Transcript The Biology of Chlamydia trachomatis

Chlamydial inclusion membrane
proteins: localization and
characterization
Nathanael Blake
HHMI Summer Internship
Mentor: Dr. Dan Rockey
The Chlamydia
The Chlamydia are an group of ubiquitous intracellular
pathogens distinguished by a unique biphasic lifecycle.
Chlamydia trachomatis
Two sites of infection: ocular and
genital.
Ocular strains cause several million cases of
blindness each year, mostly in poor nations.
Genital strains common in Western nations.
4 to 5 million cases per year in the US.
Chlamydia pneumoniae
Infects the lungs.
Ubiquitous, majority of humans are infected.
All effects of disease not known.
Asthma, chronic bronchitis?
Also, it has recently been linked to heart
disease and atherosclerosis.
Chlamydophila caviae
Infects guinea pigs.
Good animal model from which to extrapolate
about C. trachomatis in humans.
Treatments
No vaccine.
Laboratory tests required to confirm infection.
Rockefeller Foundation offered 1 million dollars for
simple test for C. trachomatis.
Closed after five years because no one succeeded.
Most infections asymptomatic.
Antibiotics easily cure almost all cases.
The Chlamydial Lifecycle
The Chlamydial inclusion
membrane
Proteins to be studied
I’m are seeking to confirm that these are localized to the inclusion membrane
and to examine their interactions with human proteins.
Hydrophilicity plots provide evidence that these proteins are
localized to the inclusion membrane.
GPIC 425
GPIC 426
Primers were designed for Ct 58, CWL 369, GPIC 425, and GPIC 426. They were ordered from SigmaGenosys and used to amplify portions of these genes from genomic DNA via PCR. Gel electrophoresis
was used to determine that they had worked and fragments corresponding to the size of the target
sequence were extracted from the gel with a QIAGEN gel extraction kit.
Ct58
CWL369
Ladder
GPIC425 Ladder GPIC426
The expression vector
PCR screen of transformed E. coli colonies
426
425
369
58
I ligated the digested plasmid (P-Mal C2) with the targeted gene fragments
and then transformed E. coli with the result. I screened for successful
transformation with LB Ampicillin plates and then ran a PCR screen on the
colonies. True positives were found for all but GPIC 426.
I then induced 58, 425, and 369 and harvested the protein.
While 58 yielded very little product, 425 and 369 provided
useable quantities.
Lad
425
369
Current Status
1. 425 and 369 are being sent off for antibody production.
2. I’m working on inducing 58.
3. 426 has not yet been transformed, despite repeated
attempts.
Moving
We left Nash and
Microbiology for Dryden and
Vet Med.
Continuing Research
1. Once 369 and 425 antibodies are received, I’ll use
fluorescent microscopy to determine their localization.
2. I will continue in my attempts to troubleshoot the
transformation of E. coli with 426 and the induction process for
58.
3. Finally, I will begin work on two-hybrid analysis of these
proteins to examine their interactions with human proteins.
This work will be carried on through the school year, via the
undergraduate research program.
Thanks to
HHMI.
Dan Rockey
Everyone in the Rockey lab.