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Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach
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Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach

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    Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach Presentation Transcript

    • Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach
    • The Human Oral Cavity
      • A great environment to do Microbiology because it is
        • important in human health
        • a complex ecosystem
        • colonized by a complex microbial community
        • an excellent niche to study
      • - microbial-microbial interactions
      • - microbial-host interactions
      • - microbial evolution
      • - lateral gene transfer
      • - microbial resistance
      • - microbial biofilms
    • The Microbial Oral Community A. H. Rickard et al ., Trends Microbiol. 2003
    • The Microbial Oral Community A. H. Rickard et al ., Trends Microbiol. 2003
    • Microbial Genome Sequencing Projects NIDCR Initial Recommendation
    • Microbial Genome Sequencing Projects Supported by NIDCR
    • Los Alamos National Laboratory The Oral Pathogen Sequence Databases
    • Status of Oral Pathogen Genomes Data obtained from: † , Genomes OnLine Databases (GOLD) *, TIGR Databases * †
    • Actinobacillus actinomycetemcomitans ( A.a. )
      • Family Pasteurellaceae
      • Gram-negative, non-sporulating
      • Non-motile, facultative anaerobe
      • Localized juvenile/aggressive periodontitis (LJP/LAP)
      • Endocarditis
    • Facts About Iron
    • Facts About Iron
    • Siderophore-dependent Main Bacterial Iron Acquisition Systems
    • Siderophore-dependent Siderophore-independent Main Bacterial Iron Acquisition Systems
    • Gene Regulation by Fur and sRNA
    • Gene Regulation by Fur and sRNA
    • Iron Acquisition by A.a. from Lactoferrin and Transferrin
      • Siderophore independent systems
      • Contain sequences related to transferrin binding systems - tbpA
      • BUT , strains have tbpA point mutations and deletions, and neither bind nor use transferrin
      • Bind human lactoferrin
      • BUT , strains do not use lactoferrin
    • Iron Acquisition A.a. from Heme, Hemoglobin, and Hemophores
      • All strain tested use heme
      • Some strains use hemoglobin via hgpA
      • Some strains have hgpA point mutations
      • Strains tested are able to grow under iron limitation in the absence of iron binding proteins
    • Ligand-Independent Iron Acquisition by A.a. Afu system Afe system
      • Strains grow under iron limitation
      • Media containing 2,2’-dipyridyl (DIP)
      • Media containing ethylenediamine-di-( o -hydroxyphenyl) acetic acid (EDDHA)
      Afu system Afe system
    • Comparative Analysis of A.a. Strains by PCR and DNA Sequencing + ND ND + hgpA + + + + tonB + + + + fur + + + + afeD + + + + afeC + + + + afeB CU1000 SUNY465 Y4 HK1651 + + + + afeA + + + + afuC + + + + afuB + + + + afuA
    • Iron Acquisition from Different Sources by CU1000(rough) and CU1060 (smooth) + + Utilization of FeCl 3 + +++ Binding of heme + + Utilization of heme ND ND Binding of hHb - - Utilization of hHb CU1060 CU1000 + + + Binding of hLf - - Utilization of hLf - - Binding of hTf - - Utilization of hTf
    • Gene Regulation by Fur Expression of Fur Expression of iron-regulated proteins
    • Cloning of Fur-Regulated Genes with Fur Titration Assays - FURTA
      • Make ~1-2 kbp library in pUC18
      • Transform E. coli H1717
      • Plate transformants on MacConkey agar containing Fe
      • Select red colonies
      • Isolated plasmid DNA
      • Sequence with universal primers
      • Compare nucleotide sequences with databases using BLASTx
    • Identification of Some Potential HK1651 Fur-Regulated Genes
      • Hemolysin
      • Hemoglobin binding protein
      • Ferritin
    • Identification of Some Potential HK1651 Fur-Regulated Genes
      • Hemolysin
      • Hemoglobin binding protein
      • Ferritin
      • Oxidoreductase
      • Formate dehydrogenase
      • Cytochrome D
    • Identification of Some Potential HK1651 Fur-Regulated Genes
      • Hemolysin
      • Hemoglobin binding protein
      • Ferritin
      • Oxidoreductase
      • Formate dehydrogenase
      • Cytochrome D
      • Cell division protein FtsA
    • Identification of Some Potential HK1651 Fur-Regulated Genes
      • Hemolysin
      • Hemoglobin binding protein
      • Ferritin
      • Oxidoreductase
      • Formate dehydrogenase
      • Cytochrome D
      • Cell division protein FtsA
      • Transmembrane protein
      • Proteins with no significant similarity in databases
    • Questions to Answer/Future Plans
      • Which system(s) are used by A.a. to acquire iron in the presence and absence of ligands?
        • Classical approaches, search for/study of one system at a time
        • or
    • Questions to Answer/Future Plans
      • Which system(s) are used by A.a. to acquire iron in the presence and absence of ligands?
