Transcript “Comparative Genomics of Chlamydia trachomatis Strains”

“Comparative Genomics of
Chlamydia trachomatis Strains”
Sonia Rajput
Dr. Dan Rockey
Biomedical Sciences
Oregon State University
October 14, 2006
Significance of Research
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An improved understanding of
chlamydial biology and diversity
New approaches to prevention
and treatment of chlamydial
disease
– Mapping of disease
associated loci
– Rapid analysis and detection
of single nucleotide
polymorphisms (SNPs)
– Model for predictive
therapies for genotyping- to
be used in clinical settings
Comparison of chlamydial genome
Chlamydia

What is Chlamydia?
– Obligate intracellular
bacterial pathogen
– Causes STD in humans and
many diseases like heart
disease, Alzheimer's,
cataracts
–
Chlamydia trachomatis:
serovars D-K are major
cause of urogenital tract
infection worldwide

What is comparative genomics?
– Comparing genomes of
different organisms to
understand their biology and
evolution
– Develop systems for
diagnostic therapies
C. trachomatis florescent
antibody stain
Problems/Solutions
Problem: Lack of a system for inactivating or inserting target
genes. Cannot “knock out” specific genes of interest
Solution: Genome sequence analysis provides information on
chlamydial genotypes and phenotypes
Problem: Little information on the relationship of the organism to
the epidemiology of chlamydial infection
Solution: Comparative genomics offers insight into the genotype of
individual strains that lead to an associated patient phenotype.
Purpose

To identify and evaluate
single nucleotide
polymorphisms (SNPs) and
insertions and deletions
between serovars/strains
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–
Strains examined: DUW3,
G9301, G9768, DS2923,
G11222, G11074 + 12 new
strains
Identify different genotypes
that might be associated
with different phenotypes
within patients
DUW3 Chlamydial genome map
Designing PCR Assays
PCR Assays:
 Designed sequence of nucleotides to discriminate
among SNPs and insertions and deletions in DUW3
& G9301
DUW3
G9301
9 10
Polymerase Chain Reaction
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PCR assays determine the beginning and end regions
amplified by annealing to DNA template
Purpose: Comparison of gene expression in different serovars
to determine if same nucleotide sequences are present
PCR gel
Primers tested
against G9301 &
DUW3 w/ 100BP
ladder
Polymerase Chain Reaction
PCR gel
Primers tested
against G9301 &
DUW3 w/ 100BP
ladder
Polymerase Chain Reaction
9 10
PCR gel
Primers tested
against G9301 &
DUW3 w/ 100BP
ladder
9, 10- CT 326 w/
G9301 & DUW3
Results- CT 326
1. std
2. G9301
3. G9768
4. G11074
5. G111
6. G566
7. G9938
8. G1243
9. G1455
10. G503
11. std
12. DuW3
13. G11222
14. G7603
15. GUW57
16. DA1
17. Gs459
18. G11178
19. std
Rectal Strains
Cervical Strains
Results- CT456
1. std
2. G9301
3. G9768
4. G11074
5. G111
6. G566
7. G9938
8. G1243
9. G1455
10. G503
11. std
12. DuW3
13. G11222
14. G7603
15. GUW57
16. DA1
17. Gs459
18. G11178
19. std
Results
Gene: CT 326
– 111 bp deletion in G9301 compared to DUW3
– Functional class- Unknown, hypothetical protein
– Primers: 5’ CCTCTCGGCAATATCCAAAA 3’,
5’ TCTCGTTTGCAACTTGTTCG 3’
 Variation among strains in band size produced
 Due to tissue tropism (rectal vs. cervical)?
BLAST: CT326 is a conserved region within these
strains of Chlamydia
Gene: CT 456
– 366 bp deletion in G9301 compared to DUW3
– Functional Class: Unknown
– Primers 5’ CACCAACGCCATCCTCTATT 3’,
5’ GCTACCACTTCCTCCTGCTG 3’
 Not much variation present between strains; wildtype
DUW3
Restriction Digest
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Design 8 primers on PMP genes in G9301 and
DUW3
Run restriction digest
Purpose:
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To observe variation or areas of conservation among strains
Statistically connect variations in SNPs with phenotypes
Long term goal: Rapid and simple detection and
analysis of SNPs to be used in novel predictive
genotyping therapies.
CT681
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Highly variable PMP gene from wildtype DUW3 to G9301
Functional class: Major outer membrane protein
Primers: F:5’ AGGCATCCTTAGTTCCTGTCGC 3’,
R:5’ CGCTTTGAGTTCTGCTTCCTCC 3’
Enzyme: MspI (New England Biolabs) cuts on SNP in G9301, not in DUW3 (12 hrs)
Estimated band size produced: G9301: 761bp; DUW3: 758bp
Perform on: Ds2923, E11023, and strain A to see variation or distinctions among
isolates
Lane:
1. 100 bp ladder
2. DUW3 w/o MspI
3. DUW3 w/ MSPI
4. G9301 w/o MspI
5. G9301w/ MSPI
6. G111 w/ MSPI (r)
7. 100 bp ladder
8. GUW57 w/ MSPI
(cx)
9. Gs459 w/ MSPI (cx)
10. G1455 w/ MSPI (r)
Conclusion
• Laboratory and clinical studies have revealed differences in
host-pathogen interactions within various Chlamydia
trachomatis serovars.
• The sequence of Chlamydial serovars displaying differences in
tissue tropism (rectal versus cervical) might lead to a
corresponding phenotype.
• Therefore, rapid analysis and detection of SNPs can be used in
novel predictive genotyping therapies, which can detect the
onset of a specific Chlamdyial strain/serovar seen in patients.
Acknowledgements
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Howard Hughes Medical
Institute Program
Dr. Dan Rockey’s Lab
Dr. Kevin Ahern
Support from the NIH