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

• 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


• Hemolysin• Hemoglobin binding protein• Ferritin• Oxidoreductase• Formate dehydrogenase• Cytochrome D


• Hemolysin• Hemoglobin binding protein• Ferritin• Oxidoreductase• Formate dehydrogenase• Cytochrome D• Cell division protein FtsA


• 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



CU100 cells caused- colonization and persistence in the oral cavity- induction of host immune response- localized bone losses




CU100 cells caused- colonization and persistence in the oral cavity- induction of host immune response- localized bone losses


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 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

Education

Genetic and Molecular Characterization of a Dental Pathogen Using a Genome-Wide Approach