Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach Luis A. Actis Miami University Oxford, Ohio

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

Siderophore-dependent Main Bacterial Iron Acquisition Systems

Siderophore-dependentSiderophore-independent Main Bacterial Iron Acquisition Systems

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 systemAfe 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 HK1651Y4SUNY465 CU1000 afuA++++ afuB++++ afuC++++ afeA++++ afeB++++ afeC++++ afeD++++ fur++++ tonB++++ hgpA+ND +

Iron Acquisition from Different Sources by CU1000(rough) and CU1060 (smooth) CU1000CU1060 Utilization of hTf-- Binding of hTf-- Utilization of hLf-- Binding of hLf++ Utilization of hHb-- Binding of hHbND Utilization of heme++ Binding of heme++++ Utilization of FeCl 3 ++

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 IGR P. J. Wilderman et al., 2003

Analysis of the P. aeruginosa IRG IGR 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

Acknowledgments The people A. Tomaras D. Dyer, Oklahoma University E. Rhodes A. Kachlany & D. Figurski, Columbia University The funds Miami University National Institutes of Health