Manipulation Of Host Innate Immunity By Listeria
Daniel A Portnoy, Professor
University Of California Berkeley
2150 Shattuck Avenue, Room 313
berkeley, Ca 947045940
Grant 5P01AI063302-050004 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: A central problem in microbial pathogenesis is how cells of the immune system integrate multiple signals to induce an appropriate response and how pathogens avoid and/or manipulate the host response. Listeria monocytogenes as a highly tractable model intracellular pathogen with which to approach this problem. Using wild-type and mutant bacteria lacking a secreted pore-forming hemolyisn (LLO), two separate pathways were discovered leading to host gene expression a vacuolar pathway characterized by Toll-like Receptor signaling and a cytosolic pathway characterized by Interferon beta (IFNb) production. IFNb expression was mediated, at least in part, by L. monocyogenes autolysins (p60 and NamA) secreted by the recently discovered SecA2 pathway. It appeas as though L. monocytogenes is manipulating a host system of innate immunity by the production of a specific peptidoglycan (PGN) cleavage product. In Aim I, the macrophage transcriptional response to vacuolar and cytosolic bacteria will be determned using wildtype L. mononcytogenes and LLO-minus mutants in combination with B. subtilis or B. subtilis expressing LLO. Using macrophages isolated from mice lacking the IFNabR, MyD88, or TLR2, a panel of genes representing the vacuolar and cytosolic signaling will be established. In Aim II, the bacterial autolysins affecting macrophage signaling will be determined by introduction of in-frame deletions and characteization of the resulting mutants in mice and macrophages from wild-type and NOD1 and NOD2 knockouts. In Aim III, the contribution of specific PGN fragments will be determined using reverse phase high pressure liquid chromatography and mass spectrometry combined with introduction of purified PGN fragments into cells using liposomes with and without LLO. In Aim IV, activated peritoneal macrophages will be used to test the hypothesis will be tested that the cytosolic pathway actually detects material (PGN) derived from a vacuolar compartment resulting from degradation in a phagosome. In the final aim, the composite transcriptional 3rogram induced by L. monocytogenes will be compared with that induced by M. tuberculosis, F. tularensis and H. capsulatum
Keywords: Listeria, Listeria infection, bacteria infection mechanism, immunity, macrophage, peptidoglycan genetic transcription, hemolysin, interferon beta, toll like receptor high performance liquid chromatography, laboratory mouse, liposome, mass spectrometry
Project start date: 2008-02-01
Project end date: 2009-01-31
Sponsored Links Excellgen http://Excellgen.com
INTRACELLULAR PATHOGENS AND INNATE IMMUNITY
Daniel A Portnoy, Professor
University Of California Berkeley, 2150 Shattuck Avenue, Room 313, Berkeley, Ca 94704-5940
Grant 5P01AI063302-07 from National Institute Of Allergy And Infectious Diseases
Abstract: Portnoy, C/1 Daniel A. 2 P01 AI063302-06 G.) ,-- the project. their commitment to ensure biosafety. The aims represent a reduction in year timeframe. Also note that the Project 5 has been deleted since Russell Vance received R01
Project start date: 2004-09-30
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
5P01AI063302-07 (2010): $2517128
5P01AI063302-05 (2008): $2060386
5P01AI063302-04 (2007): $2049695
5P01AI063302-03 (2006): $2008683
5P01AI063302-02 (2005): $2014030
Manipulation Of Host Innate Immunity By Listeria
Daniel A Portnoy, Professor
University Of California Berkeley 2150 Shattuck Avenue, Room 313 Berkeley, Ca 947045940
Grant 5P01AI063302-040004 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: A central problem in microbial pathogenesis is how cells of the immune system integrate multiple signals to induce an appropriate response and how pathogens avoid and/or manipulate the host response. Listeria monocytogenes as a highly tractable model intracellular pathogen with which to approach this problem. Using wild-type and mutant bacteria lacking a secreted pore-forming hemolyisn (LLO), two separate pathways were discovered leading to host gene expression a vacuolar pathway characterized by Toll-like Receptor signaling and a cytosolic pathway characterized by Interferon beta (IFNb) production. IFNb expression was mediated, at least in part, by L. monocyogenes autolysins (p60 and NamA) secreted by the recently discovered SecA2 pathway. It appeas as though L. monocytogenes is manipulating a host system of innate immunity by the production of a specific peptidoglycan (PGN) cleavage product. In Aim I, the macrophage transcriptional response to vacuolar and cytosolic bacteria will be determned using wildtype L. mononcytogenes and LLO-minus mutants in combination with B. subtilis or B. subtilis expressing LLO. Using macrophages isolated from mice lacking the IFNabR, MyD88, or TLR2, a panel of genes representing the vacuolar and cytosolic signaling will be established. In Aim II, the bacterial autolysins affecting macrophage signaling will be determined by introduction of in-frame deletions and characteization of the resulting mutants in mice and macrophages from wild-type and NOD1 and NOD2 knockouts. In Aim III, the contribution of specific PGN fragments will be determined using reverse phase high pressure liquid chromatography and mass spectrometry combined with introduction of purified PGN fragments into cells using liposomes with and without LLO. In Aim IV, activated peritoneal macrophages will be used to test the hypothesis will be tested that the cytosolic pathway actually detects material (PGN) derived from a vacuolar compartment resulting from degradation in a phagosome. In the final aim, the composite transcriptional 3rogram induced by L. monocytogenes will be compared with that induced by M. tuberculosis, F. tularensis and H. capsulatum.
