Structure Of The Multidrug Efflux Protein AcrA
Partho Ghosh, Associate Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R03AI064312-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Increasing numbers of bacterial pathogens pose serious human health threats due to their acquisition of multidrug resistance. A common mechanism of resistance involves extrusion of drugs through multidrug efflux pumps. The Resistance-Nodulation-Cell Division (RND) multidrug efflux system is crucial to antibiotic resistance in numerous Gram-negative bacterial pathogens, including the opportunistic pathogens Pseudomonas aeruginosa and Haemophilus influenzae. RND efflux systems are also encoded by Escherichia coli O157H7, Salmonella enterica, Shigella flexneri, and Yersinia pestis. The most thoroughly characterized RND system is the E. coli AcrAB-TolC system, which promotes efflux of novobiocin, erythromycin, fusidic acid, tetracycline, and chloramphenicol among others. Like all RND systems, AcrAB-TolC consists of an inner membrane protein, AcrB; a periplasmic protein, AcrA, which belongs to the so called family of membrane fusion proteins (MFP), although MFPs do not fuse membranes; and an outer membrane protein, TolC. Significantly, the structures of the inner and outer membrane proteins AcrB and TolC, respectively, are known, but there is no atomic resolution structure of the MFP AcrA to explain its essential and active role in drug efflux. A key next step in pursuing longer-term mechanistic studies on the role of the MFP AcrA and RND systems in general is to determine the structure of AcrA, and thereby fill in the structural picture of the AcrAB-TolC system. Our specific aims are to complete the X-ray crystal structure determination of AcrA, and to use this structural information to identify regions of AcrA that interact with AcrB and TolC through in vitro site-directed, cross-linking experiments. These short-term goals will enable the design of inhibitors of RND efflux pumps, which have the potential of rendering useful many antibiotics that have become clinically ineffective.
Keywords: Escherichia coli, bacterial protein, membrane protein, multidrug resistance, protein structure function, computer simulation, cysteine, model design /development, molecular dynamics, mutant, protein protein interaction, X ray crystallography, crosslink, protein purification
Project start date: 2005-03-01
Project end date: 2007-02-28
5R03AI064312-02 (2006): $68225
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Partho Ghosh
CRYSTAL STRUCTURES OF BACTERIAL PATHOGEN PROTEINS
Partho Ghosh, Associate Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01AI047163-05 from National Institute Of Allergy And Infectious Diseases IRG: BBCB
Abstract: Verbatim from s ) The broad, long-term objectives of this proposal are to understand the structural steps required for bacterial pathogens to invade mammalian host cells. Host cell invasion is a critical step in the life cycle of intracellular pathogens and parasites, which are major causes of human morbidity and mortality. The mechanism of cell invasion is being investigated in the pathogen Listeria monocytogenes, a cause of recent outbreaks of human illness and death. A protein attached to the cell wall of L. monocytogenes, internalin B (67 kD), is solely responsible for triggering the uptake of the bacterium into several nonphagocytic mammalian cell types. Included among these are hepatocytes, which are the major locus of bacterial proliferation in vivo. Internalin B acts by binding to a mammalian receptor, gClq receptor (gClq-R), and activating host phosphoinositide (PI) 3-kinase, leading to induction of phagocytosis. A 25 kD mammalian cell effector domain of internalin B is necessary and sufficient to activate PI-3 kinase, whereas the intact molecule is required for bacterial uptake. How internalin B activates host signaling pathways and causes uptake is not understood structurally. The specific aims of the proposal are to (1) Determine the structure of the 25 kD effector domain of internalin B. Crystals of the 25 kD effector domain that diffract x-rays to 1.5 A resolution have been grown and heavy-atom phasing has been achieved, allowing an interpretable electron density map to be calculated. (2) Determine the structure of intact internalin B. Crystals of intact internalin B that diffract x-rays to 3.15 A resolution have been grown and phasing information is being sought. (3) Co-crystallize and determine the structure of internalin B bound to gCIq-R. For these studies, a number of internalin B constructs are available in milligram quantities, as is gCIq-R in a form that crystallizes. The proposed structures are important to revealing the stereochemical basis by which internalin B induces phagocytosis and causes host cell invasion. This knowledge will be generally applicable to devising strategies to combat L. monocytogenes and other intracellular pathogens.
