Michael Stephen Glickman
Sloan-kettering Institute For Cancer Res
Project start date: 2002-12-01
Project end date: 2013-06-30
Sponsored Links Excellgen http://Excellgen.com
CYCLOPROPANE SYNTHETASES AND M.TUBERCULOSIS PATHOGENESIS
Michael Stephen Glickman, Associate Member
Sloan-kettering Institute For Cancer Res, 1275 York Ave, New York, Ny 10065
Grant 5R01AI053417-08 from National Institute Of Allergy And Infectious Diseases
Abstract: Mycobacterium tuberculosis infection remains a major global health crisis. A multidisciplinary plan to control this epidemic must include new antibiotics and effective vaccination, but both of these therapeutic options are not presently available. Abundant recent evidence has implicated the M. tuberculosis cell envelope as an important pathogenesis determinant and therefore cell envelope biosynthetic pathways are an attractive therapeutic target. Data gathered during the prior award period of this grant defined the biosynthetic function and pathogenic importance of the mycolic acid methyltransferase enzyme family which modify the mycolic acids of M. tuberculosis with cyclopropane rings and methyl branches. Whereas loss of pcaA attenuates Mtb virulence, loss of cmaA2 and trans cyclopropanation causes hypervirulence and exacerbated granulomatous inflammation. While these results implicate cyclopropanation as an important pathogenesis determinant, they do not clearly recommend mycolic acid methyltransferase as a target for antibiotic development due to the unclear benefit to the host of loss of cyclopropanation. In this application we present preliminary data indicating that mycolic acid methyltransferases as an enzyme class are essential for slow growing mycobacterial viability in vitro. Genetic ablation of cmaA2 and mmaA3 in M. bovis BCG is synthetically lethal, suggesting a novel physiologic role for this lipid modification. Furthermore, we show that a chemical inhibitor of E. coli cyclopropane fatty acid synthase inhibits multiple pathways of mycolic acid cyclopropanation and inhibits mycobacterial growth at the same concentration. These results indicate that mycolic acid methyltransferases have a previously unrecognized essential function and that chemical inhibition of this enzyme class may be an attractive strategy for M. tuberculosis drug development. In this application we will build on these findings using genetics and biochemistry to substantiate mycolic acid methyltransferases as a target for antibiotic development and to understand the essential role of cyclopropanation in slow growing mycobacteria. This project seeks to understand the function of a family of lipid modifying enzymes (mycolic acid cyclopropane synthases) in Mycobacterium tuberculosis, the causative bacterium of the disease Tuberculosis. Tuberculosis is a major global health problem and there is an urgent need for new antibiotics to treat this infection. The experiments in this application are designed to validate this enzyme family as a target for antibiotic development and to understand their role in causing disease
Keywords: Ablation; Active Sites; Aerosols; Anabolism; Antibiotic Agents; Antibiotic Drugs; Antibiotics; Attenuated; Award; Bacteria; Bilayer Fluidity; Biochemistry; CFA synthase; Chemicals; Chemistry, Biological; Cyclopropanes; Data; Detergents; Development; Disease; Disorder; E coli; EC 2.1.1; Enzyme Inhibition; Enzymes; Epidemic; Escherichia coli; Family; Generalized Growth; Genetic; Genetic Techniques; Genetics-Mutagenesis; Genus Mycobacterium; Grant; Granulomatous; Growth; INFLM; In Vitro; Infection; Inflammation; Lipids; M. tb; M. tuberculosis; M.tb; M.tuberculosis; Measures; Membrane Fluidity; Methyltransferase; Methyltransferase Gene; Miscellaneous Antibiotic; Modeling; Modification; Molecular Biology, Mutagenesis; Mutagenesis; Mycobacterium; Mycobacterium tuberculosis; Mycolic Acid; Pathogenesis; Pathway interactions; Physiologic; Physiological; Role; Technics, Genetic; Temperature; Testing; Therapeutic; Tissue Growth; Tuberculosis; Vaccination; Virulence; biosynthesis; cell envelope; chemical function; cyclopropane; cyclopropane fatty acid synthase; cyclopropane synthetase; design; designing; disease/disorder; disseminated TB; disseminated tuberculosis; drug development; experiment; experimental research; experimental study; global health; in vivo; inhibitor; inhibitor/antagonist; methylase; mouse model; multidisciplinary; mutant; mycobacterial; novel; ontogeny; overexpression; pathway; protein complex; public health relevance; research study; social role; therapeutic target; transmethylase; trimethylene; tuberculosis drugs; tuberculous spondyloarthropathy
