Chlamydial inclusion membrane proteins: localization and characterization

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Chlamydial inclusion membrane proteins
localization and characterization

• Nathanael Blake
• HHMI Summer Internship
• Mentor Dr. Dan Rockey

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The Chlamydia
The Chlamydia are an group of ubiquitous
intracellular pathogens distinguished by a unique
biphasic lifecycle.
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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.

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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.

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Chlamydophila caviae

• Infects guinea pigs.
• Good animal model from which to extrapolate about
  C. trachomatis in humans.

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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.

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The Chlamydial Lifecycle
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The Chlamydial inclusion membrane
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Proteins to be studied
Im are seeking to confirm that these are
localized to the inclusion membrane and to
examine their interactions with human proteins.
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Hydrophilicity plots provide evidence that these
proteins are localized to the inclusion membrane.
GPIC 425
GPIC 426
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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
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The expression vector
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PCR screen of transformed E. coli colonies
426
369
425
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.
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I then induced 58, 425, and 369 and harvested the
protein. While 58 yielded very little product,
425 and 369 provided useable quantities.
425
Lad
369

• Current Status
• 425 and 369 are being sent off for antibody
  production.
• Im working on inducing 58.
• 426 has not yet been transformed, despite
  repeated attempts.

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Moving
We left Nash and Microbiology for Dryden and Vet
Med.
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Continuing Research
1. Once 369 and 425 antibodies are received,
Ill 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.
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Thanks to
HHMI. Dan Rockey Everyone in the Rockey lab.