Keywords: Listeria, bacteria infection mechanism, bacterial protein, protein structure function, phosphatidylinositol 3 kinase, X ray crystallography
Project start date: 2000-05-01
Project end date: 2005-12-31
5R01AI047163-05 (2004): $185527
5R01AI047163-04 (2003): $185740
5R01AI047163-03 (2002): $185943
5R01AI047163-02 (2001): $185989
1R01AI047163-01 (2000): $175430
PROTEIN TRANSLOCATION BY THE TYPE III SECRETION SYSTEM
Partho Ghosh, Professor
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5R01AI061452-05 from National Institute Of Allergy And Infectious Diseases
Abstract: The type III secretion system (TTSS) is essential to virulence in a large group of medically important Gram-negative bacterial pathogens, including Yersinia spp., Salmonella spp., and Escherichia coli O157H7 among others. The TTSS exerts its role in virulence through translocation of bacterial proteins termed effectors into the interior of host cells. Most effectors are crucial to virulence and have deleterious consequences in host cells. Our long-term goal is to understand the steps required for transport of proteins by the TTSS. This proposal focuses on TTSS chaperone proteins. TTSS chaperones form a large and distinctive family of functionally important and conserved proteins. Members of this family have been found to be required for translocation of corresponding effector proteins into host cells, explaining why chaperones are as crucial to virulence as their corresponding effectors. Recent structural and biochemical advances suggest that TTSS chaperones have a conserved mechanism of action in promoting translocation of effectors. However, this process is still not understood in detail. This proposal seeks to build on these recent structural and biochemical advances to determine the mechanism of action of TTSS chaperones. Our specific aims are focused on the well characterized Yersinia effector YopE and its corresponding chaperone SycE. Our first aim is to determine directly whether SycE promotes localized YopE unfolding for transport through the narrow needle-like TTSS apparatus. Our second aim is to map functionally important sites on the surface of the SycE-YopE complex, which are likely to be responsible for conferring association with bacterial components involved in the TTSS process, such as dissociation factors. Our third aim is to identify bacterial components that associate with SycE-YopE and to understand the functional consequence of association. These aims are synergistic, in that structurally altered or functionally important surface regions of SycE-YopE, as identified by the first two aims, respectively, will be understood in the context of interacting bacterial components, as identified by the third aim. The combination of these three aims is likely to enable mechanistic dissection of steps required for TTSS transport of proteins. This multidisciplinary approach, which combines structure, genetics, and biochemistry, is likely to be valuable in the design of antimicrobial strategies aimed at combating the large class of bacterial pathogens that use the TTSS in virulence. A large number of bacterial pathogens use a needle-like apparatus to inject bacterial proteins, which are often toxic, into human cells. Our proposal is aimed at understanding how this injection process occurs, and results from our studies are likely to be valuable in the design of antimicrobial strategies aimed at combating these pathogens
Keywords: ATP phosphohydrolase; ATPase; Adenosine Triphosphatase; Adenosinetriphosphatase; Affinity; Affinity Chromatography; Amino Acids; Animals; Bacterial Gene Proteins; Bacterial Proteins; Binding; Binding (Molecular Function); Biochemical; Biochemistry; Biological; Carrier Proteins; Cell Function; Cell Process; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Chaperone; Chemistry, Biological; Chromatography, Affinity; Complex; Cues; Cytoplasm; DISSEC; Dissection; Dissociation; E coli O157; Escherichia coli O157; Family; Family member; Figs; Figs - dietary; Gene Products, Bacterial; Genes; Genetic Alteration; Genetic Change; Genetic Structures; Genetic defect; Goals; Human; Human, General; Injection of therapeutic agent; Injections; Life; Lymphatic System; Lymphatic system (all sites); Macromolecular