Project start date: 2002-12-01
Project end date: 2013-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01AI053417-08 (2010): $470003
5R01AI053417-07 (2009): $474750
Grants awarded to Michael Stephen Glickman
CYCLOPROPANE SYNTHETASES AND M.TUBERCULOSIS PATHOGENESIS
Michael Stephen Glickman, Associate Member
Sloan-kettering Institute For Cancer Res, 1275 York Ave, New York, Ny 10065
Grant 3R01AI053417-06A1S1 from Office Of The Director, National Institutes Of Health
Abstract: This award is issued in response to Notice OD-09-060, Recovery Act Administrative Supplements Providing Summer Research Experiences for Students and Science Educators. Mycobacterium tuberculosis infection remains a major global health crisis. A multidisciplinary plan to control this epidemic must include new antibiotics and effective vaccination, but both of these therapeutic options are not presently available. Abundant recent evidence has implicated the M. tuberculosis cell envelope as an important pathogenesis determinant and therefore cell envelope biosynthetic pathways are an attractive therapeutic target. Data gathered during the prior award period of this grant defined the biosynthetic function and pathogenic importance of the mycolic acid methyltransferase enzyme family which modify the mycolic acids of M. tuberculosis with cyclopropane rings and methyl branches. Whereas loss of pcaA attenuates Mtb virulence, loss of cmaA2 and trans cyclopropanation causes hypervirulence and exacerbated granulomatous inflammation. While these results implicate cyclopropanation as an important pathogenesis determinant, they do not clearly recommend mycolic acid methyltransferase as a target for antibiotic development due to the unclear benefit to the host of loss of cyclopropanation. In this application we present preliminary data indicating that mycolic acid methyltransferases as an enzyme class are essential for slow growing mycobacterial viability in vitro. Genetic ablation of cmaA2 and mmaA3 in M. bovis BCG is synthetically lethal, suggesting a novel physiologic role for this lipid modification. Furthermore, we show that a chemical inhibitor of E. coli cyclopropane fatty acid synthase inhibits multiple pathways of mycolic acid cyclopropanation and inhibits mycobacterial growth at the same concentration. These results indicate that mycolic acid methyltransferases have a previously unrecognized essential function and that chemical inhibition of this enzyme class may be an attractive strategy for M. tuberculosis drug development. In this application we will build on these findings using genetics and biochemistry to substantiate mycolic acid methyltransferases as a target for antibiotic development and to understand the essential role of cyclopropanation in slow growing mycobacteria. This project seeks to understand the function of a family of lipid modifying enzymes (mycolic acid cyclopropane synthases) in Mycobacterium tuberculosis, the causative bacterium of the disease Tuberculosis. Tuberculosis is a major global health problem and there is an urgent need for new antibiotics to treat this infection. The experiments in this application are designed to validate this enzyme family as a target for antibiotic development and to understand their role in causing disease
