EFFECT OF CRANBERRY CONSTITUENTS ON UTI PATHOGENESIS

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 5R01AT002058-04 from National Center For Complementary And Alternative Medicine, IRG: ZAT1

Abstract: Urinary tract infections (UTIs) are among the most common bacterial infections, with symptoms that can include dysuria, urgency and frequency of urination, flank pain and fever. Clinical diagnosis is based on symptoms and the laboratory finding of bacteriuria. Women of all ages are especially susceptible to acute and recurrent infections. Because of the rise in antibiotic-resistant bacteria and the deleterious effects of long-term antibiotic prophylaxis, alternative therapies for UTIs are needed. Alternative therapies proposed for treatment of acute and recurrent UTIs have included the ingestion of cranberries or cranberry juice. The actions ascribed to cranberries include acidification of the urine, inhibition of kinases, and anti-inflammatory, anti-adhesive and anti-oxidant effects. The Vorsa laboratory has fractionated cranberry fruit into the major flavonoid classes of proanthocyanidins, flavonols and anthocyanins. Through bioassay directed fractionation, the cranberry proanthocyanidin fraction was shown to contain compounds that can inhibit the aggregation of human erythrocytes by bacteria expressing PapG, the bacterial adhesin associated with pyelonephritis. In addition, the flavonol extract exhibited high anti-inflammatory activity in a TPA-induced mouse ear edema model. The Hultgren laboratory has utilized a mouse model of cystitis to elucidate the structure, function and mechanism of action of bacterial and host factors involved in the establishment and persistence of UTIs. The steps of the pathogenic cascade that occurs upon introduction of uropathogenic E. coli (UPEC) into the urinary tract has been delineated. This cascade includes bacterial binding and invasion into bladder epithelial cells, intracellular growth, fluxing, reservoir formation and reemergence leading to recurrence. The host responds by mounting an immune response that includes the release of cytokines and infiltration of PMNs. In collaboration, the Vorsa and Hultgren laboratories will investigate the bioavailability of specific cranberry constituents and their effects on each of the steps of UPEC pathogenesis and the host response. We will utilize compound and protein HPLC and FPLC purification, affinity chromatography, mouse models, extensive in vitro and tissue culture models, high resolution EM, DNA microarrays, mass spectroscopy, X-ray crystallography and videomicroscopy. This work will lead to a molecular under standing of the bioactivities of defined cranberry constituents on UPEC pathogenesis and may lead to better treatments for UTIs.

Keywords: fruit, host organism interaction, medicinal plant, urinary tract infection, Escherichia coli, bacteria infection mechanism, cytokine, flavonoid, gene expression, glycoside, inflammation, intermolecular interaction, nutrient bioavailability, X ray crystallography, affinity chromatography, electron microscopy, high performance liquid chromatography, laboratory mouse, microarray technology, polymerase chain reaction, protein purification, tissue /cell culture, video microscopy

Project start date: 2004-01-01

Project end date: 2008-11-30

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PATHOGENIC FIBER FORMATION IN BACTERIA: STRUCTURAL BASIS

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 5R01AI048689-05 from National Institute Of Allergy And Infectious Diseases, IRG: BM