Keywords: Listeria, Listeria infection, bacteria infection mechanism, immunity, macrophage, peptidoglycan, genetic transcription, hemolysin, interferon beta, toll like receptor, high performance liquid chromatography, laboratory mouse, liposome, mass spectrometry
Grants awarded to Daniel A Portnoy
Daniel A Portnoy, Professor
University Of California Berkeley, 2150 Shattuck Avenue, Room 313, Berkeley, Ca 94704-5940
Abstract: Seeinstructions) The purpose of Core A is to ensure scientific progress by providing adequate scientific and administrative leadership to the Program and to supply adequate financial oversight. This will be accomplished by extensive review of scientific progress through monthly meetings of all the lab groups and smaller meetings held by subgroups. Scientific direction and research data will be subject to intense, yet constructive, criticism by investigators within the P01 and by an outside advisory board. The Specific Aims are I. Ensure Scientific Progress; II. Implement Financial Management and Administrative Support. To facilitate interactions among investigators, 2h joint lab meetings will be held monthly at the UCSF Mission Bay Campus. The monthly meetings consist of introductory discussions, two scientific presentations, and post-meeting discussions. A scientific advisory board will be established to oversee research progress and to offer objective criticism of the program progress. The board will meet once every other and provide a report. To facilitate interactions between cores and projects, each of the labs will designate one person as representative to Core B (transcriptional profiling core) and another to Core A (animal core). A website (Meebo Wiki) will help coordinate sharing of data, protocols, and PowerPoint presentations from the monthly meetings. Additional mechanisms to promote interactions include joint interviews of potential post-doctoral fellows; serving on each others doctoral student´s thesis committees, and a joint seminar program between UCB and UCSF. Core A will provide the financial oversight for the Program Project and for each research plan and core. DC Berkeley will act as the central administrator of the Program´s financial activities through the campus´s Sponsored Projects Office. The Administrative Assistant will facilitate communication via the appropriate contacts at UCSF and Stanford such that UC Berkeley can monitor and expedite financial arrangements and also attend to any problems. Principal Investigators will receive monthly reports of their financial activities and therefore will have regular opportunities to resolve questions and assess the planning of purchases for their research. RELEVANCE (See instructions) The proposed studies will lead to the characterization of a host system of innate immunity that will lead to vaccines and/or therapeutics to treat disease, with relevance to biodefense, emerging infections and global health
Keywords: Administrator; Animals; Articulation; Communication; Data; Disease; Disorder; Ensure; Financial Activity; Financial Management; Health; Immunity, Innate; Immunity, Native; Immunity, Natural; Immunity, Non-Specific; Infection; Instruction; Interview; Investigators; Joints; Lead; Leadership; Mission; Monitor; Natural Immunity; Occupational activity of managing finances; Pb element; Persons; Postdoc; Postdoctoral Fellow; Principal Investigator; Programs (PT); Programs [Publication Type]; Protocol; Protocols documentation; Reporting; Research; Research Associate; Research Personnel; Researchers; SUBGP; Students; Subgroup; System; System, LOINC Axis 4; Therapeutic; Vaccines; biodefense; disease/disorder; heavy metal Pb; heavy metal lead; meetings; pathogen; post-doc; post-doctoral; programs; sharing data; web site; wiki
Project start date: 2009-06-05
Project end date: 2011-05-31
Budget start date: 5-JUN-2009
Budget end date: 31-MAY-2010
2P01AI063302-06_8625 (2009): $133152
INTRACELLULAR PATHOGENS AND INNATE IMMUNITY
Daniel A Portnoy, Professor
University Of California Berkeley, 2150 Shattuck Avenue, Room 313, Berkeley, Ca 94704-5940
Grant 2P01AI063302-06 from National Institute Of Allergy And Infectious Diseases
Abstract: Portnoy, C/1 Daniel A. 2 P01 AI063302-06 G.) ,-- the project. their commitment to ensure biosafety. The aims represent a reduction in year timeframe. Also note that the Project 5 has been deleted since Russell Vance received R01
Project start date: 2004-09-30
Project end date: 2011-05-31
Budget start date: 5-JUN-2009
Budget end date: 31-MAY-2010
2P01AI063302-06 (2009): $2567353
3P01AI063302-03S1 (2006): $57580
1P01AI063302-01 (2004): $700494
MOLECULAR BIOLOGY OF LISTERIA-HOST CELL INTERACTION
Daniel A Portnoy, Professor
