Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach
The Human Oral Cavity <ul><li>A great environment to do Microbiology because it is </li></ul><ul><ul><li>important in huma...
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. ) <ul><li>Family  Pasteurellaceae </li></ul><ul><li>Gram-negative, non-sporul...
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 <ul><li>Siderophore independent systems </li></ul><ul><li>Con...
Iron Acquisition  A.a.   from Heme, Hemoglobin, and Hemophores   <ul><li>All strain tested use heme </li></ul><ul><li>Some...
Ligand-Independent Iron Acquisition by  A.a.   Afu   system Afe  system <ul><li>Strains grow under iron limitation </li></...
Comparative Analysis of  A.a.  Strains by PCR and DNA Sequencing + ND ND + hgpA + + + + tonB + + + + fur + + + + afeD + + ...
Iron Acquisition from Different Sources by CU1000(rough) and CU1060 (smooth) + + Utilization of FeCl 3 + +++ Binding of he...
Gene Regulation by Fur Expression of Fur Expression of iron-regulated proteins
Cloning of Fur-Regulated Genes with Fur Titration Assays - FURTA <ul><li>Make ~1-2 kbp library in pUC18 </li></ul><ul><li>...
Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein...
Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein...
Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein...
Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein...
Questions to Answer/Future Plans <ul><li>Which system(s) are used by  A.a.   to acquire iron in the presence and absence o...
Questions to Answer/Future Plans <ul><li>Which system(s) are used by  A.a.   to acquire iron in the presence and absence o...
Reconstruction of  S. mutans   metabolic pathways and transport systems
<ul><li>What are the components of the  A.a.  Fur and iron regulons? </li></ul><ul><ul><li>Classical and genetic approache...
<ul><li>What are the components of the  A.a.  Fur and iron regulons? </li></ul><ul><ul><li>Classical and genetic approache...
Analysis of the  P. aeruginosa  Iron Regulon Analysis of gene expression in cells cultured under iron-rich and iron-limiti...
Analysis of the  P. aeruginosa  Iron Regulon Analysis of gene expression in cells cultured under iron-rich and iron-limiti...
Analysis of the  P. aeruginosa  Fur Regulon <ul><li>Development of computer algorithms to detect in intergenic regions (IG...
Analysis of the  P. aeruginosa  Fur Regulon <ul><li>Development of computer algorithms to detect in intergenic regions (IG...
Analysis of the  P. aeruginosa  IRG4704-4705 <ul><li>IGR4704-4705 codes for two tandem transcripts that are 95%  identical...
Where are we with  A.a. ? <ul><li>The genome of strain HK1651 has been sequenced and is being annotated </li></ul><ul><ul>...
Where are we with  A.a. ? <ul><li>Classification of predicted genes based on similarities with genes and gene products in ...
Where are we with  A.a. ? <ul><li>A rat animal model in which lesions similar to those described in human patients has bee...
Where are we with  A.a. ? <ul><li>A rat animal model in which lesions similar to those described in human patients has bee...
Where are we with  A.a. ? <ul><li>A rat animal model in which lesions similar to those described in human patients has bee...
What are some of next/future the steps? <ul><li>Use genomics to study </li></ul><ul><ul><li>basic biological functions </l...
What are some of next/future the steps? <ul><li>Use genomics to study </li></ul><ul><ul><li>basic biological functions </l...
What are some of next/future the steps? <ul><li>Use genomics to study </li></ul><ul><ul><li>basic biological functions </l...