        • Classical approaches, search for/study of one system at a time
        • or
        • Genome-wide approach using information such as that generated from the Streptococcus mutans UA159 genome sequencing project
      Ajdic et al., 2002
    • Reconstruction of S. mutans metabolic pathways and transport systems
      • What are the components of the A.a. Fur and iron regulons?
        • Classical and genetic approaches, one gene at a time and more FURTA
        • or
      Questions to Answer/Future Plans
      • What are the components of the A.a. Fur and iron regulons?
        • Classical and genetic approaches, one gene at a time and more FURTA
        • or
        • Genome-wide approach using information such as that generated from the Pseudomonas aeruginosa PAO1 genome sequencing project
      Questions to Answer/Future Plans Genome-wide transcriptional analysis with DNA microarrays
    • Analysis of the P. aeruginosa Iron Regulon Analysis of gene expression in cells cultured under iron-rich and iron-limiting conditions using GeneChip ® arrays
    • Analysis of the P. aeruginosa Iron Regulon Analysis of gene expression in cells cultured under iron-rich and iron-limiting conditions using GeneChip ® arrays U. A. Ochsner et al ., 2002
    • Analysis of the P. aeruginosa Fur Regulon
      • Development of computer algorithms to detect in intergenic regions (IGRs)
        • Fur boxes
        • structures similar to RyhB
    • Analysis of the P. aeruginosa Fur Regulon
      • Development of computer algorithms to detect in intergenic regions (IGRs)
        • Fur boxes
        • structures similar to RyhB
      Computer screening of IGRs IGR4704-4705 P. J. Wilderman et al ., 2003
    • Analysis of the P. aeruginosa IRG4704-4705
      • IGR4704-4705 codes for two tandem transcripts that are 95% identical
      • Both transcripts are iron-regulated
      • One of the transcripts is also regulated by haem
      • The cognate promoter regions contain Fur-boxes and bind Fur
      • Analysis of isogenic mutants proved that the two sRNA control expression of genes required for
          • iron storage
          • resistance to oxidative stress
      P. J. Wilderman et al ., 2003
    • Where are we with A.a. ?
      • The genome of strain HK1651 has been sequenced and is being annotated
        • Information obtained after the initial automated annotation
          • Genome size: 2,105,503 bp
          • G+C content: 44.4%
          • Number of open reading frames: 2,345
          • Average gene length: 791 nt
      D. Dyer, OUHSC
    • Where are we with A.a. ?
      • Classification of predicted genes based on similarities with genes and gene products in databases
      D. Dyer, OUHSC
    • Where are we with A.a. ?
      • A rat animal model in which lesions similar to those described in human patients has been developed
      • Feeding Sprague-Dawley rats with food containing A.a. CU1000 cells caused
          • - colonization and persistence in the oral cavity
      D. Fine & D. Figurski Labs
    • Where are we with A.a. ?
      • A rat animal model in which lesions similar to those described in human patients has been developed
      • Feeding Sprague-Dawley rats with food containing A.a. CU100 cells caused
          • - colonization and persistence in the oral cavity
          • - induction of host immune response
          • - localized bone losses
      D. Fine & D. Figurski Labs
    • Where are we with A.a. ?
      • A rat animal model in which lesions similar to those described in human patients has been developed
      • Feeding Sprague-Dawley rats with food containing A.a. CU100 cells caused
          • - colonization and persistence in the oral cavity
          • - induction of host immune response
          • - localized bone losses
      D. Fine & D. Figurski Labs
    • What are some of next/future the steps?
      • Use genomics to study
        • basic biological functions
        • genetic differences and variations among virulent and non-virulent strains
        • the role of potential bacterial virulence factors involved in the pathogenesis of LJP/LAP
        • gene transfer and genome evolution
    • What are some of next/future the steps?
      • Use genomics to study
        • basic biological functions
        • genetic differences and variations among virulent and non-virulent strains
        • the role of potential bacterial virulence factors involved in the pathogenesis of LJP/LAP
        • gene transfer and genome evolution
      • Use DNA arrays to study
        • regulation of gene expression in the bacterial pathogen
        • regulation of gene expression in the host
    • What are some of next/future the steps?
      • Use genomics to study
        • basic biological functions
        • genetic differences and variations among virulent and non-virulent strains
        • the role of potential bacterial virulence factors involved in the pathogenesis of LJP/LAP
        • gene transfer and genome evolution
      • Use DNA arrays to study
        • regulation of gene expression in the bacterial pathogen
        • regulation of gene expression in the host
      • Use genomics and DNA arrays to
        • design and generate isogenic mutants with a more rational approach
        • study the the host-pathogen interactions that result in in the pathogenesis of infectious diseases
        • develop new antimicrobial compounds and therapies to prevent and treat infectious diseases
    •