Microbiologyuniversity Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104
Grant 5R01AI029619-05 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Listeria monocytogenes is a model facultative intracellular pathogen which has been widely used for the study of cell-mediated immunity. In preliminary studies on the growth of this bacterium in tissue culture cells, the sequence of events involved with intracellular growth and cell to cell spread have been defined. These studies have provided a cell- biological explanation for the absolute requirement for cell-mediated immunity, i.e. the bacteria never leave the host cytoplasm yet are able to spread cell to cell. The goal of the proposed research is to define, in molecular terms, bacterial determinants required for intracellular growth and cell to cell spread. The strategy is to isolate transposon Tn916 and Tn917-lac insertion mutations in L. monocytogenes which are either defective or altered in intracellular growth and/or cell to cell spread. Novel delivery systems of Tn917-lac will be exploited which will assist in the isolation and characterization of mutations. Three different selection schemes will be utilized to isolate mutants 1) A novel technique called intracellular methicillin selection of mon-growing mutants; 2) isolation of small-plaque mutants; and 3) isolation of mutants which fail to plaque. The mutants will be characterized with respect to virulence in mice, cell biological defect, and/or nutritional defect. Genes encoding essential determinants of pathogenicity will be cloned and their nucleotide sequence will be determined. These genes will be cloned into an E. coli-L. monocytogenes shuttle plasmid and reintroduced into L. monocytogenes. Expression of essential determinants of pathogenesis will be studied inside cells by monitoring Beta-galactosidase expression of Tn917-lac insertion mutants. This research represents a model system to study the molecular biology of intracellular parasitism and should provide insight into the molecular requirement to induce cell-mediated immunity
Keywords: Listeria, bacterial genetics, host organism interaction, microorganism growth RNA splicing, beta galactosidase, intracellular parasitism, mutant, virulence Escherichia coli, genetic manipulation, laboratory mouse, methicillin, molecular cloning, nucleic acid sequence
Project start date: 1990-04-01
Project end date: 1995-03-31
5R01AI029619-05 (1994): $178811
5R01AI029619-04 (1993): $177735
5R01AI029619-03 (1992): $173868
Listeria Hemolysis And Escape From A Vacuole
Daniel A Portnoy, Professor
University Of California Berkeley 2150 Shattuck Avenue, Room 313 Berkeley, Ca 947045940
Grant 5R01AI027655-20 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Listeria monocytogenes is a highly tractable intracellular pathogen and a cause of serious food-borne illness in humans. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO mediates escape of L.monocytogenes from a phagocytic vacuole and is absolutely essential for virulence. LLO has a number of unique properties that prevent its activity in the host cytosol including an acidic pH optimum and a PEST-like sequence. The goals of the current proposal are to identify both bacterial and host components that control LLO compartmentalization, and to determine why mutants that fail to properly compartmentalize LLO activity are avirulent. In Aim I, two genetic selection screens are proposed to identify bacterial proteins that control LLO secretion in a vacuole, and two other screens are proposed to identify bacterial proteins that prevent LLO secretion in the cytosol. The intracellular phenotypes of the mutants will be characterized in a variety of tissue culture assays, and secretion defects examined biochemically. The role of two recently identified autolysins whose synthesis or activation appears to be vacuole-specific will be examined. In Aim II, it will be determined why cytotoxic mutants are less virulent. One hypothesis that will be directly tested is that cytotoxic mutants become extracellular and are targeted by neutrophils. The role of neutrophils and neutrophil chemotaxis will be addressed in neutropenic and knockout mice. In Aim III, a functional genomics approach will be used to identify host proteins that control escape of L.monocytogenes from a vacuole and that prevent LLO toxicity in the host cytosol. The host cells to be used in these studies are a phagocytic Drosophila cell line that is extremely sensitive to double-stranded RNA interference (RNAi). RNAi specific for 7800 Drosophila genes will be produced and used to screen, microscopically, for host genes that encode proteins controlling LLO compartmentalization.