 
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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

  1. 1. Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach
  2. 2. The Human Oral Cavity <ul><li>A great environment to do Microbiology because it is </li></ul><ul><ul><li>important in human health </li></ul></ul><ul><ul><li>a complex ecosystem </li></ul></ul><ul><ul><li>colonized by a complex microbial community </li></ul></ul><ul><ul><li>an excellent niche to study </li></ul></ul><ul><li>- microbial-microbial interactions </li></ul><ul><li>- microbial-host interactions </li></ul><ul><li>- microbial evolution </li></ul><ul><li>- lateral gene transfer </li></ul><ul><li>- microbial resistance </li></ul><ul><li>- microbial biofilms </li></ul><ul><li> </li></ul>
  3. 3. The Microbial Oral Community A. H. Rickard et al ., Trends Microbiol. 2003
  4. 4. The Microbial Oral Community A. H. Rickard et al ., Trends Microbiol. 2003
  5. 5. Microbial Genome Sequencing Projects NIDCR Initial Recommendation
  6. 6. Microbial Genome Sequencing Projects Supported by NIDCR
  7. 7. Los Alamos National Laboratory The Oral Pathogen Sequence Databases
  8. 8. Status of Oral Pathogen Genomes Data obtained from: † , Genomes OnLine Databases (GOLD) *, TIGR Databases * †
  9. 9. Actinobacillus actinomycetemcomitans ( A.a. ) <ul><li>Family Pasteurellaceae </li></ul><ul><li>Gram-negative, non-sporulating </li></ul><ul><li>Non-motile, facultative anaerobe </li></ul><ul><li>Localized juvenile/aggressive periodontitis (LJP/LAP) </li></ul><ul><li>Endocarditis </li></ul>
  10. 10. Facts About Iron
  11. 11. Facts About Iron
  12. 12. Siderophore-dependent Main Bacterial Iron Acquisition Systems
  13. 13. Siderophore-dependent Siderophore-independent Main Bacterial Iron Acquisition Systems
  14. 14. Gene Regulation by Fur and sRNA
  15. 15. Gene Regulation by Fur and sRNA
  16. 16. Iron Acquisition by A.a. from Lactoferrin and Transferrin <ul><li>Siderophore independent systems </li></ul><ul><li>Contain sequences related to transferrin binding systems - tbpA </li></ul><ul><li>BUT , strains have tbpA point mutations and deletions, and neither bind nor use transferrin </li></ul><ul><li>Bind human lactoferrin </li></ul><ul><li>BUT , strains do not use lactoferrin </li></ul>
  17. 17. Iron Acquisition A.a. from Heme, Hemoglobin, and Hemophores <ul><li>All strain tested use heme </li></ul><ul><li>Some strains use hemoglobin via hgpA </li></ul><ul><li>Some strains have hgpA point mutations </li></ul><ul><li>Strains tested are able to grow under iron limitation in the absence of iron binding proteins </li></ul>
  18. 18. Ligand-Independent Iron Acquisition by A.a. Afu system Afe system <ul><li>Strains grow under iron limitation </li></ul><ul><li>Media containing 2,2’-dipyridyl (DIP) </li></ul><ul><li>Media containing ethylenediamine-di-( o -hydroxyphenyl) acetic acid (EDDHA) </li></ul>Afu system Afe system
  19. 19. 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
  20. 20. 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
  21. 21. Gene Regulation by Fur Expression of Fur Expression of iron-regulated proteins
  22. 22. Cloning of Fur-Regulated Genes with Fur Titration Assays - FURTA <ul><li>Make ~1-2 kbp library in pUC18 </li></ul><ul><li>Transform E. coli H1717 </li></ul><ul><li>Plate transformants on MacConkey agar containing Fe </li></ul><ul><li>Select red colonies </li></ul><ul><li>Isolated plasmid DNA </li></ul><ul><li>Sequence with universal primers </li></ul><ul><li>Compare nucleotide sequences with databases using BLASTx </li></ul>
  23. 23. Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein </li></ul><ul><li>Ferritin </li></ul>
  24. 24. Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein </li></ul><ul><li>Ferritin </li></ul><ul><li>Oxidoreductase </li></ul><ul><li>Formate dehydrogenase </li></ul><ul><li>Cytochrome D </li></ul>
