Molecular Interactions: Oral Bacteria & Matrix Proteins
Keith P Mintz, Research Associate Professor
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 5R01DE013824-07 from National Institute Of Dental And Craniofacial Research IRG: ODCS
Abstract: Actinobacillus actinomycetemcomitans is a Gram-negative, facultative anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis (LAP) and with cases of adult periodontitis. This pathogen is the causative agent for other serious infections including infectious endocarditis, soft tissue abscesses, pneumonia, and may contribute to cardiovascular disease. The periodontium is believed to be the source for these non-oral diseases, but little is known about the tropism used by A. actinomycetemcomitans to colonize the oral cavity and to infiltrate and disseminate in tissues. Pathogens have developed diverse strategies to be successful in colonization of host tissues. A common theme amongst these pathogens is the ability to initiate infection by adhesion to specific host macromolecules under stringent or hostile conditions. These molecules include proteins secreted by host cells that form the extracellular matrix (ECM). A. actinomycetemcomitans is found in the connective tissue of the periodontium and in close association with collagen fibers in infected tissues. The bacterium also binds to the ECM proteins, collagen, fibronectin and laminin. Using a genetic approach, we have identified the first A. actinomycetemcomitans collagen adhesin, Ema (extracellular matrix protein adhesin) A and multiple genes involved in regulating the expression of ECM protein adhesin activity. EmaA is structurally related to YadA, a multipurpose ECM protein adhesin of the enteropathogenic bacterium Yersinia enterocolitica, and is associated with bacterial cell surface appendages. These EmaA structures are proposed to be fundamental for collagen adhesion. To elucidate the role of EmaA in colonization and pathogenicity of the bacterium, we propose to 1) map the functional domains of EmaA by determining the collagen binding, subcellular localization, and assembly of surface structures in A. actinomycetemcomitans, 2) investigate the surface structures associated with EmaA by transmission electron microscopy and the role of these structures in resistance to the innate immune response of the host, and 3) determine the number of EmaA molecules required for the assembly of structures on the surface of A. actinomycetemcomitans. A long term goal of the proposed research is to identify and characterize bacterial adhesins that are required for the colonization of the oral cavity and non-oral tissues. These adhesins may serve as targets for future drug development involving small molecules or vaccines that disrupt host-pathogen interactions. Lay statement Binding to host tissues is the initial phase of all infectious diseases. Understanding how bacteria interact with host cells or tissue constituents will aid in the development of novel therapeutics to prevent initiation or progression of the disease.
Project start date: 2000-07-01
Project end date: 2011-06-30
5R01DE013824-07 (2007): $350532
Sponsored Links Excellgen http://Excellgen.com
MOLECULAR INTERACTIONS: ORAL BACTERIA & MATRIX PROTEINS
Keith P Mintz, Research Assistant Professor
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Grant 5R01DE013824-10 from National Institute Of Dental & Craniofacial Research
Abstract: Actinobacillus actinomycetemcomitans is a Gram-negative, facultative anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis (LAP) and with cases of adult periodontitis. This pathogen is the causative agent for other serious infections including infectious endocarditis, soft tissue abscesses, pneumonia, and may contribute to cardiovascular disease. The periodontium is believed to be the source for these non-oral diseases, but little is known about the tropism used by A. actinomycetemcomitans to colonize the oral cavity and to infiltrate and disseminate in tissues. Pathogens have developed diverse strategies to be successful in colonization of host tissues. A common theme amongst these pathogens is the ability to initiate infection by adhesion to specific host macromolecules under stringent or hostile conditions. These molecules include proteins secreted by host cells that form the extracellular matrix (ECM). A. actinomycetemcomitans is found in the connective tissue of the periodontium and in close association with collagen fibers in infected tissues. The bacterium also binds to the ECM proteins, collagen, fibronectin and laminin. Using a genetic approach, we have identified the first A. actinomycetemcomitans collagen adhesin, Ema (extracellular matrix protein adhesin) A and multiple genes involved in regulating the expression of ECM protein adhesin activity. EmaA is structurally related to