Structure; Maintenance; Maintenances; Man (Taxonomy); Man, Modern; Maps; Modeling; Molecular; Molecular Chaperones; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Molecular Structure; Mutagenesis, Site-Directed; Mutate; Mutation; NMR Spectroscopy; Needles; Physiologic; Physiological; Principal Investigator; Process; Programs (PT); Programs [Publication Type]; Proliferating; Protein Trafficking; Protein translocation; Proteins; Radiation, X-Rays; Radiation, X-Rays, Gamma-Rays; Reticuloendothelial System, Lymphatic System; Roentgen Rays; Role; Salmonella; Site; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Spectroscopy, NMR; Structure; Subcellular Process; Surface; System; System, LOINC Axis 4; Targeted DNA Modification; Targeted Modification; Testing; Traffickings, Protein; Transmembrane Protein Transport; Transport Proteins; Transporter Protein; Two Hybrid; Type III Secretion System; Type III Secretion System Pathway; Virulence; X-Radiation; X-Rays; Xrays; Yeast One Hybrid System; Yeast One/Two-Hybrid System; Yersinia; affinity purification; aminoacid; anti-microbial; antimicrobial; combat; conformation; conformational state; design; designing; experiment; experimental research; experimental study; gene product; genome mutation; in vivo; interdisciplinary approach; mutant; nuclear magnetic resonance spectroscopy; pathogen; positional cloning; programs; protein transport; research study; reverse genetics; social role; tool; yeast two hybrid system
Project start date: 2006-05-01
Project end date: 2011-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PA-04-119
5R01AI061452-05 (2010): $414192
5R01AI061452-04 (2009): $419377
5R01AI061452-02 (2007): $359750
1R01AI061452-01A2 (2006): $385115
Host Activation Of The Pseudomonas Aeruginosa Cytoxin ExoU
Partho Ghosh, Associate Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R21AI069342-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: The highly cytotoxic protein ExoU (74 kDa) from the opportunistic bacterial pathogen Pseudomonas aeruginosa is a major contributor to severe and acute infections. ExoU has been shown to be important for virulence in animal models of pneumonia, and expression of ExoU is closely linked with severe disease and mortality in patients. ExoU is delivered through the bacterial type III secretion system directly into the cytosol of host cells, where it exerts its destructive action. Our long-term objectives are to uncover the mechanism of action of type III secretion effectors, in order to devise means to combat infections caused by type III secretion system-utilizing bacterial pathogens. Recent evidence indicates that the swift and profound cytotoxicity caused by ExoU is due to its action as a phospholipase. However, ExoU is found to be inactive prior to entry into eukaryotic cells, and instead requires a eukaryotic host cell factor for activation as a membrane-lytic, cytotoxic phospholipase. The ExoU-activating host cell factor appears to be conserved from yeast to humans, but to be absent in bacteria. Furthermore, this activation mechanism appears generalizable to the ExoU-related protein RP534 expressed by the bacterial pathogen Rickettsia prowazekii, the cause of epidemic typhus, and may be generalizable to ExoU-related proteins of other bacterial pathogens, such as Legionella pneumophila. This R21 proposal is aimed at identifying the host cell factor responsible for activation of ExoU as a cytotoxic phospholipase. If successful, these studies will be the basis for a longer-term, more extensive project focused on mechanistic principles underlying ExoU activation and possible routes to inhibition of this cytotoxin and related proteins from bacterial pathogens. Our specific aims are to (1) biochemically identify and validate the host cell factor responsible for ExoU activation, and to (2) use yeast genetics to identify and characterize host genes involved in ExoU activation and cytotoxicity. The proposed combination of biochemical and genetic approaches provides a powerful means for understanding steps required for activation of ExoU. The work described here will have potential impact in treating acute infections caused by the opportunistic bacterial pathogen Pseudomonas aeruginosa, and may also have impact on treatment of other bacterial infections.
