Transcript Production of plasmid-encoded chlamydial proteins in host cells Jeffrey Burnett

Production of plasmid-encoded
chlamydial proteins in host cells
Jeffrey Burnett
Dr. Dan Rockey Lab
Chlamydia trachomatis
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most common STI
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causes:
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2.8 million cases annually
Pelvic Inflammatory Disease
Ectopic pregnancy
Infertility
Preventable blindness
asymptomatic
C. trachomatis
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Gram (-) bacterium
Obligate intracellular
aerobe
▫ Cannot make its own ATP
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Inclusion
Biphasic lifecycle
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Non-infectious
metabolically active form
Infectious “spore” form
Background knowledge
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Horoschak and Moulder
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Bose and Liebhaber
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Decrease in amount of cells
No decrease in DNA synthesis
Greene and Zhong
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Interactions between chlamydia and host cell cycle
Background knowledge
McCoy cells – blue (nuclei), green
(cytoskeleton)
McCoy cells – blue (nuclei), green
(cytoskeleton), red (chlamydial protein
transfection - CT223)
Background information
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Rockey et al
Inclusion membrane proteins (inc)
 CT119 - incA
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Identification of CT 223 as inc gene
My Project
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Two projects:
Find active domain of CT 223
Double transfection with genes in inc
operon
Project #1
Construct
p23f
Structure of construct
1
96
p23cL1/2
96
DA97/DA116
25%
DA71/DA116
8%
DA71/DA121
15%
DA122/DA116
158
270
214
p23f2/3
Hydrophobic domain
23%
270
159
1
Oligos
270
p23c
p23cR1/2
PN
7%
Hydrophilic regions
DA97/DA119
Project #1 - conclusion
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Active site of CT223 is c-terminus 56 AA
sequence
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To do:
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What is the relevance?
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Note: Chlamydia blocks cytokinesis
Find binding partners
Project #2 - part one
gene # / restriction site
Sequence
CT223 C. trachomatis / Eco RI
agcaGAATTCttgagatctagaaaaatagaagc
CT223 C. trachomatis / KpnI
agcaGGTACCaatggtgagtttagcattagg
CT224 C. trachomatis / EcoRI
agcaGAATTCatgagttttgttggagatagt
CT224 C. trachomatis / XhoI
agcaCTCGAGctaatcattgggaaaaattga
CT225 C. trachomatis / EcoRI
agcaGAATTCatggtggctaacaactcctttatt
CT225 C. trachomatis / EcoRV
agcaGATATCttaatcccacccatgaaattt
CT226 C. trachomatis / EcoRI
agcaGAATTCatgttggccttttttttgcga
CT226 C. trachomatis / EcoRV
agcaGATATCttatatcagactttcttccaa
CT227 C. trachomatis / EcoRI
agcaGAATTCatgtcttatcttttttgttcc
CT227 C. trachomatis / EcoRV
agcaGATATCtcatgagacacttatagtcac
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Make expression
vectors
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Transformation
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Send for sequencing
Project #2 - part one
Project #2 - part one
Project #2 - part two
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Goal of part two:
1.
Attain double transfection of same host mammalian cell
2.
Conclude whether or not there is an effect on polynuclear
cells
Project #2 - part two
224/223
227/223
233 only
RFP/GFP
Mock
Transfected
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
Polynuclear
Average
6
6
7
5
5
4
7
4
6
6
3
5
3
2
1
0
2
1
0
1
% of total
Total
24
28
20
20
23
100/24
20
100/20
20
100/20
6
100/6
4
100/4
20
16
28
16
24
24
12
20
12
8
4
0
8
4
0
4
Relative number of polynuclear cells (%)
Project #2 - part two
30
25
20
15
223/224
227/223
223
10
RFP/GFP
Mock
5
0
Results
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No effect on polynuclear morphology between
single transfection CT223 and double
transfections
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To do:
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Transfections with CT225, and CT226 to see if same
conclusion can be drawn
Summary
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Project #1
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Found active domain of CT223 - last 56 aa
To Do: relevance / active partner in host
Project #2
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Found no increase in rate of polynuclear cells in doubletransfected cells
To Do: look at other genes (CT225, CT226)
Acknowledgements
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Howard Hughes Medical Institute
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Dr. Dan Rockey and his Lab
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Dr. Kevin Ahern
Damir Alzhanov
Department of Biomedical Sciences in the
college of Veterinary Medicine
Thank you for your
time.
Questions?