Transcript Production of plasmid-encoded chlamydial proteins in host cells Jeffrey Burnett Dr. Dan Rockey Lab.

Production of plasmid-encoded
chlamydial proteins in host cells
Jeffrey Burnett
Dr. Dan Rockey Lab
Chlamydia trachomatis
▫
most common STI
▫
▫
causes:
▫
▫
▫
▫
▫
2.8 million cases annually
Pelvic Inflammatory Disease
Ectopic pregnancy
Infertility
Preventable blindness
asymptomatic
C. trachomatis
▫
▫
Gram (-) bacterium
Obligate intracellular
aerobe
▫ Cannot make its own ATP
▫
▫
Inclusion
Biphasic lifecycle
▫
▫
Non-infectious
metabolically active form
Infectious “spore” form
Background knowledge

Horoschak and Moulder


Bose and Liebhaber


Decrease in amount of cells
No decrease in DNA synthesis
Greene and Zhong

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

Rockey et al
Inclusion membrane proteins (inc)
 CT119 - incA


Identification of CT 223 as inc gene
My Project



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

Active site of CT223 is c-terminus 56 AA
sequence

To do:

What is the relevance?


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

Make expression
vectors

Transformation

Send for sequencing
Project #2 - part one
Project #2 - part one
Project #2 - part two

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
2 2 4 /2 2 3
2 2 7 /2 2 3
2 3 3 only
RFP /G FP
M oc k
T rans fec ted P olynuc lear
25
6
25
6
25
7
25
5
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
5
4
7
4
6
6
3
5
3
2
1
0
2
1
0
1
A verage
24
28
20
20
% of total
T otal
23
1 0 0 /2 4
20
1 0 0 /2 0
20
1 0 0 /2 0
6
1 0 0 /6
4
1 0 0 /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

No effect on polynuclear morphology between
single transfection CT223 and double
transfections

To do:

Transfections with CT225, and CT226 to see if same
conclusion can be drawn
Summary

Project #1



Found active domain of CT223 - last 56 aa
To Do: relevance / active partner in host
Project #2


Found no increase in rate of polynuclear cells in doubletransfected cells
To Do: look at other genes (CT225, CT226)
Acknowledgements

Howard Hughes Medical Institute


Dr. Dan Rockey and his Lab


Dr. Kevin Ahern
Damir Alzhanov
Department of Biomedical Sciences in the
college of Veterinary Medicine
Thank you for your
time.
Questions?