Keywords: Pseudomonas aeruginosa, cell, heat, protein, Legionella, Pseudomonas, Rickettsia prowazekii, bacteria, bacterial protein, base, bias, cytoplasm, cytotoxicity, disease /disorder model, disease outbreak, enzyme, forensic medicine, fungal genetics, gene, genetics, genome, human, identity, infection, lead, magnetism, mass spectrometry, membrane, model, molecular genetics, oligosaccharide, opportunistic infection, phospholipase inhibitor, pneumonia, role, secretion, sectioning, small molecule, typhus, virulence, yeast
Project start date: 2006-04-01
Project end date: 2008-03-31
5R21AI069342-02 (2007): $180636
Sponsored Links Excellgen http://Excellgen.com
1R21AI069342-01 (2006): $224920
Streptococcus M Protein And Antibody Cross-reactivity In Rheumatic Fever
Partho Ghosh, Associate Professor
Chemistry And Biochemistryuniversity Of California San Diego
9500 Gilman Dr, Dept 0934
la Jolla, Ca 920930934
Grant 5R21AI071167-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Rheumatic fever is a severe inflammatory disease that affects the heart, brain, and connective tissues. A substantial body of evidence indicates that rheumatic fever is an autoimmune disease triggered by Streptococcus pyogenes (Group A Strep) infection. While the incidence of rheumatic fever has declined in the US over the last several decades for reasons that are not entirely clear, the potential of rheumatic fever being triggered by streptococcal vaccines has limited the development of such vaccines. A more thorough understanding for the basis of antibody cross-reactivity between streptococcal and self-antigens is required for making progress on streptococcal vaccines. One of the dominant hypotheses for antibody cross-reactivity centers on M proteins, which are major streptococcal antigens and virulence factors. M proteins are predicted to form dimeric ?-helical coiled-coils and hypothesized to elicit cross-reactive antibodies through molecular mimicry of self proteins having similar coiled-coil structures, such as laminin, myosin, and tropomyosin. However, no atomic-resolution structure of any M protein exists to evaluate this hypothesis. Furthermore, protein structure modeling of M proteins is problematic due to the non-canonical nature of their coiled-coil sequences. The major impediment to evaluating the molecular mimicry hypothesis has been difficulty in crystallizing M proteins. We have overcome this problem and have obtained crystals that diffract to ~2.9 resolution. Our first specific aim is to complete the structure determination of a functional, physiologically produced fragment of M1, a highly prevalent M protein genotype associated with rheumatic fever and invasive bacterial infections. A portion of this M1 fragment constitutes part of a promising 26-valent streptococcal vaccine currently in clinical trials. Our second specific aim is to determine the basis for recognition of M1 by cross-reactive antibodies We propose to determine the structure of M1 in complex with a Fab fragment from a cross-reactive antibody. Detailed knowledge of potential molecular mimicry and cross-reactivity in M1 protein will be applicable to considerations of safety and efficacy in streptococcal vaccines. A major problem in developing vaccines to combat infection by Streptococcus pyogenes is the possibility that such vaccines will bring about rheumatic fever, which is an autoimmune condition affecting the heart, brain, and joints. We seek to understand the basis for such autoimmune reactions more thoroughly by studying the structure of M1 protein, a major streptococcal antigen that has been hypothesized to resemble certain human proteins and therefore to elicit autoimmune antibodies. The results from our studies are likely to indicate which features of M1 elicit such antibodies, and this information may be applicable to the design of safe and effective streptococcal vaccines