Keywords: DNA damage, Listeria, Listeria infection, bacterial genetics, cell cycle, cytogenetics, hemolysin, host organism interaction, pore forming protein, vesicle /vacuole, antigen presentation, biological signal transduction, cell autolysis, cell growth regulation, chemotaxis, neutrophil, phosphorylation, virulence, RNA interference, flow cytometry, genetic mapping, genetic screening, genetically modified animal, laboratory mouse, polymerase chain reaction, tissue /cell culture
Project start date: 1988-06-15
Project end date: 2008-05-31
5R01AI027655-20 (2007): $353188
5R01AI027655-19 (2006): $364387
5R01AI027655-18 (2005): $373764
Sponsored Links Excellgen http://Excellgen.com
5R01AI027655-17 (2004): $374317
2R01AI027655-16 (2003): $374721
LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE
Daniel A Portnoy, Professor
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01AI027655-08 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Listeria monocytogenes is a gram-positive, rapidly growing food-borne human pathogen which has been used extensively as a model facultative intracellular pathogen both in mice and tissue culture cells. The cell biology of intracellular growth can be divided into two broad stages; life in a vacuole and life in the host cytoplasm. The overall goal of the proposed research is to define the specific roles and regulation of gene-products which are required for the successful passage of L. monocytogenes through the vacuolar environment. The roles of four gene-products will be specifically evaluated; 1) Listeriolysin O (LLO), encoded by hly, a pore-forming hemolysin previously shown to be an essential determinant of pathogenicity necessary for lysis of the host vacuole; 2) PI-PLC, encoded by plcA, a phosphatidylinositol specific phospholipase C; 3) PC-PLC, encoded by plcB, a broad spectrum phospholipase C which hydrolyzes phosphatidylcholine; and 4) PrfA, encoded by prfA, a positive transcriptional activator of hly, plcA, and plcB. In Aim I, two hemolysins related to LLO (streptolysin O and perfringolysin O) will be evaluated to determine their capacity to complement a hlyTn9l7 mutation. Characterization of these strains in animals and in tissue culture should provide insight into the specialized features of LLO which allow it to lyse the vacuole without damaging the host cell. In Aim II, the roles of PI-PLC and PC-PLC will be fully evaluated. In each case, in-frame deletion mutations will be introduced into the L. monocytogenes chromosome by homologous recombination and allelic exchange. Strains deleted for one or both genes will be constructed and evaluated by quantitative electron microscopy for their ability to lyse the host vacuole in both primary macrophages and cell lines. In Aim III, the role of the two prfA promoters in mediating vacuolar gene expression will be explored by constructing mutants lacking either or both promoters. hly transcription will be monitored using a hlyTn9l7-lac gene fusion. In Aim IV, other genes required for the escape of L. monocytogenes from a vacuole will be isolated using intracellular methicillin selection.
Keywords: Listeria, Listeria infection, hemolysin, host organism interaction, microorganism growth, vesicle /vacuole, Bacillus subtilis, fusion gene, gene mutation, genetic promoter element, genetic transcription, intracellular parasitism, macrophage, nucleic acid sequence, phospholipase C, transcription factor, virulence, electron microscopy, genetic strain, laboratory mouse, methicillin, molecular cloning, tissue /cell culture
Project start date: 1988-06-01
Project end date: 1998-05-31
5R01AI027655-08 (1995): $204685
5R01AI027655-07 (1994): $199941
2R01AI027655-06 (1993): $200442
7R01AI027655-10 (1997): $202399
ACTA AND THE CELL TO CELL SPREAD OF LISTERIA
Daniel A Portnoy, Professor
University Of California Berkeley 2150 Shattuck Avenue, Room 313 Berkeley, Ca 947045940
Grant 5R37AI029619-11 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Listeria monocytogenes is a facultative intracytoplasmic pathogen which primarily infects pregnant women and immunocompromised individuals, including AIDS patients, and is a leading cause of death from food-borne illness. Murine listeriosis is a premier model for the study of cell- mediated immunity (CMI) in which antibody plays no role in protection. Previous studies have provided a molecular and cell biological explanation for the requirement for CMI, as intracytoplasmic bacteria are able to exploit a host system of actin-based motility to move from cell-to-cell without exposure to the humoral immune system. The primary bacterial determinant which governs cell-to-cell spread is ActA, a surface protein which mediates actin polymerization and profilin binding. ActA is a 70- kDa protein which contains four proline-rich repeats with the consensus sequence DFPPPPTDEEL. In Aim I, the role of the proline-rich repeats for ActA function will be characterized by successive deletion of individual repeats and by mutagenesis of residues within the repeats. Mutations will be constructed using PCR-based methods and introduced into the L. monocytogenes chromosome by allelic exchange. The mutants will be fully characterized for actin-based motility, profilin binding, actin nucleation, cell-to-cell spread, and virulence in mice. These studies will establish the role of the repeats and the importance of the prolines and charged residues within the repeat. ActA is phosphorylated inside of mammalian cells resulting in a dramatic shift in apparent molecular mass upon SDS-PAGE. In Aim II, the sites of phosphorylation will be identified by mutagenesis of serine and threonine residues, and the effect of these mutations will be fully evaluated in tissue culture models of infection. ActA will be phosphorylated in vitro using purified kinases in an attempt to reproduce its intracellular properties. In Aim III, the precise biochemical activity of Act A will be determined. First, a secreted form of ActA lacking its transmembrane domain will be purified to homogeneity using conventional methods of protein purification. Purified ActA and mutated derivatives, either soluble or coupled to sepharose, will be evaluated for actin nucleation and profilin binding. If ActA alone has no detectable activity, it will be activated by incubation in Xenopus egg and HeLa cell extracts. The component(s) of the extracts necessary for ActA activation will be identified and characterized. The proposed studies should further our understanding of an essential determinant of bacterial pathogenesis and basic mechanisms of host cell actin-based motility.
