genetic and molecular characterization of a dental pathogen using a genome-wide approach
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
Essential nutrient for almost all living cells
Very abundant on earth's crust
Insoluble complexes at physiological conditions
Fe(III) + 3OH- -- Fe(OH)3 - Ks = 10-38
Free iron at pH 7.00 = 10-18 M
Requirement for bacterial growth is 10-7 M
Catalyst of the Haber-Weiss reaction
FeO
2H
2O
2OH OH
Lipid peroxidation Cell damage
H2
O
Facts About Iron
Essential nutrient for almost all living cells
Very abundant on earth's crust
Insoluble complexes at physiological conditions
Fe(III) + 3OH- -- Fe(OH)3 - Ks = 10-38
Free iron at pH 7.00 = 10-18 M
Requirement for bacterial growth is 10-7 M
Catalyst of the Haber-Weiss reaction
FeO
2H
2O
2OH OH
Lipid peroxidation Cell damage
H2
O
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 Acquisitionby A.a.
A
Inner membrane
Periplasmic space
Outer membrane
Fe
A
B
C
A B C
A
B
C D
afu afe
B
B
C
Fe
Fur
D C B 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
HK1651 Y4 SUNY465 CU1000
afuA + + + +
afuB + + + +
afuC + + + +
afeA + + + +
afeB + + + +
afeC + + + +
afeD + + + +
fur + + + +
tonB + + + +
hgpA + ND ND +
Iron Acquisition from Different Sources by CU1000(rough) and CU1060 (smooth)
CU1000 CU1060
Utilization of hTf - -
Binding of hTf - -
Utilization of hLf - -
Binding of hLf + + +
Utilization of hHb - -
Binding of hHb ND ND
Utilization of heme + +
Binding of heme +++ +
Utilization of FeCl3 + +
Gene Regulation by FurExpression 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
Cellular mainrole No. of predicted genes
Amino acid 65Biosynthesis of cofactors, prosthetic groups, and carriers 74Cell envelope 97Cellular processes 64Central intermediary metabolism 28DNA metabolism 79Energy metabolism 184Fatty acid and phospholipid metabolism 40Hypothetical proteins 671Other categories 16Protein fate 77Protein synthesis 124Purines, pyrimidines, nucleosides, and nucleotides 42Regulatory functions 54Signal transduction 7Transcription 34Transport and binding proteins 181Unclassified 460Unknown function 67
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