Keywords: Streptococcus, antibody, protein, rheumatic fever Streptococcus infection, Streptococcus pyogenes, X ray, antigen, autoantibody, autoantigen, binding protein, brain, cell, cell line, cell membrane, clinical trial, conditioning, connective tissue, crystallization, cytotoxicity, emotion, extracellular matrix, family, genotype, hand, health, heart, human, hybridoma, hyperthermia, immunization, immunoglobulin structure, infection, insight, intracellular, joint, laminin, lead, macrophage, model, monoclonal antibody, myosin, neutrophil, protein binding, protein structure, solvent, sound frequency, success, tropomyosin, university, vaccine, vaccine development, virulence
Project start date: 2007-07-01
Project end date: 2009-06-30
5R21AI071167-02 (2008): $182606
1R21AI071167-01A1 (2007): $225100
VARIABLE PROTEINS OF DIVERSITY-GENERATING RETROELEMENTS
Partho Ghosh
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5R01AI072504-04 from National Institute Of Allergy And Infectious Diseases
Abstract: Only three biological examples of massive protein sequence variation are known to exist. The most extensively characterized example occurs in the immune system of jawed vertebrates (i.e., antibodies and T-cell receptors, whose immunoglobulin-fold accommodates ~1014-1016 possible sequences), and the second, less characterized example in the immune system of jawless vertebrates (leucine-rich repeat-fold proteins with ~1014 sequences). The third example was recently discovered in a class of prokaryotic retroelements, termed diversity-generating retroelements (DGR), found in diverse bacterial species, including the human pathogen Bordetella and members of the human periodontal and intestinal microbiota. The central feature of DGRs is production of massive protein sequence variation through a unique adenine-specific, template-based mechanism. The most extensively characterized DGR-encoded variable protein is Mtd (~1013 sequences), which functions as the receptor-binding protein of Bordetella bacteriophage; variation in Mtd enables host tropism switching by the phage. To understand how Mtd accommodates massive sequence variation, we determined the structures of a number of Mtd variants, and characterized the interaction of Mtd with one of its prevalent Bordetella receptors, pertactin. The structures revealed that Mtd uses a C-type lectin fold as a novel scaffold for massive sequence variation, and that the receptor-binding site of Mtd is remarkably stable to sequence variation. Our long-term objective is to understand how DGR-encoded variable proteins accommodate massive sequence variation and bind diverse targets. We specifically aim to use the power of phage genetics combined with detailed insights from biochemistry and structural biology to determine the basis for (1) receptor recognition by Mtd and (2) accommodation of massive sequence variation by other DGR-encoded variable proteins. The justification for these studies comes from the fact that massive protein sequence variation is extremely rare in biology. An understanding of DGR-encoded variable proteins will likely provide basic insight into modes of macromolecular recognition, a fundamental process in all biological systems, as well as place limits on the biological function of DGR-encoded variable proteins. The results from our studies may also have practical applications. Antibodies are nearly unparalleled in the natural work in their ability to vary in sequence and bind almost any target antigen. Recently bacteria were found to encode proteins that vary in sequence at a scale comparable to antibodies. Our project is aimed at providing basic knowledge on how these variable bacterial proteins recognize diverse targets, with the expectation that this knowledge will have practical applications
Keywords: 1H-Purin-6-amine; Adenine; Adopted; Affinity; Amino Acid Sequence; Antibodies; Antigen Targeting; Antigenic Determinants; Bacteria; Bacterial Gene Proteins; Bacterial Proteins; Bacteriophages; Binding; Binding (Molecular Function); Binding Determinants; Binding Proteins; Binding Sites; Bio-Informatics; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Function; Biological Process; Biology; Bordetella; C-Type Lectins; C-terminal; Chemistry, Biological; Combining Site; Crystallization; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Epitopes; Equilibrium; Evolution; Gene Products, Bacterial; Genetic; Human; Human, General; Ig Somatic Hypermutation; Immune