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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

The Human Oral Cavity A great environment to do Microbiology because it is • 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

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 NIDCR Initial Recommendation

Microbial Genome Sequencing Projects Supported by NIDCR Microbial Genome Sequencing Projects Supported by NIDCR

Los Alamos National Laboratory The Oral Pathogen Sequence Databases Los Alamos National Laboratory The Oral Pathogen Sequence Databases

Status of Oral Pathogen Genomes † * Data obtained from: †, Genomes On. Line 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 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

Facts About Iron Facts About Iron

Main Bacterial Iron Acquisition Systems Siderophore-dependent Main Bacterial Iron Acquisition Systems Siderophore-dependent

Main Bacterial Iron Acquisition Systems Siderophore-dependent Siderophore-independent Main Bacterial Iron Acquisition Systems Siderophore-dependent Siderophore-independent

Gene Regulation by Fur and s. RNA Gene Regulation by Fur and s. RNA

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 • 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 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 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 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 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 Gene Regulation by Fur Expression of iron-regulated proteins

Cloning of Fur-Regulated Genes with Fur Titration Assays - FURTA • Make ~1 -2 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 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 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 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 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 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 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 Reconstruction of S. mutans metabolic pathways and transport systems

Questions to Answer/Future Plans • What are the components of the A. a. Fur 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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