Keywords: Listeria, actin, cell motility, host organism interaction, polymerization, actin binding protein, bacterial genetics, beta galactosidase, intracellular parasitism, microorganism growth, mutant, virulence, SDS polyacrylamide gel electrophoresis, Xenopus oocyte, laboratory mouse, molecular cloning, nucleic acid sequence, polymerase chain reaction
Project start date: 1990-04-01
Project end date: 2000-06-30
5R37AI029619-11 (1999): $296780
5R37AI029619-10 (1998): $287949
5R37AI029619-08 (1997): $303937
LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE
Daniel A Portnoy, Professor
University Of California Berkeley, 2150 Shattuck Avenue, Room 313, Berkeley, Ca 94704-5940
Grant 5R01AI027655-23 from National Institute Of Allergy And Infectious Diseases
Abstract: Listeria monocytogenes is a facultative intracellular pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. Importantly, L. monocytogenes is also a clinically relevant food-borne pathogen that causes a high rate of mortality in pregnant women and the immunocompromised. A primary determinant of L. monocytogenes pathogenesis and a target of the host´s immune response is Listeriolysin O (LLO). LLO is a member of a large family of pore-forming cytolysins that is largely responsible for mediating escape of L. monocytogenes from a phagosome and for virulence; LLO-minus mutants are 5-logs less virulent in animal models of infection. LLO activity is a double-edged sword as its activity must be restricted to an acidic phagosome or the host cell will die due to LLO-mediated cell death (referred to as compartmentalization). In Aim I of this proposal the molecular determinants mediating compartmentalization will be determined by using a combination of mutagenesis, cell biology and biochemistry. LLO mutants will be characterized with respect to phosphorylation, ubiquitylation, proteolysis, half-life, and aggregation. In Aim II, the role played by autophagy will be examined with respect to escape from a phagosome and the fate of LLO secreted into the host cytosol. Macrophages that are defective for autophagy will provide an excellent system with which to examine these questions. In Aim III, a newly developed, mariner-based, transposon mutagenesis system will be used to identify the role played by gene-products, other than LLO, on LLO expression, synthesis, secretion and toxicity. These studies should identify the hypothetical host protein(s) that interact with LLO and LLO mRNA to prevent toxicity, and open up new areas of investigations pertaining to LLO expression, synthesis, secretion and toxicity. Lastly, the role of post-translational modifications and autophagy will be examined with respect to the presentation of LLO epitopes in both the MHC Class I and Class II pathways of antigen processing and presentation. Novel in vivo assays will be used to select LLO mutants that are not recognized by the host´s acquired immunity, thereby providing basic information on properties of foreign proteins that leads to immunogenicity. Diseases caused by intracellular pathogens, for example, tuberculosis, AIDS and Malaria, remain one of the largest challenges facing the international biomedical community. The proposed studies on Listeria monocytogenes will provide insight into the molecular biology, cell biology and immunology relevant to the treatment and prevention of diseases caused by intracellular pathogens
Keywords: 20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; AIDS; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Affect; Animal Model; Animal Models and Related Studies; Antigen Presentation Pathway; Antigen Processing and Presentation; Antigenic Determinants; Area; Assay; Autophagocytosis; Binding Determinants; Bioassay; Biochemical; Biochemistry; Biologic Assays; Biological Assay; CD8; CD8B; CD8B1; CD8B1 gene; Cancers; Cell Death; Cell membrane; Cells; Cellular biology; Chemistry, Biological; Communities; Cytolysins; Cytolysis; Cytoplasmic Membrane; Cytosol; DNA Molecular Biology; Data; Detection; Disease; Disorder; Epitopes; Family; Generalized Growth; Genes, Class I; Genes, MHC Class I; Genetics-Mutagenesis; Growth; Half-Life; Half-Lifes; Hemalysins; Hemolysin; Immune response; Immune system; Immunocompromised; Immunocompromised Host; Immunocompromised Patient; Immunologic Deficiency Syndrome, Acquired; Immunology; Immunology (Including BRMP); Immunology (NCI Program); Immunosuppressed Host; Infection; International; Investigation; L. monocytogenes; LYT3; Lead; Light; Listeria; Listeria monocytogenes; Listeria monocytogenes hlyA protein; Listeriolysin; Lysis; MHC Class I; MHC Class I Genes; Macropain; Macroxyproteinase; Malaria; Malignant Neoplasms; Malignant Tumor; Mediating; Messenger RNA; Modeling; Molecular; Molecular Biology; Molecular Biology, Mutagenesis; Mortality; Mortality Vital