YadA, a multipurpose ECM protein adhesin of the enteropathogenic bacterium Yersinia enterocolitica, and is associated with bacterial cell surface appendages. These EmaA structures are proposed to be fundamental for collagen adhesion. To elucidate the role of EmaA in colonization and pathogenicity of the bacterium, we propose to 1) map the functional domains of EmaA by determining the collagen binding, subcellular localization, and assembly of surface structures in A. actinomycetemcomitans, 2) investigate the surface structures associated with EmaA by transmission electron microscopy and the role of these structures in resistance to the innate immune response of the host, and 3) determine the number of EmaA molecules required for the assembly of structures on the surface of A. actinomycetemcomitans. A long term goal of the proposed research is to identify and characterize bacterial adhesins that are required for the colonization of the oral cavity and non-oral tissues. These adhesins may serve as targets for future drug development involving small molecules or vaccines that disrupt host-pathogen interactions. Lay statement Binding to host tissues is the initial phase of all infectious diseases. Understanding how bacteria interact with host cells or tissue constituents will aid in the development of novel therapeutics to prevent initiation or progression of the disease
Keywords: 21+ years old; A. actinomycetemcomitans; Abscess; Actinobacillus actinomycetemcomitans; Adhesins, Bacterial; Adhesions; Adult; Amino Acid Sequence; Anaerobic Bacteria; Antibodies; Antigenic Determinants; Bacteria; Bacteria, Anaerobic; Bacterial Adhesins; Bacterium actinomycetem comitans; Bacterium comitans; Binding; Binding (Molecular Function); Binding Determinants; Binding Proteins; Biological; Body Tissues; Buccal Cavity; C-terminal; Cardiovascular Diseases; Cavitas Oris; Cell surface; Cell-Extracellular Matrix; Cells; Chronic; Chronic Periodontitis; Cold-Insoluble Globulins; Collagen; Collagen Fiber; Communicable Diseases; Complex; Connective Tissue; Development; Disease; Disease Progression; Disorder; ECM; Elements; Endocarditis; Epitopes; Extracellular Matrix; Extracellular Matrix Proteins; FN1; FNZ; Fibrillar Collagen; Fibronectin 1; Fibronectins; Future; Genes; Genetic; Gingiva; Gingival; Glycoprotein GP-2; Goals; Gram-Negative Anaerobic Bacteria; Head; Head and Neck, Buccal Cavity; Human; Human, Adult; Human, General; Immune response; Immunoelectron Microscopy; Infection; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Inflammatory; LETS Proteins; Laminin; Large External Transformation-Sensitive Protein; Ligand Binding Protein; Man (Taxonomy); Man, Modern; Maps; Mediating; Membrane; Microscopy, Immuno-Electron; Microscopy, Immunoelectron; Molecular; Molecular Interaction; Mouth; Mouth Diseases; N-terminal; NH2-terminal; Neck; Opsonic Glycoprotein; Opsonic alpha(2)SB Glycoprotein; Oral; Oral Cavity Disease; Oral Cavity Disorder; Oral Disease; Oral Disorder; Oral cavity; Paradentium; Paradentiums; Parodontosis; Pathogenicity; Pathogenicity Factors; Peptide Signal Sequences; Peptides; Periodontal Diseases; Periodontitis; Periodontium; Phase; Pneumonia; Pneumonitis; Position; Positioning Attribute; Protein Structure, Primary; Proteins; Pulmonary Inflammation; Regulation; Research; Resistance; Role; Signal Peptide; Signal Sequences; Signal Sequences, Peptide; Source; Structure; Surface; TEM; TM Domain; Testing; Tissues; Tooth Supporting Structures; Transmembrane Domain; Transmembrane Region; Transmission Electron Microscopy; Tropism; Upper respiratory tract; VESCL; Vaccines; Variant; Variation; Vesicle; Virulence Factors; Y. enterocolitica; Y.enterocolitica; Yersinia enterocolitica; adhesin; adult human (21+); alpha 2-Surface Binding Glycoprotein; anaerobe; anti-microbial; antimicrobial; appendage; bactericidal; bactericide; base; cardiovascular disorder; disease/disorder; drug development; gene product; host response; immunoresponse; insight; macromolecule; membrane structure; mouth disorder; mutant; new therapeutics; next generation therapeutics; novel; novel therapeutics; oral bacteria; oral flora; oral tissue; pathogen; pathogenic bacteria; periodontal disorder; periodontium disease; periodontium disorder; prevent; preventing; protein sequence; protein signal sequence; resistant; small molecule; social role; soft tissue
Project start date: 2000-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5R01DE013824-10 (2010): $343209
5R01DE013824-09 (2009): $346676
5R01DE013824-08 (2008): $346676
5R01DE013824-05 (2005): $304000
Grants awarded to Keith P Mintz
Molecular Interactions: Oral Bacteria & Matrix Proteins
Keith P Mintz, Research Assistant Professor
Microbiol & Molecular Geneticsuniversity Of Vermont & St Agric College
85 South Prospect Street
burlington, Vt 05405
