
84fdc8d2d1eb41d4529a16818a2c27ee.ppt
- Количество слайдов: 45
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 On. Line 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
Main Bacterial Iron Acquisition Systems Siderophore-dependent
Main Bacterial Iron Acquisition Systems Siderophore-dependent Siderophore-independent
Gene Regulation by Fur and s. RNA
Gene Regulation by Fur and s. RNA
Iron Acquisition by A. a. from Lactoferrin and Transferrin • Siderophore independent systems • Contain sequences related to transferrin binding systems - tbp. A • BUT, strains have tbp. A 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 hgp. A • Some strains have hgp. A 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)
Comparative Analysis of A. a. Strains by PCR and DNA Sequencing HK 1651 Y 4 SUNY 465 CU 1000 afu. A + + afu. B + + afu. C + + afe. A + + afe. B + + afe. C + + afe. D + + fur + + ton. B + + hgp. A + ND ND +
Iron Acquisition from Different Sources by CU 1000(rough) and CU 1060 (smooth) CU 1000 CU 1060 Utilization of h. Tf - - Binding of h. Tf - - Utilization of h. Lf - - Binding of h. Lf + ++ Utilization of h. Hb - - ND ND + + +++ + Binding of h. Hb Utilization of heme Binding of heme Utilization of Fe. Cl 3
Gene Regulation by Fur Expression of iron-regulated proteins
Cloning of Fur-Regulated Genes with Fur Titration Assays - FURTA • Make ~1 -2 kbp library in p. UC 18 • Transform E. coli H 1717 • Plate transformants on Mac. Conkey agar containing Fe • Select red colonies • Isolated plasmid DNA • Sequence with universal primers • Compare nucleotide sequences with databases using BLASTx
Identification of Some Potential HK 1651 Fur-Regulated Genes • Hemolysin • Hemoglobin binding protein • Ferritin
Identification of Some Potential HK 1651 Fur-Regulated Genes • Hemolysin • Hemoglobin binding protein • Ferritin • Oxidoreductase • Formate dehydrogenase • Cytochrome D
Identification of Some Potential HK 1651 Fur-Regulated Genes • Hemolysin • Hemoglobin binding protein • Ferritin • Oxidoreductase • Formate dehydrogenase • Cytochrome D • Cell division protein Fts. A
Identification of Some Potential HK 1651 Fur-Regulated Genes • Hemolysin • Hemoglobin binding protein • Ferritin • Oxidoreductase • Formate dehydrogenase • Cytochrome D • Cell division protein Fts. A • 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 UA 159 genome sequencing project Ajdic et al. , 2002
Reconstruction of S. mutans metabolic pathways and transport systems
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
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 PAO 1 genome sequencing project 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 Gene. Chip® arrays
Analysis of the P. aeruginosa Iron Regulon Analysis of gene expression in cells cultured under iron-rich and iron-limiting conditions using Gene. Chip® 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 Ryh. B
Analysis of the P. aeruginosa Fur Regulon • Development of computer algorithms to detect in intergenic regions (IGRs) – Fur boxes – structures similar to Ryh. B Computer screening of IGRs IGR 4704 -4705 P. J. Wilderman et al. , 2003
Analysis of the P. aeruginosa IRG 4704 -4705 • IGR 4704 -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 s. RNA 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 HK 1651 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. CU 1000 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. CU 100 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. CU 100 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 host-pathogen interactions that result in in the pathogenesis of infectious diseases – develop new antimicrobial compounds and therapies to prevent and treat infectious diseases