Abstract: Verbatim from Applicant s ) The biogenesis of diverse fibrous organelles by Gram-negative bacteria plays a critical role in the pathogenesis of many diseases. Uropathogenic E. coli assembles P and type 1 pili on their surfaces via the conserved chaperone-usher pathway. These pili mediate attachment to host tissues, a key early event in the development of disease. Pilus subunits have immunoglobulin-like (Ig) folds that lack their canonical C-terminal b-strand. The Ig-like periplasmic chaperone transiently donates a b-strand to complete the fold of the subunit, via a mechanism termed donor strand complementation. Subunits do not fold in the absence of a chaperone and are proteolytically degraded. During pilus assembly, the Ig fold of a subunit is thought to be more permanently completed by the exposed N-terminal extension of its neighbor, which displaces the chaperone in a mechanism termed donor strand exchange. Pilus subunits with complete Ig folds will be generated by genetically linking the relevant N-terminal extension to the C-terminus of the subunit to create stable, monomeric donor strand complemented (Dsc) subunits. Dsc subunits will be tested for their ability to fold in the absence of a chaperone to determine the role of the missing strand in subunit folding. Dsc and N-terminal deleted (Ntd) subunits, which have their N-terminal extensions removed and therefore cannot interact with other subunits, will be used with x-ray crystallography and in vivo and in vitro assembly systems to elucidate the structural basis of donor strand exchange. Stable Dsc adhesins will be crystallized with their saccharide receptors to determine the structural basis of microbial colonization and will be tested as vaccines to treat and prevent urinary tract infections. Gram-negative pathogens also assemble surface fibers termed curli via the nucleation-precipitation pathway. Curli share the diagnostic properties of amyloid fibers, which characterize a variety of human diseases, including Alzheimer s disease. The nucleation, formation, and structure of amyloid-like curli fibers will be studied. These studies will reveal general principles that govern the fundamental processes of protein folding and organelle biogenesis, shed light on bacterial attachment and its role in pathogenesis, and contribute to the development of new methods to treat and prevent a variety of diseases.

Keywords: Escherichia coli, bacteria infection mechanism, cell component structure /function, pilus, adhesin, amyloid protein, carbohydrate receptor, immunoglobulin, molecular assembly /self assembly, molecular chaperone, organelle, protein folding, X ray crystallography

Project start date: 2001-02-01

Project end date: 2006-01-31

MOLECULAR BASIS OF E. COLI ADHESINS IN BLADDER DISORDERS

Scott J Hultgren, Professor
Molecular Microbiologywashington University
1 Brookings Dr, Campus Box 1054
saint Louis, Mo 631304899

Grant 2R01DK051406-11A2 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: UKGD

Abstract: Urinary tract infections (UTIs) result in considerable morbidity and health care costs. The most common cause is the Gram-negative bacteria, uropathogenic Escherichia coli (UPEC). Human infections are characterized by bacteriuria and the release of cytokines, exfoliated cells, and polymorphonuclear leukocytes (PMN) into the urine. These clinical manifestations of disease are all seen in mice. Using qRT-PCR, microarray analyses, microscopy, and immunohistochemistry, we have further described the response to UPEC infection. We have demonstrated that type 1 pili-mediated UPEC binding to and invasion of bladder epithelial cells potentiates toll-like receptor (TLR) 4-mediated signaling and results in rapid upregulation of genes involved in cell differentiation, proliferative and immediate-early responses, pro-inflammatory responses, apoptosis, stress responses, signal transduction, cell-cell contacts, and metabolic changes. Further, we have shown that TLR4-mediated signaling on both stromal and hematopoietic cells is required for efficient clearance of bacteria. Hematopoietic cells include macrophages (M), dendritic cells (DC), granulocytes, and B and T cells. In this proposal we describe experiments to build on our previous work to understand host processes important in determining the outcome of an encounter between pathogens and the normally sterile host urinary tract. We will delineate the host response to UPEC, including the kinetics of cytokine production within the infected bladder, the hierarchy of infiltration of immune cells, and real time visualization of early interactions between immune cells and infecting bacteria. We will also broaden our understanding of UTIs and host response by characterizing the host response to two Gram-positive bacteria that also cause UTIs in humans, Staphylococcus saprophyticus and Enterococcus faecalis. Differing host response mechanisms and bacterial tropisms between Gram-negative and Gram- positive UTIs may have implications for disease symptoms, progression, and treatment. Using mouse mutant backgrounds and cell and cytokine depletion experiments, we will examine the roles of host immune cells and defense molecules in inflammation, bacterial clearance, reservoir maintenance, and epithelial exfoliation and regeneration. A detailed understanding of the host response to UTIs may lead to elucidation of new and better ways to evaluate, treat, and prevent these common infections. UTIs occur frequently in otherwise healthy women and result in an estimated ~$2.5 billion annually in health care costs. In this proposal we describe experiments to broaden our understanding of UTIs by characterizing the soluble mediators and host immune cells involved in the host response to the most common cause of UTI (uropathogenic Escherichia coli) and two Gram-positive bacteria (Staphylococcus saprophyticus and Enterococcus faecalis). A better understanding of the host factors involved in determining the outcome of infection is needed to better diagnosis, treat and prevent this common disease