Globulins; Immune system; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Immunoglobulins / Antibodies; Infection; Intestinal; Intestines; Investigators; Jaw; Knowledge; LRR; Leucine-Rich Repeat; Ligand Binding Protein; MHC Receptor; Major Histocompatibility Complex Receptor; Man (Taxonomy); Man, Modern; Membrane Proteins; Membrane-Associated Proteins; Methods and Techniques; Methods, Other; Molecular Biology, Protein Sequencing; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Nature; Nostoc; Peptide Sequence Determination; Phage Receptors; Phages; Pressure; Pressure- physical agent; Process; Production; Programs (PT); Programs [Publication Type]; Protein Sequencing; Protein Structure, Primary; Proteins; Radiation, X-Rays; Radiation, X-Rays, Gamma-Rays; Reactive Site; Receptor Protein; Receptors, Antigen, T-Cell; Research Personnel; Researchers; Retroelements; Right-Handed Beta-Alpha Superhelix; Roentgen Rays; Scaffolding Protein; Screening procedure; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Single Crystal Diffraction; Solvents; Somatic Hypermutation, Immunoglobulin; Structure; Surface Proteins; T-Cell Receptor; Techniques; Testing; Tropism; Variant; Variation; Vertebrate Animals; Vertebrates; Vitamin B4; Work; X Ray Crystallographies; X-Radiation; X-Ray Crystallography; X-Rays; Xrays; application in practice; bacterial virus; balance; balance function; base; biological systems; body system, allergic/immunologic; bowel; conformation; conformational state; expectation; experiment; experimental research; experimental study; gene product; insight; member; novel; organ system, allergic/immunologic; pathogen; pertactin; practical application; pressure; programs; protein folding; protein sequence; receptor; receptor binding; research study; scaffold; scaffolding; screening; screenings; somatic hypermutation; structural biology; vertebrata
Project start date: 2007-09-15
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
5R01AI072504-04 (2010): $292431
5R01AI072504-03 (2009): $295754
5R01AI072504-02 (2008): $296105
1R01AI072504-01 (2007): $315805
Structure Of The Multidrug Efflux Protein AcrA
Partho Ghosh, Associate Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 1R03AI064312-01 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Increasing numbers of bacterial pathogens pose serious human health threats due to their acquisition of multidrug resistance. A common mechanism of resistance involves extrusion of drugs through multidrug efflux pumps. The Resistance-Nodulation-Cell Division (RND) multidrug efflux system is crucial to antibiotic resistance in numerous Gram-negative bacterial pathogens, including the opportunistic pathogens Pseudomonas aeruginosa and Haemophilus influenzae. RND efflux systems are also encoded by Escherichia coli O157H7, Salmonella enterica, Shigella flexneri, and Yersinia pestis. The most thoroughly characterized RND system is the E. coli AcrAB-TolC system, which promotes efflux of novobiocin, erythromycin, fusidic acid, tetracycline, and chloramphenicol among others. Like all RND systems, AcrAB-TolC consists of an inner membrane protein, AcrB; a periplasmic protein, AcrA, which belongs to the so called family of membrane fusion proteins (MFP), although MFPs do not fuse membranes; and an outer membrane protein, TolC. Significantly, the structures of the inner and outer membrane proteins AcrB and TolC, respectively, are known, but there is no atomic resolution structure of the MFP AcrA to explain its essential and active role in drug efflux. A key next step in pursuing longer-term mechanistic studies on the role of the MFP AcrA and RND systems in general is to determine the structure of AcrA, and thereby fill in the structural picture of the AcrAB-TolC system. Our specific aims are to complete the X-ray crystal structure determination of AcrA, and to use this structural information to identify regions of AcrA that interact with AcrB and TolC through in vitro site-directed, cross-linking experiments. These short-term goals will enable the design of inhibitors of RND efflux pumps, which have the potential of rendering useful many antibiotics that have become clinically ineffective.