Grant 1R01DE013824-01A1 from National Institute Of Dental & Craniofacial Research IRG: OBM
Abstract: Gram-negative bacteria are associated with periodontal diseases, which are a group of chronic inflammatory diseases of the gingiva and the supporting structures of the periodontium. Actinobacillus actinomycetemcomitans (Aa) is a Gram-negative, facultative coccobacillus that colonizes the human oral cavity and upper respiratory tract. This bacterium is closely associated with periodontitis in young individuals and with cases of adult periodontitis. This pathogen has been associated with other serious human infections such as endocarditis, soft tissue abscesses, and more recently cardiovascular disease. Although the periodontium is believed to be the source of these extraoral infections, little is known about the tropisms used by Aa to maintain itself within the oral cavity and to infiltrate and disseminate in tissues. Pathogens have evolved diverse strategies to be successful in colonization of the host tissue. A common theme amongst these pathogens is the ability to initiate infection by adhesion to specific host macromolecules under stringent or hostile conditions. These molecules include proteins that are secreted by host cells that form the extracellular matrix. This matrix is usually composed of collagen and specific noncollagenous proteins, e.g. fibronectin. The major protein found in the periodontium is collagen and we have demonstrated that Aa binds to both collagen and fibronectin. In this proposal, we plan to identify the genes coding for matrix binding proteins and determine the amino acid sequences of these proteins required for binding. It is our hypothesis that the synthesis of matrix binding proteins is involved in Aa colonization of the periodontal pocket and underlying tissues. In order to realize these goals, we propose to 1) isolate the genes coding for collagen and fibronectin binding proteins by constructing a transposon mutagenesis and a phage display library; 2) determine the gene sequences and generate isogenic mutants; 3) determine the regions(s) of the protein essential for binding activity; 4) determine the immunoreactivity of LJP patient sera to these proteins. This information can be used for the development of therapeutic agents of vaccines for periodontal disease
Keywords: Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, bacteria infection mechanism, bacterial protein, binding protein, extracellular matrix protein, oral bacteria, periodontitis bacterial genetics, cell adhesion molecule, cell type, collagen, epithelium, fibronectin, genome, mutant, periodontium, protein sequence, protein structure, transposon /insertion element genetic library, human tissue, polymerase chain reaction, protein purification
Project start date: 2001-08-01
Project end date: 2004-06-30
1R01DE013824-01A1 (2001): $187475
ENVIRONMENTAL ADAPTATION AND MEMBRANE PHYSIOLOGY OF AN ORAL PATHOGEN
Keith P Mintz, Research Assistant Professor
University Of Vermont & St Agric College, 85 South Prospect Street, Burlington, Vt 05405
Grant 5R01DE018889-02 from National Institute Of Dental & Craniofacial Research
Abstract: Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative, facultative anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis (LAP). The organism is an opportunistic pathogen causing serious systemic diseases including pneumonia, infective endocarditis, and may contribute to cardiovascular disease. The pathogenicity of the organism has been ascribed to virulence determinants including adhesins, invasins and toxin secretion. These factors must translocate two distinct membranes to be functionally active. In A. actinomycetemcomitans, the biogenesis and protein composition of the inner and outer membranes remain largely undefined. We have identified a novel gene morC, encoding a 141 kDa membrane protein with previously unknown function. The null mutant showed changes in the morphology of the outer membrane, membrane permeability and a marked decreased secretion of leukotoxin. The importance of MorC to maintain membrane morphology and function is supported by these findings. The morphogenesis protein (MorC) is highly conserved among gram-negative human pathogens. In Aggregatibacter and related genera, the gene is located in a three-gene operon under the control of a promoter responsive to environmental changes. This operon may be regulated by a two-component signal transduction system. The conservation of this gene and the operon structure among a variety of bacterial species implies an important role of this protein in the biology of these microorganisms. To better understand the role of MorC in membrane biogenesis and pathogenicity, we propose the following aims 1) molecular characterization of the morC operon; 2) determination of the functional domain(s) of MorC; and 3) determination of the role of MorC in leukotoxin secretion and membrane biogenesis. The information obtained from the accomplishment of the stated aims can be applied to the development of therapeutics directly targeted to the MorC protein or synergistically with existing therapies t reduce the pathogenic potential of A. actinomycetemcomitans and other pathogens. Bacteria are the cause of serious, life threatening infections worldwide. Understanding the fundamental properties of the physiology and structure of these pathogens will aid in the development of medicines which interrupt the life cycle of these microorganisms and prevent bacterial infections