Keywords: Ablation; Active Sites; Aerosols; Anabolism; Antibiotic Agents; Antibiotic Drugs; Antibiotics; Attenuated; Award; Bacteria; Bilayer Fluidity; Biochemistry; CFA synthase; Chemicals; Chemistry, Biological; Cyclopropanes; Data; Detergents; Development; Disease; Disorder; E coli; EC 2.1.1; Enzyme Inhibition; Enzymes; Epidemic; Escherichia coli; Family; Generalized Growth; Genetic; Genetic Techniques; Genetics-Mutagenesis; Genus Mycobacterium; Grant; Granulomatous; Growth; Health; INFLM; In Vitro; Infection; Inflammation; Lipids; M. tb; M. tuberculosis; M.tb; M.tuberculosis; Measures; Membrane Fluidity; Methyltransferase; Methyltransferase Gene; Miscellaneous Antibiotic; Modeling; Modification; Molecular Biology, Mutagenesis; Mutagenesis; Mycobacterium; Mycobacterium tuberculosis; Mycolic Acid; Pathogenesis; Pathway interactions; Physiologic; Physiological; Role; Sites, Active; Technics, Genetic; Temperature; Testing; Therapeutic; Tissue Growth; Tuberculosis; Vaccination; Virulence; biosynthesis; cell envelope; chemical function; cyclopropane; cyclopropane fatty acid synthase; cyclopropane synthetase; design; designing; disease/disorder; disseminated TB; disseminated tuberculosis; drug development; experiment; experimental research; experimental study; in vivo; inhibitor; inhibitor/antagonist; methylase; mouse model; multidisciplinary; mutant; mycobacterial; novel; ontogeny; overexpression; pathway; protein complex; public health relevance; research study; social role; therapeutic target; transmethylase; trimethylene; tuberculosis drugs; tuberculous spondyloarthropathy
Project start date: 2009-06-05
Project end date: 2010-09-30
Budget start date: 5-JUN-2009
Budget end date: 30-SEP-2010
PFA/PA: PA-07-070
3R01AI053417-06A1S1 (2009): $15168
2R01AI053417-06A1 (2008): $474750
MOLECULAR ANALYSIS OF THE RIP1 VIRULENCE PATHWAY OF M. TUBERCULOSIS
Michael Stephen Glickman, Associate Member
Sloan-kettering Institute For Cancer Res, 1275 York Ave, New York, Ny 10065
Grant 5R01AI080628-02 from National Institute Of Allergy And Infectious Diseases
Abstract: M. tuberculosis infection is an ongoing global health crisis that requires new drugs or vaccines for effective control. Although many individual genes have been shown to be important for M. tuberculosis pathogenesis in the mouse, we still understand relatively little about how M. tuberculosis responds to varied host environments at the molecular level. The Rip1 (Rv2869c) intramembrane protease is a member of the Site two protease (S2P) class of intramembrane metalloproteases which cleave substrate proteins within transmembrane domains. We have previously shown that Rip1 is a critical determinant of M. tuberculosis growth and persistence in mice and controls cell envelope composition through transcriptional regulation of lipid biosynthetic genes. Further preliminary data presented here identifies three anti-sigma factors as Rip1 substrates anti-SigK (RskA), anti-SigL (RslA), and anti-SigM (RsmA). Accordingly, we show that SigK, SigL, and SigM target genes are not activated in the rip1 strain, demonstrating that loss of Rip1 inactivates three downstream transcriptional programs. In addition, we show that Rip1 is required for the regulation of iron responsive genes in response to iron limitation, a response that also requires SigL and SigM. Based on this data, we advance and test the major hypothesis that the severe attenuation of the Rip1 null strain is due to simultaneous inactivation of SigK, SigL, SigM regulons. To test this hypothesis, we propose a two year project to define the genetic and biochemical characteristics of the Rip1 pathway outlined in the specific aims below. We will determine the contribution of SigK/anti-sigK, SigL/anti-sigL, and SigM/anti-sigM to Rip1 dependent transcriptional regulation by transcriptional profiling of defined mutant strains in high and low iron. We will analyze anti-sigma factor cleavage in the same conditions using western blotting using anti-sigma factor antibodies in wild type and Rip1 deficient strains. Finally, we will define the molecular basis for site one cleavage of anti-sigma factors by genetically ablating candidate site one proteases and testing anti-sigma factor cleavage in these mutant strains.
