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.

GPIC 425

GPIC 426

Hydrophilicity plots provide evidence that these proteins are localized to the inclusion membrane.

Primers were designed for Ct 58, CWL 369, GPIC 425, and GPIC 426. They were ordered from Sigma-Genosys 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

426 425 369 58

PCR screen of transformed E. coli colonies

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.

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.

425 369Lad

Moving

We left Nash and Microbiology for Dryden and Vet Med.

Continuing Research1. 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 toHHMI.

Dan Rockey

Everyone in the Rockey lab.