Project start date: 1997-01-01

Project end date: 2012-01-31

EFFECT OF CRANBERRY CONSTITUENTS ON UTI PATHOGENESIS

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 1R01AT002058-01 from National Center For Complementary And Alternative Medicine, IRG: ZAT1

Abstract: Urinary tract infections (UTIs) are among the most common bacterial infections, with symptoms that can include dysuria, urgency and frequency of urination, flank pain and fever. Clinical diagnosis is based on symptoms and the laboratory finding of bacteriuria. Women of all ages are especially susceptible to acute and recurrent infections. Because of the rise in antibiotic-resistant bacteria and the deleterious effects of long-term antibiotic prophylaxis, alternative therapies for UTIs are needed. Alternative therapies proposed for treatment of acute and recurrent UTIs have included the ingestion of cranberries or cranberry juice. The actions ascribed to cranberries include acidification of the urine, inhibition of kinases, and anti-inflammatory, anti-adhesive and anti-oxidant effects. The Vorsa laboratory has fractionated cranberry fruit into the major flavonoid classes of proanthocyanidins, flavonols and anthocyanins. Through bioassay directed fractionation, the cranberry proanthocyanidin fraction was shown to contain compounds that can inhibit the aggregation of human erythrocytes by bacteria expressing PapG, the bacterial adhesin associated with pyelonephritis. In addition, the flavonol extract exhibited high anti-inflammatory activity in a TPA-induced mouse ear edema model. The Hultgren laboratory has utilized a mouse model of cystitis to elucidate the structure, function and mechanism of action of bacterial and host factors involved in the establishment and persistence of UTIs. The steps of the pathogenic cascade that occurs upon introduction of uropathogenic E. coli (UPEC) into the urinary tract has been delineated. This cascade includes bacterial binding and invasion into bladder epithelial cells, intracellular growth, fluxing, reservoir formation and reemergence leading to recurrence. The host responds by mounting an immune response that includes the release of cytokines and infiltration of PMNs. In collaboration, the Vorsa and Hultgren laboratories will investigate the bioavailability of specific cranberry constituents and their effects on each of the steps of UPEC pathogenesis and the host response. We will utilize compound and protein HPLC and FPLC purification, affinity chromatography, mouse models, extensive in vitro and tissue culture models, high resolution EM, DNA microarrays, mass spectroscopy, X-ray crystallography and videomicroscopy. This work will lead to a molecular under standing of the bioactivities of defined cranberry constituents on UPEC pathogenesis and may lead to better treatments for UTIs.

Keywords: fruit, host organism interaction, medicinal plant, urinary tract infection, Escherichia coli, bacteria infection mechanism, cytokine, flavonoid, gene expression, glycoside, inflammation, intermolecular interaction, nutrient bioavailability, X ray crystallography, affinity chromatography, electron microscopy, high performance liquid chromatography, laboratory mouse, microarray technology, polymerase chain reaction, protein purification, tissue /cell culture, video microscopy