Statistics; Multicatalytic Proteinase; Mutagenesis; Paludism; Pathogenesis; Pathway interactions; Pb element; Phagosomes; Phosphorylation; Photoradiation; Plasma Membrane; Plasmodium Infections; Play; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Pregnant Women; Process; Property; Property, LOINC Axis 2; Prosome; Proteasome; Proteasome Endopeptidase Complex; Protein Cleavage; Protein Modification; Protein Modification, Post-Translational; Protein Phosphorylation; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Proteolysis; Proteosome; RNA, Messenger; Role; System; System, LOINC Axis 4; T-Cells; T-Lymphocyte; Testing; Thymus-Dependent Lymphocytes; Tissue Growth; Toxic effect; Toxicities; Toxin; Tuberculosis; Vacuole; Virulence; Virulent; acquired immunity; autophagy; base; body system, allergic/immunologic; cell biology; cell mediated immune response; clinical relevance; clinically relevant; cytolysin; disease prevention; disease/disorder; disorder prevention; disseminated TB; disseminated tuberculosis; food born pathogen; food borne pathogen; foodborn pathogen; foodborne pathogen; gene product; heavy metal Pb; heavy metal lead; hlyA protein, Listeria monocytogenes; host response; immunogenicity; immunoresponse; immunosuppressed patient; in vivo; insight; lisA protein, Listeria monocytogenes; listeriolysin O; lymphocyte pore-forming protein; mRNA; macrophage; malignancy; member; microbial; model organism; multicatalytic endopeptidase complex; mutant; necrocytosis; neoplasm/cancer; novel; ontogeny; organ system, allergic/immunologic; pathogen; pathway; perforin; plasmalemma; prevent; preventing; public health relevance; social role; thymus derived lymphocyte; tuberculous spondyloarthropathy; vaccine development
Project start date: 1988-06-15
Project end date: 2013-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-070
5R01AI027655-23 (2010): $431791
Sponsored Links Excellgen http://Excellgen.com
5R01AI027655-22 (2009): $423451
MANIPULATION OF HOST INNATE IMMUNITY BY LISTERIA
Daniel A Portnoy, Professor
University Of California Berkeley, 2150 Shattuck Avenue, Room 313, Berkeley, Ca 94704-5940
Abstract: Listeria monoctyogenes is a facultative intracellular human pathogen and a model organism with which to explore basic mechanisms of pathogenesis and host defense. This research proposed in this application relies heavily on the use of bacterial and host mutants to examine a macrophage cytosolic surveillance pathway of innate immunity that detects intracellular pathogens. Activation of the cytosolic response leads to the expression of IFN-p and co-regulated genes. In Aim I,forward genetic screens will be used to identify L monocytogenes molecular determinants that contribute to stimulating the host cytosolic host pathway of innate immune recognition. The screen has been validated by the identification and characterization of a bacterial multidrug resistance transporter (MDR) required to activate the host cytosolic pathway. In Aim II, the genetic results in Aim I will guide studies to identify the bacterial ligand(s) and cognate host receptor(s). The precise identity of the ligands will be determined using analytical tools and take advantage of a pair of strains that vary 60-fold in their induction of the host cytosolic surveillance pathway. In Aim III,the relevance of the bacterial determinants that govern the host innate immune response will be evaluated by examining mutants in four animal models of infection including the systemic murine listeriosis model in naive and immunized mice, a pregnant Guinea Pig model that monitors trafficking to the placenta, a newly developed oral model of listeriosis in immunocompromised mice that leads to infection of the brain, and finally, the capacity to induce immunity to L. monocytogenes and foreign antigens. The last model has relevance to vaccine development. If successful, these studies will lead to the discovery of a novel microbial molecule that stimulates a host pathway that contributes to the development of innate and acquired immunity. RELEVANCE (See instructions) The proposed studies on Listeria monocytogenes will provide insight into the pathogenic mechanisms of a lethal food-borne pathogen that causes infection of the brain and miscarriages. Further, these studies will lead to the characterization of a host system of innate immunity that will lead to vaccines and/or therapeutics to treat disease, with relevance to biodefense, emerging infections and global health. PROJECT/PERFORMANCE S1TE(