  25. 25. Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein </li></ul><ul><li>Ferritin </li></ul><ul><li>Oxidoreductase </li></ul><ul><li>Formate dehydrogenase </li></ul><ul><li>Cytochrome D </li></ul><ul><li>Cell division protein FtsA </li></ul>
  26. 26. Identification of Some Potential HK1651 Fur-Regulated Genes <ul><li>Hemolysin </li></ul><ul><li>Hemoglobin binding protein </li></ul><ul><li>Ferritin </li></ul><ul><li>Oxidoreductase </li></ul><ul><li>Formate dehydrogenase </li></ul><ul><li>Cytochrome D </li></ul><ul><li>Cell division protein FtsA </li></ul><ul><li>Transmembrane protein </li></ul><ul><li>Proteins with no significant similarity in databases </li></ul>
  27. 27. Questions to Answer/Future Plans <ul><li>Which system(s) are used by A.a. to acquire iron in the presence and absence of ligands? </li></ul><ul><ul><li>Classical approaches, search for/study of one system at a time </li></ul></ul><ul><ul><li>or </li></ul></ul>
  28. 28. Questions to Answer/Future Plans <ul><li>Which system(s) are used by A.a. to acquire iron in the presence and absence of ligands? </li></ul><ul><ul><li>Classical approaches, search for/study of one system at a time </li></ul></ul><ul><ul><li>or </li></ul></ul><ul><ul><li>Genome-wide approach using information such as that generated from the Streptococcus mutans UA159 genome sequencing project </li></ul></ul>Ajdic et al., 2002
  29. 29. Reconstruction of S. mutans metabolic pathways and transport systems
  30. 30. <ul><li>What are the components of the A.a. Fur and iron regulons? </li></ul><ul><ul><li>Classical and genetic approaches, one gene at a time and more FURTA </li></ul></ul><ul><ul><li>or </li></ul></ul>Questions to Answer/Future Plans
  31. 31. <ul><li>What are the components of the A.a. Fur and iron regulons? </li></ul><ul><ul><li>Classical and genetic approaches, one gene at a time and more FURTA </li></ul></ul><ul><ul><li>or </li></ul></ul><ul><ul><li>Genome-wide approach using information such as that generated from the Pseudomonas aeruginosa PAO1 genome sequencing project </li></ul></ul>Questions to Answer/Future Plans Genome-wide transcriptional analysis with DNA microarrays
  32. 32. Analysis of the P. aeruginosa Iron Regulon Analysis of gene expression in cells cultured under iron-rich and iron-limiting conditions using GeneChip ® arrays
  33. 33. 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
  34. 34. Analysis of the P. aeruginosa Fur Regulon <ul><li>Development of computer algorithms to detect in intergenic regions (IGRs) </li></ul><ul><ul><li>Fur boxes </li></ul></ul><ul><ul><li>structures similar to RyhB </li></ul></ul>
  35. 35. Analysis of the P. aeruginosa Fur Regulon <ul><li>Development of computer algorithms to detect in intergenic regions (IGRs) </li></ul><ul><ul><li>Fur boxes </li></ul></ul><ul><ul><li>structures similar to RyhB </li></ul></ul>Computer screening of IGRs IGR4704-4705 P. J. Wilderman et al ., 2003
  36. 36. Analysis of the P. aeruginosa IRG4704-4705 <ul><li>IGR4704-4705 codes for two tandem transcripts that are 95% identical </li></ul><ul><li>Both transcripts are iron-regulated </li></ul><ul><li>One of the transcripts is also regulated by haem </li></ul><ul><li>The cognate promoter regions contain Fur-boxes and bind Fur </li></ul><ul><li>Analysis of isogenic mutants proved that the two sRNA control expression of genes required for </li></ul><ul><ul><ul><li>iron storage </li></ul></ul></ul><ul><ul><ul><li>resistance to oxidative stress </li></ul></ul></ul>P. J. Wilderman et al ., 2003
  37. 37. Where are we with A.a. ? <ul><li>The genome of strain HK1651 has been sequenced and is being annotated </li></ul><ul><ul><li>Information obtained after the initial automated annotation </li></ul></ul><ul><ul><ul><li>Genome size: 2,105,503 bp </li></ul></ul></ul><ul><ul><ul><li>G+C content: 44.4% </li></ul></ul></ul><ul><ul><ul><li>Number of open reading frames: 2,345 </li></ul></ul></ul><ul><ul><ul><li>Average gene length: 791 nt </li></ul></ul></ul>D. Dyer, OUHSC