Project start date: 2004-01-01

Project end date: 2007-11-30

ORWH: SCORE On Sex And Gender Factors Affecting Women´s Health

Scott J Hultgren, Professor
Molecular Microbiologywashington University

Grant 5P50DK064540-07 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

Abstract: This interdisciplinary SCOR program seeks to understand the epidemiology, pathogenic strategies, and resultant host responses of urinary tract infections (DTI) caused by uropathogenic E. coli (UPEC), one of the most common diseases affecting women. This knowledge will be applied to critically evaluate all aspects of clinical UTI management, including diagnosis, treatment, and prevention. We have proposed an integrative and translational set of experiments that capitalizes on the complementary expertise found in each of the three projects. Basic scientists in Projects 1 and 3 have access to uropathogenic strains collected from women in Project 2 at different clinical stages of UTI. Working together, Projects 1, 2, and 3 will identify genetic and molecular markers and correlates of the different clinical UTI syndromes associated with UPEC infection. These will be pursued both in humans (Project 2) and mice (Project 1) for prognostic indicators of disease outcome bacterial clearance, asymptomatic infection, chronic colonization, or recurrence. Genotypic and phenotypic profiles of UPEC strains from well-characterized UTI cases will be generated in Project 1 and 3 by blending a powerful genetic system with functional and comparative genomics, defined in vitro and murine models, comparative immunoproteomics, biochemistry, cell biology, laser capture microdissection, antigen discovery techniques, and high resolution electron microscopy. The host response to intracellular bacterial communities (IBCs) and quiescent intracellular reservoirs (QIRs) formed by different UPEC isolates will be examined in detail both in a mouse model (Project 1), using gene and cytokine expression profiling, and in humans (Project 2), by monitoring the adaptive immune response and metabolite profiles in human urine. In addition, exfoliated bladder epithelial cells in mouse and human urine will be screened for evidence of IBC formation, allowing parallel correlation of microscopic assays with clinical outcome in both mice and humans. These efforts promise to connect specific measurements made at the bench to clinical outcomes observed at the bedside. Project 3 will also address primary prevention of UTI by using comparative pan-genomics to study the mechanism by which UPEC emerge from the distal gastrointestinal tract and traverse the perineum to the urethra to cause infection. Completion of these interwoven projects promises to address questions in the clinical management of this ubiquitous disease

Keywords: bacteria infection mechanism, bacterial cytopathogenic effect, bacterial genetics, host organism interaction, pathologic process, relapse /recurrence, urinary tract infection clinical research, female, human subject, laboratory mouse, patient oriented research, women`s health

Project start date: 2002-09-30

Project end date: 2012-07-31

Pathogenic Fiber Formation In Bacteria: Structural Basis

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 5R01AI048689-07 from National Institute Of Allergy And Infectious Diseases, IRG: BACP

Abstract: The rise in antibiotic resistant pathogens, emergence of new diseases, and involvement of bacterial pathogens in diseases formerly thought to be due to non-infectious agents has rekindled the need to understand the "molecular logic" of virulent bacteria. Extracellular fibers made by Gram-negative bacteria can effect bacterial interactions with their environment or host tissue. This grant focuses on the mechanisms of assembly of two such fibers, adhesive pili and curli. Pili are composite structures consisting of an adhesive heteropolymeric distal tip fibrillum joined to a rigid helical rod. We discovered that periplasmic chaperones serve as folding templates for pilus subunits, actively shaping the final structure of pilus subunits. Further, we discovered that pilus assembly occurs by a mechanism that we termed donor strand exchange wherein the N terminal extension of every subunit completes the immunoglobulin fold of its neighboring subunit. The resulting pilus structures initiate host-pathogen interactions critical in the pathogenic processes of a wide range of bacteria by binding to host receptors. We found that type 1 pilus mediated entry of uropathogenic Escherichia coli into superficial cells lining the bladder activates a complex genetic cascade leading to the formation of intracellular bacterial communities that undergo a defined maturation and differentiation program necessary to subvert innate host defenses. We will use a multidisciplinary approach including molecular genetics, biochemistry, microscopy, physical techniques and in vivo and in vitro systems to better understand pilus assembly and function. We will investigate the molecular basis of the donor strand exchange interactions that determine the specificity of the subunit types in the pilus and the mechanism by which pilus assembly is initiated and terminated. We will investigate the function of the specific architecture of the plus fiber in pilus strength and function, including mediating biofilm formation and host pathogen interactions. We will also investigate the biogenesis of another type of fiber called curli. Curli fibers are assembled via a nucleation/precipitation pathway and we have shown that they share all the biochemical characteristics of amyloid proteins involved in human diseases such as Alzheimer s, Huntington s, and Parkinson s. We will elucidate the structural basis of how two periplasmic chaperone-like proteins, CsgE and CsgF, facilitate the protein-protein interactions necessary for amyloid formation. This work is spawning new insights into the most basic principles of molecular biology related to protein folding and macromolecular assembly and is providing a paradigm to understand infectious diseases that will lead to better strategies for treatment and prevention.