Keywords: A. actinomycetemcomitans; Actinobacillus actinomycetemcomitans; Adhesins, Bacterial; Alveolodental Ligament; Alveolodental Membrane; Amino Acid Sequence; Amino Acids; Anaerobic Bacteria; Appearance; Assay; Bacteria; Bacteria, Anaerobic; Bacterial Adhesins; Bacterial Gene Proteins; Bacterial Infections; Bacterial Proteins; Bacterium actinomycetem comitans; Bacterium comitans; Bioassay; Biochemical Genetics; Biogenesis; Biologic Assays; Biological Assay; Biological Function; Biological Process; Biology; Buccal Cavity; Cardiovascular Diseases; Causality; Cavitas Oris; Cell Communication and Signaling; Cell Membrane Permeability; Cell Signaling; Characteristics; Chronic Periodontitis; Communicable Diseases; Complement; Complement Proteins; Data; Defect; Development; Disease; Disorder; Etiology; Family; Gene Products, Bacterial; Gene Proteins; Generalized Growth; Genes; Genes, LacZ; Genetic, Biochemical; Gram-Negative Bacteria; Growth; Habitats; Head and Neck, Buccal Cavity; Health; Human; Human, General; Hydrophobicity; Infection; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Infective endocarditis; Intercistronic Sequence; Intergenic Sequence; Intracellular Communication and Signaling; LacZ; LacZ Genes; Life; Life Cycle; Life Cycle Stages; Man (Taxonomy); Man, Modern; Medicine; Membrane; Membrane Proteins; Membrane-Associated Proteins; Method LOINC Axis 6; Methodology; Microbial Biofilms; Microorganisms, General; Molecular; Molecular Biology, Protein Sequencing; Morphogenesis; Morphology; Mouth; Nitrates; Nitrites; Operon; Oral; Oral cavity; Organism; Organism-Level Process; Organismal Process; Origin of Life; Outcome; Parents; Parodontosis; Pathogenicity; Peptide Sequence Determination; Periodontal Diseases; Periodontal Infection; Periodontal Ligament; Periodontal Membrane; Periodontitis; Permeability; Phase; Phenotype; Physiologic; Physiologic Processes; Physiological; Physiological Processes; Physiology; Pneumonia; Pneumonitis; Promoter; Promoter Regions; Promoter Regions (Genetics); Promoters (Genetics); Promotor; Promotor (Genetics); Promotor Regions; Promotor Regions (Genetics); Property; Property, LOINC Axis 2; Protein Gene Products; Protein Region; Protein Sequencing; Protein Structure, Primary; Proteins; Pulmonary Inflammation; Regulation; Reporter; Role; Science of Medicine; Sequence Alignment; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Sequence Homology; Sequence, Intergenic; Signal Transduction; Signal Transduction Systems; Signaling; Simulate; Sodium Chloride; Sodium chloride (NaCl); Starvation; Stress; Structure; Surface Proteins; System; System, LOINC Axis 4; Systemic disease; Systemic infection; TEM; Taxon; Tissue Growth; Tooth Loss; Toxin; Transmission Electron Microscopy; Two Hybrid; Upper respiratory tract; Virulence; Yeast One Hybrid System; Yeast One/Two-Hybrid System; adhesin; alveolar bone; alveolar bone of maxilla; alveolar process of maxilla; alveolar supporting bone; aminoacid; anaerobe; bacterial disease; base; biofilm; biological adaptation to stress; biological signal transduction; cardiovascular disorder; combat; deletion analysis; disease causation; disease etiology; disease/disorder; disease/disorder etiology; disorder etiology; environmental adaptation; environmental change; experiment; experimental research; experimental study; gene conservation; gene product; genetic promoter element; histidine kinase; homology (molecular); leukotoxin; life course; living system; membrane biogenesis; membrane permeability; membrane structure; microorganism; mutant; new therapeutics; next generation therapeutics; novel; novel therapeutics; ontogeny; oral pathogen; pathogen; periodontal disorder; periodontium disease; periodontium disorder; prevent; preventing; protein protein interaction; protein sequence; protein structure function; protein-histidine kinase; public health relevance; reaction; crisis; research study; response; restoration; salt; social role; socket wall; stress response; stress; reaction; therapeutic development; yeast two hybrid system