  38. 38. Where are we with A.a. ? <ul><li>Classification of predicted genes based on similarities with genes and gene products in databases </li></ul>D. Dyer, OUHSC
  39. 39. Where are we with A.a. ? <ul><li>A rat animal model in which lesions similar to those described in human patients has been developed </li></ul><ul><li>Feeding Sprague-Dawley rats with food containing A.a. CU1000 cells caused </li></ul><ul><ul><ul><li>- colonization and persistence in the oral cavity </li></ul></ul></ul>D. Fine & D. Figurski Labs
  40. 40. Where are we with A.a. ? <ul><li>A rat animal model in which lesions similar to those described in human patients has been developed </li></ul><ul><li>Feeding Sprague-Dawley rats with food containing A.a. CU100 cells caused </li></ul><ul><ul><ul><li>- colonization and persistence in the oral cavity </li></ul></ul></ul><ul><ul><ul><li>- induction of host immune response </li></ul></ul></ul><ul><ul><ul><li>- localized bone losses </li></ul></ul></ul>D. Fine & D. Figurski Labs
  41. 41. Where are we with A.a. ? <ul><li>A rat animal model in which lesions similar to those described in human patients has been developed </li></ul><ul><li>Feeding Sprague-Dawley rats with food containing A.a. CU100 cells caused </li></ul><ul><ul><ul><li>- colonization and persistence in the oral cavity </li></ul></ul></ul><ul><ul><ul><li>- induction of host immune response </li></ul></ul></ul><ul><ul><ul><li>- localized bone losses </li></ul></ul></ul>D. Fine & D. Figurski Labs
  42. 42. What are some of next/future the steps? <ul><li>Use genomics to study </li></ul><ul><ul><li>basic biological functions </li></ul></ul><ul><ul><li>genetic differences and variations among virulent and non-virulent strains </li></ul></ul><ul><ul><li>the role of potential bacterial virulence factors involved in the pathogenesis of LJP/LAP </li></ul></ul><ul><ul><li>gene transfer and genome evolution </li></ul></ul>
  43. 43. What are some of next/future the steps? <ul><li>Use genomics to study </li></ul><ul><ul><li>basic biological functions </li></ul></ul><ul><ul><li>genetic differences and variations among virulent and non-virulent strains </li></ul></ul><ul><ul><li>the role of potential bacterial virulence factors involved in the pathogenesis of LJP/LAP </li></ul></ul><ul><ul><li>gene transfer and genome evolution </li></ul></ul><ul><li>Use DNA arrays to study </li></ul><ul><ul><li>regulation of gene expression in the bacterial pathogen </li></ul></ul><ul><ul><li>regulation of gene expression in the host </li></ul></ul>
  44. 44. What are some of next/future the steps? <ul><li>Use genomics to study </li></ul><ul><ul><li>basic biological functions </li></ul></ul><ul><ul><li>genetic differences and variations among virulent and non-virulent strains </li></ul></ul><ul><ul><li>the role of potential bacterial virulence factors involved in the pathogenesis of LJP/LAP </li></ul></ul><ul><ul><li>gene transfer and genome evolution </li></ul></ul><ul><li>Use DNA arrays to study </li></ul><ul><ul><li>regulation of gene expression in the bacterial pathogen </li></ul></ul><ul><ul><li>regulation of gene expression in the host </li></ul></ul><ul><li>Use genomics and DNA arrays to </li></ul><ul><ul><li>design and generate isogenic mutants with a more rational approach </li></ul></ul><ul><ul><li>study the the host-pathogen interactions that result in in the pathogenesis of infectious diseases </li></ul></ul><ul><ul><li>develop new antimicrobial compounds and therapies to prevent and treat infectious diseases </li></ul></ul>

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