Keywords: bacteria, pilus, Escherichia, Escherichia coli, X ray crystallography, X ray microscopy, acid, adhesin, adhesion, amyloid protein, amyloidosis, antibiotic, atomic force microscopy, bacterial disease, base, binding site, biochemistry, biofilm, cell, cell line, cell membrane, cognition, communicable disease, community, conformation, crystallization, cysteine, electron microscopy, emotion, environment, epithelium, extracellular, gene expression, gene mutation, genetics, gram negative bacteria, health /scientific organization, heat, heat shock protein, human, immunoglobulin, infection, information system, insight, intracellular, lead, medicine, membrane, microscopy, model, molecular biology, molecular chaperone, molecular dynamics, molecular genetics, monomer, motivation, mutant, operon, organism, peptide, pilin, play, precipitation, prevention, proline, protein, protein folding, protein protein interaction, receptor, receptor binding, role, stress, thinking, tissue, trypsin, urinary tract, urinary tract infection, urine, virulence, wound

Project start date: 2001-02-01

Project end date: 2011-01-31

ORWH: SCOR--Sex /Gender Factors Affecting Women s Health

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 5P50DK064540-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZAR1

Abstract: On a global basis, urinary tract infections (UTIs), which are among the most common infectious diseases in the United States, occur -150 million times annually and account for more than $6 billion in direct health care costs. UTIs primarily affect women because of the female- anatomy and the ascending nature of the disease. The Gram-negative bacterium Escherichia coli is the most common cause of these infections which account for significant morbidity. In addition, to the original infection one of the major clinical problems is recurrence. A woman treated for an uncomplicated UTI has a 25-50% chance of developing a recurrent infection. It has generally been assumed that UTIs are caused by non-invasive bacteria, that they are acute in nature, and are self-limited. It has also been assumed that recurrent infections are due to re-inoculation of the urinary tract with E.coli from an intestinal or vaginal reservoir. Recent evidence suggests that this dogma may in some cases be incorrect and misleading and could be interfering with the proper evaluation and treatment of these infections. This proposal seeks to elucidate the molecular and epidemiologic basis of acute and recurrent UTIs. Special emphasis will be placed on better defining the epidemiology of UTIs, determining the presence of persistent bacterial bladder and vaginal reservoirs following acute symptomatic UTI in women and elucidating the molecular factors involved in the host-pathogen interaction. The temporal, associations between asymptornatic and symptomatic bladder infection and vaginal colonization in the clinical setting will be assessed. Molecular characterization of the virulence determinants in well-characterized uropathogenic strains will be elucidated by blending a powerful genetic system with functional genomics, defined in vitro and murine models, biochemistry, cell biology, and high resolution electron microscopy. The mechanistic details of how the bladder responds to infection, and how specific virulence. factors affect this host response will be determined using microarray, quantitative RT-PCR and laser capture microdis section. This proposal represents an intricate and integrated network between three projects and will lead to a new understanding of the host-pathogen interactions that occur throughout the infectious cycle including the host defense response in the bladder and the virulence mechanisms by which bacterial pathogens subvert these defenses. Results of this research could revolutionize the way UTIs are evaluated and lead to new and better ways to treat and prevent this infection that primarily affects women.

Keywords: bacteria infection mechanism, bacterial cytopathogenic effect, bacterial genetics, host organism interaction, pathologic process, relapse /recurrence, urinary tract infection, clinical research, female, human subject, laboratory mouse, patient oriented research, women s health

Project start date: 2002-09-30

Project end date: 2007-08-31

Host-Pathogen Interactions In Acute And Chronic UTI

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 1P50DK064540-010001 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZAR1