Keywords: Aerosols; Anabolism; Antibodies; Biochemical; Blotting, Western; Candidate Disease Gene; Candidate Gene; Cell Communication and Signaling; Cell Signaling; Characteristics; Cleaved cell; Data; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Drugs; Environment; Esteroproteases; Fe element; Gene Expression; Gene Targeting; Gene Transcription; Generalized Growth; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Growth; Individual; Infection; Intracellular Communication and Signaling; Iron; Lipids; M. tb; M. tuberculosis; M.tb; M.tuberculosis; Mammals, Mice; Medication; Metallopeptidases; Metalloproteases; Metalloproteinases; Mice; Molecular; Molecular Analysis; Murine; Mus; Mutation; Mycobacterium tuberculosis; Pathogenesis; Pathway interactions; Peptidases; Peptide Hydrolases; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenotype; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteases; Protein Cleavage; Proteinases; Proteins; Proteolysis; Proteolytic Enzymes; RNA Expression; Regulation; Regulon; Relative; Relative (related person); Role; Siderochromes; Siderophores; Sigma Element; Sigma Factor; Sigma Initiation Factor; Sigma Subunit; Signal Transduction; Signal Transduction Systems; Signaling; Site; Starvation; TM Domain; Targetings, Gene; Testing; Tissue Growth; Transcription; Transcription Activation; Transcription Regulation; Transcription, Genetic; Transcriptional Activation; Transcriptional Control; Transcriptional Regulation; Transmembrane Domain; Transmembrane Region; Tuberculosis; Up-Regulation; Vaccines; Virulence; Western Blotting; Western Blottings; Western Immunoblotting; attenuation; base; biological signal transduction; biosynthesis; cell envelope; cleaved; deprivation; disseminated TB; disseminated tuberculosis; drug/agent; experiment; experimental research; experimental study; gene product; genome mutation; global health; in vivo; insight; member; metalloproteinase (general); mouse model; mutant; mycobactins; ontogeny; pathogen; pathway; programs; protein blotting; research study; response; social role; tuberculous spondyloarthropathy
Relevance: Relevance This project seeks to understand the function of the Rip1 protease of M. tuberculosis in the pathogenesis of Tuberculosis. Detailed understanding of the Rip1 pathway will validate this protease as a drug target and further our understanding of this important global pathogen
Project start date: 2009-07-17
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01AI080628-02 (2010): $474750
1R01AI080628-01A1 (2009): $467558
DNA LIGASES IN MYCOBACTERIAL DNA REPAIR & PATHOGENESIS
Michael Stephen Glickman, Associate Member
Sloan-kettering Institute For Cancer Res, 1275 York Ave, New York, Ny 10065
Grant 5R01AI064693-05 from National Institute Of Allergy And Infectious Diseases
Abstract: DNA ligases catalyze the final common step of DNA repair and replication by restoring the integrity of the DNA phosphodiester backbone. All known bacterial genomes encode an NAD dependent DNA ligase (LigA) which is essential for viability. Some bacterial genomes encode an additional ATP dependent ligase of unknown function. Mycobacteria, including the major human pathogen Mycobacterium tuberculosis (Mtu) and the genetically tractable M. smegmatis (MSm), encode 3 putative ATP dependent DNA ligases (LigB, LigC, LigD) in addition to LigA. The function of these ATP dependent ligases in mycobacterial DNA repair systems and mycobacterial pathogenesis is unknown. Our preliminary data shows that these 3 ligases are ATP dependent ligases in vitro, but with distinct catalytic properties. Null mutants of each ligase in MSm and Mtu indicate that these ligases are ssential for growth individually and in combination