Keywords: ATGN; Affect; Animal Model; Animal Models and Related Studies; Antigens; Assay; Bioassay; Biologic Assays; Biological; Biological Assay; Brain; Cavia; Cell Communication and Signaling; Cell Signaling; Cells; Cytosol; DISSEC; Data; Development; Disease; Disorder; Dissection; Drug Resistance, Multiple; Drug Resistant, Multiple; Embryonic Tissue, Placenta; Encephalon; Encephalons; Family; Genes; Genetic; Genetic Screening; Guinea Pigs; Health; Host Defense; Human; Human, General; Immune; Immune response; Immune system; Immunity; Immunity, Innate; Immunity, Native; Immunity, Natural; Immunity, Non-Specific; Immunocompromised; Immunocompromised Host; Immunocompromised Patient; Immunosuppressed Host; Infection; Infectious Diseases / Laboratory; Infectious Diseases Research; Instruction; Intracellular Communication and Signaling; Investigation; Knockout Mice; L. monocytogenes; Laboratories; Lead; Ligands; Listeria; Listeria Infections; Listeria monocytogenes; Listeriosis; Mammals, Guinea Pigs; Mammals, Mice; Man (Taxonomy); Man, Modern; Mass Spectrum; Mass Spectrum Analysis; Metabolic; Mice; Mice, Knock-out; Mice, Knockout; Miscarriage; Modeling; Molecular; Monitor; Multi-Drug Resistance; Multidrug Resistance; Murine; Mus; Natural Immunity; Nervous System, Brain; Null Mouse; Oral; Pathogenesis; Pathway interactions; Pb element; Performance; Photometry/Spectrum Analysis, Mass; Placenta; Placenta-Tissue, Cells; Placentoma, Normal; Placentome; Publishing; Receptor Protein; Research; Resistance to Multi-drug; Resistance to Multidrug; Resistance to Multiple Drug; Resistant to Multiple Drug; Resistant to multi-drug; Resistant to multidrug; Resolution; Role; Signal Transduction; Signal Transduction Systems; Signaling; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Spontaneous abortion; System; System, LOINC Axis 4; Therapeutic; Vaccines; acquired immunity; analytical tool; biodefense; biological signal transduction; body system, allergic/immunologic; cytokine; disease/disorder; food born pathogen; food borne pathogen; foodborn pathogen; foodborne pathogen; heavy metal Pb; heavy metal lead; host response; immunogen; immunoresponse; immunosuppressed patient; insight; macrophage; microbial; model organism; mouse model; multi-drug resistant; multidrug resistant; mutant; novel; organ system, allergic/immunologic; pathogen; pathway; pregnant; radiolabel; radiotracer; receptor; response; social role; tissue culture; trafficking; vaccine development
Project start date: 2009-06-05
Project end date: 2011-05-31
Budget start date: 5-JUN-2009
Budget end date: 31-MAY-2010
2P01AI063302-06_8623 (2009): $407852
LISTERIA HEMOLYSIN AND INTRACELLULAR GROWTH
Daniel A Portnoy, Professor
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R29AI027655-05 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Intracellular pathogens are responsible for an extensive amount of morbidity and mortality world-wide. Our current understanding of host reponse to intracellular pathogens stems mainly from extensive analysis of murine cell-mediated immunity to the facultative intracellular bacterial pathogen Listeria monocytogenes. Athough the immune response to L. monocytogenes has received enormous attention surprising little research has been devoted to understanding the cell biology of intracellular growth or to bacteria determinants of pathogenicity. The overall goal of the proposed research is to define in molecular terms listerial determinants required for intracellular growth. One likely determinant of L. monocytogenes pathogenesis is the elaboration of a sulfhydryl-activated hemolysin. In preliminary studies, conjugative transposons were used to isolate non- hemolytic mutants. The mutants fail to grow in the mouse macrophage like cell line J774. In the proposed study, the mutants will be evaluated with respect to growth in vivo as well as intracellularly, in vitro. Preliminary data support that L. monocytogenes grows freely in the eucaryotic cell cytoplasm. Electron microscopy of thin-sectioned infected cells will be used to evaluated the role of hemolysin for intracellular localization. As an initial step in structure-function analysis of the hemolysin, the hemolysin gene will be cloned from L. monocytogenes cosmid DNA libraries in E. coli. The primary amino acid sequnce will be deduced from the nucleotide sequence and compared with other pore-forming proteins. The cloned gene will also be used as a hybridization probe to examine its conservation among Listeria species. The hemolysin gene will be cloned into a streptococcal shuttle vector, transformed into a streptococcal strain and conjugated to a non-hemolytic strain of L. monocytogenes. This methodology will facilitate future work in which in vitro constructed hemolysin mutations can be studied in their normal background. Genes other than hemolysin are likely to contribute to listerial infectivity. An attempt will be made to isolate tansposon insertions in genes other than hemolysin which are required for intracellular multiplication. Mutants will be selected inside tissue culture cells in the presence of ampicillin, an antibiotic that kills only growing bacteria.