Abstract: Millions of women suffer from recurrent urinary tract infections (UTIs) every year despite current antimicrobial treatments. UTIs are usually caused by the Gram-negative bacterium Escherichia coIi. The iGram positive pathogen Enterococcus faecalis is also an important cause of UTIs, particularly in the inosocomial setting. This project will use a blend of genetics, high-resolution electron and immunoflourescent microscopy, mutagenesis and well-established in vitro tissue culture and in vivo mouse cystitis models to gain an understanding of the bacterial factors which are involved in the molecular cross-talk between the host and the pathogen. This interaction determines whether an infection occurs and if the infection is quickly cleared with a return to sterility in the bladder or a persistent reservoir results. The genetic differences in sequence and regulation between K12 and clinical strains of E. coil (both symptomatic and ASB) will be determined, using sequencing and microarrays. These studies will provide the basis for both targeted and global mutagenesis analyses. The mechanisms by which UPEC proceeds through each step of pathogenesis will then be determined and the contributions of specific bacterial genes to virulence will be determined. The triggers for innate host defenses, specifically the interactions between bacterial LPS with the host TLR4 isignalling system will be analyzed. In addition, mutations that are developed in this Project, that are deficient in pathogenesis will be used in other projects to gain a detailed understanding of the host response and the molecular cross-talk that occurs as a consequence of host-pathogen interactions. These studies will provide insight into possible downstream sequelae, such as recurrent UTIs. The knowledge of general and !specific mechanisms of infection will be increased by investigating the molecular basis of how Enterococcus interacts with the urinary tract and causes disease. The specific knowledge that will be gained in this project will i. lead to a better understanding of the mechanisms by which bacteria causes acute, recurrent and chronic cystitis, ii. lead to improved methods of treating and preventing this ubiquitous disease and iii. establish a coordinated set of analyses and model systems which can be used to understand the fine molecular details of both the early and long-term consequences of the interaction between a pathogen and its host tissue. In addition, since a wide variety of diseases are caused by Gram-negative bacteria that infect or invade mucos surfaces, the knowledge gained will be applicable to many infectious d!seases.

Keywords: acute disease /disorder, bacteria infection mechanism, bacterial genetics, chronic disease /disorder, host organism interaction, urinary tract infection, Escherichia coli, Escherichia coli k12, Streptococcus enterococcus group, bacterial cytopathogenic effect, bacteriuria, disease /disorder model, gene expression, genotype, pathologic process, relapse /recurrence, urinary bladder disorder, virulence, enzyme linked immunosorbent assay, female, immunocytochemistry, laboratory mouse, microarray technology, microorganism culture, nucleic acid sequence

Project start date: 2002-09-01

Project end date: 2007-08-31

Structure Of Proteins Involved In Bacterial Pathogenesis

Scott J Hultgren, Professor
Molecular Microbiologywashington University
1 Brookings Dr, Campus Box 1054
saint Louis, Mo 631304899

Grant 5R01AI049950-07 from National Institute Of Allergy And Infectious Diseases, IRG: MSFC

Abstract: Many of the adhesins of Gram-negative bacteria are incorporated into heteropolymeric fibers assembled by the chaperone/usher pathway. These fibers range in morphology from composite pili assembled by members of the FGS (F1G1 short) subfamily of chaperones, to fibrous capsule-like structures assembled by the FGL (F1G1 long) subfamily of chaperones. Here we propose to build on our work on P and type 1 pili, which has provided a paradigm for the assembly of hundreds of virulence fibers in diverse Gram-negative organisms. Using biochemistry, genetics, electron microscopy and X-ray crystallography, we have defined the structure and mechanism of action of the periplasmic chaperones in significant detail. We have solved the structure of the PapD periplasmic chaperone in complex with the tip adaptor subunit, PapK, and with the major tip subunit, PapE. We have also solved the structure of the PapE subunit in complex with the N-terminal extension of PapK and the structure of the receptor binding domain of the PapG adhesin with and without its host carbohydrate ligand. Pilus chaperones are comprised of two immunoglobulin (Ig)-like domains. These structures showed that pilin subunits also have an Ig-like fold, but they are missing their seventh (G) strand, thus exposing the hydrophobic core. In a process we termed donor-strand complementation, the chaperone´s G1 strand serves as the pilin´s seventh strand, catalyzing the folding of the subunit and preventing non-productive subunit aggregation. At the usher, pilus assembly occurs by donor-strand exchange, in which the G1 strand of the chaperone is replaced by an N-terminal extension that is present on every subunit and exposed on incoming chaperone-subunit complexes. The pilus subunit then undergoes a topological transition that triggers the closure of its groove, cementing its neighbor´s NTE as part of its own Ig fold. Thus, the final pilus structure is a series of Ig-like domains, each of which is formed from parts of two individual subunit monomers. Each subunit has distinct specificity for other interactive subunits and distinct roles in pilus assembly. We propose to expand our knowledge of the structures of the pilus adhesins and further study the structure of the subunits and assembly machinery of two such assembly systems the prototypical FGS P pilus system of uropathogenic Escherichia coli and the Saf FGL system of Salmonella. In addition, we will perform structural studies on anti-chaperone compounds