but that three alternative ATP dependent ligases does not rescue the essentiality of LigA in M. smegmatis. In addition both ligC and ligD participate in a novel pathway of prokaryotic Non homologous end joining (NHEJ) which is efficient but low fidelity. Sequence analysis of NHEJ junctions indicates involvement of polymerase and nuclease activities in blunt end mycobacterial NHEJ. The purpose of the experiments proposed herein is to elucidate the molecular mechanisms, physiologic role, and pathogenetic importance of Non homologous end joining (NHEJ) mediated by ATP dependent DNA ligases in M. smegmatis and M. tuberculosis. We propose a multidisciplinary research program encompassing genetics, biochemistry and microbial pathogenesis that will address the hypothesis that the DNA ligases of mycobacteria are a novel DNA repair system that defends the mycobacterial chromosome against double strand breaks during in vivo growth and persistence
Keywords: Address; Antibiotic Resistance; Bacteria; Bacterial Genome; Biochemical; Biochemistry; Bleo; Bleomycin; Bleomycin Antibiotic; Chemistry, Biological; Chromosomes; Closure by Ligation; DNA; DNA Binding; DNA Binding Interaction; DNA Damage; DNA Damage Repair; DNA Double Strand Break; DNA Injury; DNA Joinases; DNA Ligases; DNA Repair; DNA Topoisomerase (ATP-Hydrolysing); DNA Topoisomerase II; DNA Topoisomerases, Type II; DNA Type 2 Topoisomerase; Data; Deoxyribonucleic Acid; Electromagnetic Radiation, Ionizing; Endogenous Nitrate Vasodilator; Endothelium-Derived Relaxing Factor; Enzymes; GeneHomolog; Generalized Growth; Genetic; Genome, Bacterial; Genotoxins; Genus Mycobacterium; Growth; H2O2; Homolog; Homologous Gene; Homologue; Human; Human, General; Hydrogen Peroxide; Hydrogen Peroxide (H2O2); Hydroperoxide; In Vitro; Interdisciplinary Research; Interdisciplinary Study; Ionizing radiation; Laboratories; Ligase; Ligation; Lundbeck Brand of Bleomycin Sulfate; M. tb; M. tuberculosis; M.tb; M.tuberculosis; Man (Taxonomy); Man, Modern; Mediating; Molecular; Mononitrogen Monoxide; Multidisciplinary Collaboration; Multidisciplinary Research; Mutagens; Mycobacterium; Mycobacterium tuberculosis; NHEJ; Nitric Oxide; Nitric Oxide, Endothelium-Derived; Nitrogen Monoxide; Nitrogen Protoxide; Nitrogen oxide; Non-Homologous End Joining; Nonhomologous DNA End Joining; Nucleotides; Outcome; Pathogenesis; Pathway interactions; Peptide Domain; Physiologic; Physiological; Play; Poisons; Polydeoxyribonucleotide Ligases; Polydeoxyribonucleotide Synthetases; Polymerase; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein Domains; Radiation-Ionizing Total; Resistance to antibiotics; Resistance, Antibiotic; Resistant to antibiotics; Role; SEQ-AN; Sequence Analyses; Sequence Analysis; Spinal Column; Spine; Staging; Study, Interdisciplinary; Synthetases; System; System, LOINC Axis 4; Tertiary Protein Structure; Testing; Tissue Growth; Topo II; Topoisomerase II; Toxic Chemical; Toxic Substance; Tuberculosis; Unscheduled DNA Synthesis; Vertebral column; antibiotic resistant; backbone; cofactor; disseminated TB; disseminated tuberculosis; endothelial cell derived relaxing factor; experiment; experimental research; experimental study; genotoxic agent; in vivo; microbial; mouse model; mutant; mycobacterial; novel; nuclease; ontogeny; pathogen; pathway; phosphodiester; poison; programs; repair; repaired; research study; seal; social role; toxic compound; tuberculous spondyloarthropathy
Project start date: 2005-02-01
Project end date: 2011-01-31
Budget start date: 1-FEB-2009
Budget end date: 31-JAN-2011
5R01AI064693-05 (2009): $424620
Michael Stephen Glickman
Sloan-kettering Institute For Cancer Res
Project start date: 2010-05-15
Project end date: 2014-04-30