Download Biofilm Production in Clinical and Environmental Strains

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
¥ 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
Fe
O
2
H O
2 2
OH
OH
Lipid peroxidation
Cell damage
H O
2
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
Fe
O
2
H O
2 2
OH
OH
Lipid peroxidation
Cell damage
H O
2
Main Bacterial Iron Acquisition Systems
Siderophore-dependent
Main Bacterial Iron Acquisition Systems
Siderophore-dependent
Siderophore-independent
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 Acquisition
by A.a.
Fe
Afu system
A
Afe system
A
A
Fe
B
B
C
A
D
C
C
B
B
D
C
afu
Afu system
C
B
B
A
afe
Afe system
Fur
Inner membrane
Periplasmic space
Outer membrane
• 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
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
-
-
ND
ND
+
+
+++
+
+
+
Binding of hHb
Utilization of heme
Binding of heme
Utilization of FeCl3
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
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 PAO1 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 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
Cellular mainrole
Ami no acid
Biosynthes is of cofactors, prosthetic groups , and ca rriers
Cell enve lope
Cellular processes
Central i ntermediary metaboli sm
DNA metaboli sm
Energy metaboli sm
Fatty a cid and phospho li pid metabolism
Hypothetical proteins
Other categor ie s
Protein fate
Protein syn thesis
Purines, pyrimidines, nuc leosides, and nuc leotides
Regul atory fun ctions
Signa l transdu ction
Transcription
Transport and bind ing proteins
Unclassified
Unknown function
No. of predicted gene s
65
74
97
64
28
79
184
40
671
16
77
124
42
54
7
34
181
460
67
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
E. Rhodes
D. Dyer, Oklahoma University
A. Kachlany & D. Figurski,
Columbia University
• The funds
Miami University
National Institutes of Health