2), which is produced or recognized by over 70 species of bacteria. Our first approach is to bring the native intracellular AI-2 signal processing mechanisms to the extracellular surroundings to quench the QS response of bacteria. Specifically we deliver the Escherichia coli AI-2 kinase, LsrK, to E. coli populations ex vivo and phosphorylate and degrade the extracellular AI-2. This significantly attenuates the native QS response in E. coli. Similar results are obtained in a tri-species synthetic ecosystem comprising E. coli, Salmonella typhimurium and Vibrio harveyi. In our second quenching strategy, we explore a panel of small synthetic molecules that are analogs of AI-2 (C1-alkyl analogs). The analogs are observed to cause species-specific and cross-species quorum quenching in the tri-species synthetic ecosystems of the aforementioned strains. Some of the AI-2 analogs quench pyocyanin (toxin production) in the opportunistic pathogen Pseudomonas aeruginosa. Based on these observations, I used analog cocktails to quench QS en masse in assembled synthetic ecosystems. Finally, I tested the efficiency of the analogs in quenching pathogenic phenotypes such as biofilm formation in E. coli. The analogs inhibit biofilm formation and act in concert with antibiotics to reduce biofilm formation even further. Our results suggest entirely new modalities for interrupting or tailoring the networks of communication among bacteria and identifying drug targets to develop the next generation of antimicrobials based on QS.Item LOCAL AND GLOBAL GENE REGULATION ANALYSIS OF THE AUTOINDUCER-2 MEDIATED QUORUM SENSING MECHANISM IN ESCHERICHIA COLI(2011) Byrd, Christopher Matthew; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The term `quorum sensing' (QS) is used to define a population density based communication mechanism which uses chemical signal molecules called autoinducers to trigger unique and varied changes in gene expression. Although several communication methods have been identified in bacteria that are unique to a particular species, one type of signal molecule, autoinducer-2 (AI-2) is linked to interspecies communication, indicating its potential as a universal signal for cueing a QS response among multiple bacterial types. In E. coli, AI-2 acts as an effector by binding to the QS repressor LsrR. As a result, LsrR unbinds and relieves repression of the lsr regulon, stimulating a subsequent QS gene expression cascade. In this dissertation, LsrR structure and in vitro binding activity are examined. Genomic binding and DNA microarray analyses are conducted and three novel sites putatively regulated by LsrR, yegE-udk, mppA and yihF, are revealed. Two cAMP receptor protein (CRP) binding locations in intergenic region of the lsr regulon are also confirmed. The role of each CRP site in divergent expression is qualified, indicating the lsr intergenic region to be a class III CRP-dependent promoter. Also, four specific DNA binding sites for LsrR in the lsr intergenic region are proposed, and reliance upon simultaneous binding to these various sites and the resulting effects on LsrR repression is presented. Finally, a complex model for regulation of the lsr regulon is depicted incorporating LsrR, CRP, DNA looping, and a predicted secondary layer of repression by an integration host factor (IHF)-like protein. Further understanding of this QS genetic mechanism may potentially be used for inhibiting bacterial proliferation and infection, modifying the natural genetic system to elicit alternate desired responses, or extracted and applied to a highly customizable and sensitive in vitro biosensor.Item Biological Nanofactories: Altering Cellular Response via Localized Synthesis and Delivery(2008-11-19) Fernandes, Rohan; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Conventional research in targeted delivery of molecules-of-interest involves either packaging of the molecules-of-interest within a delivery mechanism or pre-synthesis of an inactive prodrug that is converted to the molecule-of-interest in the vicinity of the targeted area. Biological nanofactories provide an alternative approach to targeted delivery by locally synthesizing and delivering the molecules-of-interest at surface of the targeted cells. The machinery for synthesis and delivery is derived from the targeted cells themselves. Biological nanofactories are nano-dimensioned and are comprised of multiple functional modules. At the most basic level, a biological nanofactory consists of a cell targeting module and a synthesis module. When deployed, a biological nanofactory binds to the targeted cell surface and locally synthesizes and delivers molecules-of-interest thus altering the response of the targeted cells. In this dissertation, biological nanofactories for the localized synthesis and delivery of the 'universal' quorum sensing signaling molecule autoinducer-2 are demonstrated. Quorum sensing is process by which bacterial co-ordinate their activities at a population level through the production, release, sensing and uptake of signaling autoinducers and plays a role in diverse bacterial phenomena such as bacterial pathogenicity, biofilm formation and bioluminescence. Two types of biological nanofactories; magnetic nanofactories and antibody nanofactories are presented in this dissertation as demonstrations of the biological nanofactory approach to targeted delivery. Magnetic nanofactories consist of the AI-2 biosynthesis enzymes attached to functionalized chitosan-mag nanoparticles. Assembly of these nanofactories involves synthesis of the chitosan-mag nanoparticles and subsequent assembly of the AI-2 pathway enzymes onto the particles. Antibody nanofactories consist of the AI-2 biosynthesis enzymes self assembled onto the targeting antibody. Assembly of these nanofactories involves creation of a fusion protein that attaches to the targeting antibody. When added to cultures of quorum sensing bacteria, the nanofactories bind to the surface of the targeted cells via the targeting module and locally synthesize and deliver AI-2 there via the synthesis module. The cells sense and uptake the AI-2 and alter their natural response. Prospects of using biological nanofactories to alter the native response of targeted cells to a 'desired' state, especially with respect to down-regulating undesirable co-ordinated bacterial response, are envisioned.Item AUTOINDUCER-2 (AI-2) MEDIATED QUORUM SENSING IN ESCHERICHIA COLI(2004-12-14) Wang, Liang; Bentley, William E; Hutcheson, Steven W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria have evolved complex genetic circuits to regulate their physiological activities and behaviors in response to extracellular signals. In a process termed "quorum sensing", or density-dependent gene regulation, bacteria produce, release and respond to certain signaling molecules termed autoinducers. The bacterial autoinducer-2 (AI-2) has received intense interest recently because the gene for its synthesis, luxS, is common in a large number of Gram-negative and Gram-positive bacterial species. In this study, the luxS controlled genes were identified in Escherichia coli K12 strain under two different growth conditions using DNA microarrays. Deletion of the luxS was shown to affect expression of genes involved in AI-2 transport (the lsr operon) and methionine biosynthesis (metE), and to a lesser degree those involved in methyl transfer, iron uptake, resistance to oxidative stress, utilization of various carbon sources, and virulence. The effects of glucose on extracellular AI-2 level were investigated further. It was shown that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cAMP-CRP complex. This complex directly stimulates transcription of the lsr (luxS regulated) operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with LsrR repressor to regulate AI-2 uptake. This study, for the first time, has shown that quorum sensing regulates specific activities in E. coli K12, and has elucidated regulatory mechanisms for AI-2 biosynthesis and transport in this organism. With a better understanding of AI-2/luxS mediated gene regulation, we may be able to develop strategies for harnessing AI-2 quorum sensing for our advantage in bioreactor studies and ultimately in control of the bacterial pathogenicity.