Project start date: 2001-06-01

Project end date: 2012-06-30

Molecular Basis Of E. Coli Adhesins In Bladder Disorders

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 5R01DK051406-10 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

Abstract: Adapted from  s ) Urinary tract infections (UTIs) are common infections that affect a large proportion of the world population and account for significant morbidity and medical expenditures. These infections are most commonly caused by Escherichia coli (E. coli). A long-term goal of this proposal is to understand the processes by which E. coli causes acute, recurrent and chronic UTIs and the sequelae of these infections. An integrated approach will be used that blends a powerful bacterial genetic system, a mouse UTI model, and x-ray crystallography with high-resolution electron microscopy (EM), protein chemistry, carbohydrate chemistry, and tissue culture systems in order to reveal the cellular, molecular, and structural basis for the pathogenesis of these infections. The FimH adhesin present at the tip of type 1 pili has been shown in animal models to mediate binding to the uroplakin-coated lumenal surface of the bladder. The uroplakin receptor complexes recognized by the FimH adhesin will be cloned and used to investigate the consequences of FimH-uroplakin interactions. Also, the three dimensional structure of the FimH adhesin will be used to design a panel of site directed mutations to delineate the mannose binding pocket of the FimH adhesin and the structural basis of bacterial colonization of the urinary tract. The adaptive responses to bladder infections and the activation of signals that lead to the release of cytokines and recruitment of neutrophils will be dissected in detail. FimH-mediated attachment to the bladder epithelial cells activates a cascade of innate defenses that leads to rapid exfoliation and proliferation of underlying epithelial cells. The molecular basis of exfoliation will be investigated and its role in protecting the bladder from infection will be studied. The molecular mechanisms by which uropathogenic E. coli are able to invade bladder epithelial cells and evade the host response will be elucidated. Uropathogenic E. coli replicate intracellularly and form "bacterial factories" in the lumenal superficial facet cells of the bladder. The virulence factors required for this process will be identified and studied. Finally, the fluxing of E. coli out of the facet cells and colonization of underlying tissue will be investigated as a mechanism to cause persistent and recurrent infections. These studies will contribute to the development of adhesin-based vaccines to treat and prevent urinary tract infections and their sequelae.

Keywords: Escherichia coli infection, adhesin, bacteria infection mechanism, host organism interaction, pathologic process, urinary tract infection, bacterial genetics, cell adhesion, disease /disorder model, protein structure function, urinary bladder epithelium, X ray crystallography, laboratory mouse, site directed mutagenesis, tissue /cell culture

Project start date: 1997-01-01

Project end date: 2007-12-31

MOLECULAR BASIS OF E COLI ADHESINS IN BLADDER DISORDERS

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 5R01DK051406-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG4

Abstract: Adapted from  s ) Even though interstitial cystitis is not associated with ongoing bacterial infections, previous infections may predispose or account for this inflammatory condition of the bladder. Understanding the molecular basis of bladder infections caused by E. coli will provide important insight into the possible sequelae of these infections such as interstitial cystis and unveil novel therapeutic and preventative strategies. This proposal represents an intensive effort combining high powered genetics, biochemistry, crystallography, and high resolution electron microscopy with a relevant primate model of cystitis to identify the molecular basis of the factors that are critical in bacterial cystitis. The exact roles played by bacterial adhesive organelles, including P pili, type 1 pili and curli, in causing bladder disorders, will be studied by testing a panel of isogenic mutant derivatives of the uropathogenic strain DS17 to i) bind to human and monkey bladder tissue in situ and ii) cause cystitis in cynomolgus monkeys. Receptor analogues will be used as inhibitors to determine the molecular basis of the adhesin-receptor interaction. Human bladder tissue obtained from patients with either bacterial cystitis or interstitial cystitis and tissue from infected monkeys will also be subjected to special staining using anti-type 1, anti-FimH, and anti-P, anti-PapG and anti-curli antisera to elucidate whether these adhesions are expressed in vivo by adherent microorganisms or possibly deposited on the bladder mucosa. The presence of these factors in interstitial cystitis tissue, or cross-reacting antigens, would suggest that bacterial infections may be a predisposing factor. Purified PapG and FimH adhesions will also be tested for their ability to confer protection against cystitis in the primate model. In order to definitively analyze the interactive surface of the adhesin molecule as it is presented to the host, the first three dimensional structure of a bacterial adhesin (FimH) will be determined. A thorough understanding of the structural basis of microbial attachment, its role in virulence, and the interactive surfaces of an adhesin that facilitate its interaction with host receptors will give us a broad understanding of this disease that primarily affects women and unveil strategies that can be developed to block the host-pathogen interaction and reveal relevant vaccine candidates that protect against infection.