Relevance: Bacteria are the cause of serious, life threatening infections world wide. Understanding the fundamental properties of the physiology and structure of these pathogens will aid in the development of medicines which interrupt the life cycle of these microorganisms and prevent bacterial infections
Project start date: 2009-06-10
Project end date: 2014-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: PA-07-070
5R01DE018889-02 (2010): $361313
Molecular Interactions: Oral Bacteria & Matrix Proteins
Keith P Mintz, Research Assistant Professor
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 2R01DE013824-06 from National Institute Of Dental And Craniofacial Research IRG: ODCS
Abstract: Actinobacillus actinomycetemcomitans is a Gram-negative, facultative anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis (LAP) and with cases of adult periodontitis. This pathogen is the causative agent for other serious infections including infectious endocarditis, soft tissue abscesses, pneumonia, and may contribute to cardiovascular disease. The periodontium is believed to be the source for these non-oral diseases, but little is known about the tropism used by A. actinomycetemcomitans to colonize the oral cavity and to infiltrate and disseminate in tissues. Pathogens have developed diverse strategies to be successful in colonization of host tissues. A common theme amongst these pathogens is the ability to initiate infection by adhesion to specific host macromolecules under stringent or hostile conditions. These molecules include proteins secreted by host cells that form the extracellular matrix (ECM). A. actinomycetemcomitans is found in the connective tissue of the periodontium and in close association with collagen fibers in infected tissues. The bacterium also binds to the ECM proteins, collagen, fibronectin and laminin. Using a genetic approach, we have identified the first A. actinomycetemcomitans collagen adhesin, Ema (extracellular matrix protein adhesin) A and multiple genes involved in regulating the expression of ECM protein adhesin activity. EmaA is structurally related to YadA, a multipurpose ECM protein adhesin of the enteropathogenic bacterium Yersinia enterocolitica, and is associated with bacterial cell surface appendages. These EmaA structures are proposed to be fundamental for collagen adhesion. To elucidate the role of EmaA in colonization and pathogenicity of the bacterium, we propose to 1) map the functional domains of EmaA by determining the collagen binding, subcellular localization, and assembly of surface structures in A. actinomycetemcomitans, 2) investigate the surface structures associated with EmaA by transmission electron microscopy and the role of these structures in resistance to the innate immune response of the host, and 3) determine the number of EmaA molecules required for the assembly of structures on the surface of A. actinomycetemcomitans. A long term goal of the proposed research is to identify and characterize bacterial adhesins that are required for the colonization of the oral cavity and non-oral tissues. These adhesins may serve as targets for future drug development involving small molecules or vaccines that disrupt host-pathogen interactions. Lay statement Binding to host tissues is the initial phase of all infectious diseases. Understanding how bacteria interact with host cells or tissue constituents will aid in the development of novel therapeutics to prevent initiation or progression of the disease.
Keywords: adhesin, collagen, oral bacteria, protein, tissue, adhesion, extracellular matrix protein
Project start date: 2000-07-01
Project end date: 2011-06-30
2R01DE013824-06 (2006): $361000
2R01DE013824-04 (2004): $304000
Invasion And Adhesion Of Periopathogens
Keith P Mintz, Research Associate Professor
Microbiol & Molecular Geneticsuniversity Of Vermont & St Agric College
85 South Prospect Street
burlington, Vt 05405
Grant 5R01DE009760-15 from National Institute Of Dental & Craniofacial Research IRG: ZRG1
Keywords: adhesion, cell, microtubule Actinobacillus actinomycetemcomitans, Saccharomyces cerevisiae, antiserum, bacteria, biopsy, cell component, cell line, cytoplasm, cytoskeleton, element, endocarditis, gene, health /scientific organization, human, infection, inflammation, intracellular, larynx, library, literature survey, macromolecule, membrane, mutant, nose, nucleic acid sequence, organelle, pathology, periodontitis, periodontium, play, polymer, protein, quality of life, receptor, role, tissue, trachea, tubulin, yeast
Project start date: 1992-03-01
Project end date: 2008-11-30
5R01DE009760-15 (2007): $359123
5R01DE009760-14 (2006): $369849
BACTERIAL ADHESINS OF PERIOPATHOGENS
Keith P Mintz, Research Assistant Professor
University Of Vermont And St Agric College 85 South Prospect Street Burlington, Vt 05405
Grant 1F32DE005626-01 from National Institute Of Dental And Craniofacial Research IRG: DSR
1F32DE005626-01 (1992): $28600