Keywords: Listeria, Listeria infection, bacterial antigen, hemolysin, intracellular parasitism, microorganism growth, virulence, bacterial genetics, fibroblast, host organism interaction, macrophage, microorganism genetics, molecular cloning, mutant, nucleic acid probe, nucleic acid sequence, population genetics, protein sequence, transposon /insertion element, Escherichia coli, Streptococcus enterococcus group, ampicillin, electron microscopy, genetic library, genetic manipulation, laboratory mouse, plasmid, streptolysin, tissue /cell culture
Project start date: 1988-06-01
Project end date: 1993-05-31
5R29AI027655-05 (1992): $118840
LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE
Daniel A Portnoy, Professor
University Of California Berkeley 2150 Shattuck Avenue, Room 313 Berkeley, Ca 947045940
Grant 5R01AI027655-15 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Adapted from s ) Listeria monocytogenes is a model facultative intracellular pathogen which primarily infects pregnant women and immunocompromised individuals. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO is largely responsible for rupture of the host vacuole which results from phagocytosis. Perfringolysin O (PFO) is a related pore-forming protein which is involved in the pathogenesis of infections by an extracellular pathogen. When expressed by L. monocytogenes, PFO mediates escape from the phagocytic vacuole, but is toxic to the cell. Normally, LLO is continually expressed during infection, but in contrast to PFO, it is proteolytically degraded in the cytosol and presented on the cell surface in association with MHC class I molecules. It is hypothesized that pH optimum and cytosolic processing are essential LLO-encoded determinants which distinguish it from PFO. The focus of the current proposal is to define the precise structural and mechanistic features of LLO which differentiate it from other members of the family of thiol-activated cytolysins, facilitate its intravacuolar activity, and direct its fate in the cytosol. In Aim I, protein sequences responsible for LLO s acidic pH optimum and processing in the host cytosol will be identified. This will be accomplished by domain and sub-domain swapping between LLO and PFO, and modified charged-to-alanine scanning mutagenesis. The LLO/PFO chimeras will be purified from E. coli and characterized biochemically. Next, the chimeras will be introduced into L. monocytogenes and characterized in tissue culture models of infection., In Aim II, the pathway of LLO processing in the host cytosol will be fully evaluated. LLO will be identified by metabolic labeling of L. monocytogenes within infected host cells, followed by immunoprecipitation. Precursor/product relationships will be determined by pulse-chase experiments. Specific inhibitors will be used to evaluate the role of the proteosome and other proteases in degradation. The role of LLO phosphorylation will be evaluated biochemically and genetically. In Aim III, the precise nature of the L. monocytogenes phagosome will be characterized with regard to pH, time of perforation, and markers of the endosome/lysosome pathway of maturation. The role of pH optimum and the L. monocytogenes phospholipases will be evaluated by using mutant and chimeric strains. In Aim IV, the investigators will evaluate the feasibility of using E. coli K12 expressing LLO and a recombinant protein as a novel system to introduce foreign proteins into the mammalian cytosol for antigen presentation.
Keywords: Listeria, Listeria infection, hemolysin, host organism interaction, pore forming protein, vesicle /vacuole, Escherichia coli, acid base balance, antigen presentation, intracellular parasitism, macrophage, phagocytosis, posttranslational modification, protein sequence, recombinant protein, virulence, molecular cloning, plasmid, site directed mutagenesis, tissue /cell culture, transfection /expression vector
Project start date: 1988-06-15
Project end date: 2003-05-31
5R01AI027655-15 (2002): $313352
5R01AI027655-14 (2001): $304268
5R01AI027655-13 (2000): $295446
5R01AI027655-12 (1999): $253985
2R01AI027655-11 (1998): $245381
ACTA AND THE CELL TO CELL SPREAD OF LISTERIA
Daniel A Portnoy, Professor
University Of California Berkeley 2150 Shattuck Avenue, Room 313 Berkeley, Ca 947045940
Grant 5R37AI029619-16 from National Institute Of Allergy And Infectious Diseases IRG: NSS
Keywords: Listeria, actin, cell motility, host organism interaction, polymerization, actin binding protein, bacterial genetics, beta galactosidase, intracellular parasitism, microorganism growth, mutant, virulence, SDS polyacrylamide gel electrophoresis, Xenopus oocyte, laboratory mouse, molecular cloning, nucleic acid sequence, polymerase chain reaction
Project start date: 1990-04-01
Project end date: 2005-06-30
5R37AI029619-16 (2004): $388684
5R37AI029619-15 (2003): $378371
Sponsored Links Excellgen http://Excellgen.com
5R37AI029619-14 (2002): $368357
5R37AI029619-13 (2001): $358631
4R37AI029619-12 (2000): $348242
LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE
Daniel A Portnoy, Professor
Microbiologyuniversity Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104
Grant 5R01AI027655-09 from National Institute Of Allergy And Infectious Diseases IRG: BM
Project start date: 1988-06-01
Project end date: 1997-05-31
5R01AI027655-09 (1996): $215269
ACTA AND THE CELL TO CELL SPREAD OF LISTERIA
Daniel A Portnoy, Professor
Microbiologyuniversity Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104
Grant 5R37AI029619-07 from National Institute Of Allergy And Infectious Diseases IRG: BM
Project start date: 1990-04-01
Project end date: 2000-03-31
5R37AI029619-07 (1996): $296592