Keywords: Escherichia coli infection, host organism interaction, interstitial cystitis, adhesin, cell adhesion, disease /disorder etiology, virulence, animal tissue, human tissue

Project start date: 1997-01-01

Project end date: 2001-12-31

Pathogenic Fiber Formation In Bacteria: Structural Basis

Scott J Hultgren, Professor
Molecular Microbiologywashington University
1 Brookings Dr, Campus Box 1054
saint Louis, Mo 631304899

Grant 5R01AI048689-08 from National Institute Of Allergy And Infectious Diseases, IRG: BACP

Keywords: bacteria, pilus Escherichia, Escherichia coli, X ray crystallography, X ray microscopy, acid, adhesin, adhesion, amyloid protein, amyloidosis, antibiotic, atomic force microscopy, bacterial disease, base, binding site, biochemistry, biofilm, cell, cell line, cell membrane, cognition, communicable disease, community, conformation, crystallization, cysteine, electron microscopy, emotion, environment, epithelium, extracellular, gene expression, gene mutation, genetics, gram negative bacteria, health /scientific organization, heat, heat shock protein, human, immunoglobulin, infection, information system, insight, intracellular, lead, medicine, membrane, microscopy, model, molecular biology, molecular chaperone, molecular dynamics, molecular genetics, monomer, motivation, mutant, operon, organism, peptide, pilin, play, precipitation, prevention, proline, protein, protein folding, protein protein interaction, receptor, receptor binding, role, stress, thinking, tissue, trypsin, urinary tract, urinary tract infection, urine, virulence, wound

Project start date: 2001-02-01

Project end date: 2011-01-31

CHAPERONE ASSISTED PILI ASSEMBLY IN PATHOGENIC E COLI

Scott J Hultgren, Professor
Molecular Microbiologywashington University
1 Brookings Dr, Campus Box 1054
saint Louis, Mo 631304899

Grant 5R37AI029549-18 from National Institute Of Allergy And Infectious Diseases, IRG: NSS

Keywords: Escherichia coli, bacterial protein, molecular assembly /self assembly, molecular chaperone, pilus adhesin, bacterial genetics, biological signal transduction, gene expression, intermolecular interaction, liposome, periplasm, protein biosynthesis, protein folding, protein structure function, receptor binding, virulence X ray crystallography, chemical synthesis, electron microscopy, laboratory mouse, mutant, site directed mutagenesis

Project start date: 1991-03-01

Project end date: 2009-03-31

Administrative Core

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 2P50DK064540-067597 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

CHAPERONE ASSISTED PILI ASSEMBLY IN PATHOGENIC E COLI

Scott J Hultgren, Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899

Grant 4R37AI029549-14 from National Institute Of Allergy And Infectious Diseases, IRG: NSS

Keywords: Escherichia coli, bacterial protein, molecular assembly /self assembly, molecular chaperone, pilus, adhesin, bacterial genetics, biological signal transduction, gene expression, intermolecular interaction, liposome, periplasm, protein biosynthesis, protein folding, protein structure function, receptor binding, virulence, X ray crystallography, chemical synthesis, electron microscopy, laboratory mouse, mutant, site directed mutagenesis

Project start date: 1991-03-01

Project end date: 2009-03-31