Keywords: Escherichia coli, bacterial protein, membrane protein, multidrug resistance, protein structure function, computer simulation, cysteine, model design /development, molecular dynamics, mutant, protein protein interaction, X ray crystallography, crosslink, protein purification
Project start date: 2005-03-01
Project end date: 2007-02-28
1R03AI064312-01 (2005): $70340
Mechanism Of Host Cell Invasion By Microbial Pathogens
Partho Ghosh, Associate Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01AI047163-07 from National Institute Of Allergy And Infectious Diseases IRG: HIBP
Abstract: A number of bacterial pathogens gain intracellular entry into host cell types that are normally non-phagocytic by inducing phagocytosis. Intracellular invasion is crucial to the virulence of these pathogens, enabling them to evade certain immune defenses and proliferate in relatively protected niches. Our long-term efforts are focused on understanding the steps required of microbial pathogens to convert host cells that are dormant phagocytes into active phagocytes in effecting intracellular invasion. Our studies have focused on InIB, a 67 kDa invasion protein produced by the Gram-positive, facultative intracellular pathogen Listeria monocytogenes, a cause of meningitis, abortion, gastroenteritis, and septicemia in humans. InIB exists in both bacterial surface-attached and soluble, released forms, and is responsible for bacterial invasion of a broad variety of host cell types, including epithelial and endothelial cells. InIB acts by binding and activating the host cell receptor tyrosine kinase Met (also called hepatocyte growth factor receptor, HGFR), thereby inducing signaling pathways that lead to actin-dependent uptake of the bacterium. As with a number of other receptor tyrosine kinases, dysfunction of Met is linked to a plethora of malignancies, such as kidney, breast, liver, and gastric carcinomas. In the last grant period, we determined the X-ray crystal structure of InIB and carried out functional studies that support the hypothesis that InIB, once released from the bacterial surface, induces phagocytosis by acting as a close functional mimic of hepatocyte growth factor (HGF). In this proposal, we seek to understand the steps required for InIB to induce phagocytosis and promote intracellular invasion. Our specific aims are to determine (1) the mode of Met binding and activation by InIB, (2) whether bacterial surface-attachment of InIB is functionally important, and (3) whether InlB-mediated invasion proceeds through localized Met activation at sites of bacterial proximity or contact, or through globally distributed Met activation across the host cell surface. This last aim seeks to understand the cell biological basis by which host cells are made permissive to invasion. Our multidisciplinary approach, which combines biochemistry, genetics, and cell and structural biology, will provide an integrated view of intracellular invasion from molecular to cellular levels. The fundamental knowledge resulting from our studies will be important in guiding antimicrobial strategies directed against intracellular pathogens. We are studying how bacterial pathogens that live within human cells initially gain entry into these cells. An understanding of this process may be applicable to combating a broad variety of intracellular pathogens. In addition, the particular pathogen we are studying, Listeria monocytogenes, affects a protein called Met that is involved in some cancers. Thus, results from our studies may also have an impact on cancer therapies.
Keywords: cell, intracellular, Listeria, X ray, acid, actin, base, behavior, biochemistry, birth, carcinoma, cell biology, cell membrane, cell proliferation, cell type, crystallization, electron microscopy, emotion, environment, fluorescence, fluorescence microscopy, gastroenteritis, gene mutation, genetics, growth factor, growth factor receptor, hand, heparin, hepatocyte growth factor, hormone, human, infection, kidney, lead, ligand, lighting, liver, liver cell, mammary gland, meningitis, model, mucopolysaccharide, mutant, neoplasm /cancer, oligosaccharide, phagocyte, phagocytosis, protein, receptor, role, septicemia, structural biology, therapy, tyrosine, virulence
Project start date: 2000-05-01
Project end date: 2010-12-31
5R01AI047163-07 (2007): $211545
2R01AI047163-06A1 (2006): $218676
Sponsored Links Excellgen http://Excellgen.com
5R01AI047163-10 (2010): $184895
CRYSTAL STRUCTURES OF BACTERIAL PATHOGEN PROTEINS
Partho Ghosh, Associate Professor
Stanford University Stanford, Ca 94305
Grant 2P41RR001209-260547 from National Center For Research Resources IRG: ZRG1
Keywords: bacterial protein, biomedical resource, protein structure, structural biology, Borrelia, Escherichia coli, Listeria, Streptococcus pyogenes, X ray crystallography
Project start date: 2005-05-01
Project end date: 2006-02-28