Untangling the Structure of Lyme Disease ESIAP FYI

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http://www.sc.doe.gov/Science_News/feature_articles_2001/may/Untangling_the_Stru\
cture/Untangling_Structure_of_Lyme_Disease.htm

Untangling the Structure of Lyme Disease

The Department of Energy's National Synchrotron Light Source at
Brookhaven National Laboratory helped researchers discover new
information about the bacterium that causes Lyme disease. Their work
may lead to an effective vaccine and new treatment protocols.

Ixodes scapularis (deer ticks) are the most common vector for Lyme
disease. Larval and nymphal ticks are no bigger than the eye of a
common sewing needle. Adult ticks are about the size of a small apple
seed. May 7-"It's the perfect stealth bacteria," says one frustrated
physician. He's talking about Borrelia burgdorferi, the bacterium that
causes Lyme disease. This illness, which is often mistaken for diseases
ranging from multiple sclerosis to Lupus, can inflict excruciating
headaches and muscle pain, affect the brain and nervous system, attack
major organs, and inflame joints. Although there have been more than
100,000 reports of the tick-borne Lyme disease in the U.S. since 1982,
researchers are still struggling to create vaccines and treatments that
are effective against B. burgdorferi.

New findings may explain problems with vaccine effectiveness, suggest
treatment approaches Investigators are particularly pleased with two
recent discoveries made using the Department of Energy's National
Synchronous Light Source (NSLS) at Brookhaven National Laboratory. The
uniquely refined images they were able to
create demonstrated the bacterium changes its outer surface protein
according to its host, and that different strains of the bacterium have
different electrical charges, which may determine their ability to
cause disease.

Outer surface, or peripheral, proteins do not penetrate the cell wall
and are easily shed, but they are significant in determining a cell's
capabilities-for example, how it attaches to other cells or survives in
specific environments.

"These findings make it clear what direction the research should take,"
says John Dunn, a biologist and principal investigator at Brookhaven
National Laboratory. "There's a lot of work left to do, but now we have
a much better sense of where we should be looking."

Researchers from Brookhaven, Stony Brook University's School of
Medicine, the University of Rochester medical Center, and Rutgers
University reported their findings on the OspC structure in the March
1, 2001 edition of the EMBO Journal.

Computer-generated image of the OspA structure found on the B.
burgdorferi bacterium. OspA is supressed when the bacterium moves from
the tick gut into mammalian blood streams. Altered surface proteins As
B. burgdorferi moves by tick bite from the gut of a tick to the
bloodstream of a mammal, it suppresses one outer surface protein (an
"Osp"), called OspA, and switches on another, called OspC. The switch
is regulated, at least partly, by temperature. OspA is expressed at
temperatures below 32 degrees C, and is synthesized in the 24 degrees C
environment of the tick gut. Between 32 to 37 degrees C, the range for
mammalian blood, OspA is suppressed and OspC is synthesized. The genes
for producing these proteins appear to be controlled by
mRNA, and the process suggests that the bacterium has developed
mechanisms that permit sustained survival in two very different hosts.

This switch may explain some of the problems encountered with the
original Lyme disease vaccine, which was developed to counteract OspA.
Vaccines that confer active immunity, such as the OspA vaccine, are
very specific and stimulate the immune system to attack invading cells
that exhibit a particular protein. Because B. burgdorferi does not
exhibit OspA in the human body (or exhibits it weakly), the immune
system of the vaccinated person doesn't "recognize" the bacterium.

Invasive bacterium may pack a negative charge

The Brookhaven researchers also found that gene sequences within
different groups of OspC itself are highly variable. To date, 19 major
groups (A-S) have been identified; they differ from each other at the
dimer interface on the surface of the cell (a dimer is molecule in the
protein chain that is made up of two identical, simpler, molecules).
Apparently only four OspC groups (A, B, I,
and K) are "invasive"-that is, are responsible for systemic human
disease.

Computer-generated image of the OspC-HB19 dimer structure found on the
B. burgdorferi bacterium. Four OspC groups are implicated in Lyme
disease in humans.

Further, the invasive bacteria appear to share a common trait: they all
have a strong negative charge in the area of the OspC dimer. The
researchers postulate that this negative charge may help the bacterium
attach to cell tissue, which carries a more positive charge.

"Understanding the correlation between surface charge and invasiveness
may be useful not only in developing an effective vaccine, but also in
predicting whether other OspC bacterium are likely to cause disease,"
said Subramanyam Swaminathan, another member of the Brookhaven research
team.

Understanding the structure is the key

The new understanding of the structure was made possible by the protein
fixation and imaging techniques at NSLS. The NSLS permits researchers
to focus and control light beams such that images can be seen at
resolutions as fine as 2 A-near atomic resolution.

It is no easy matter to concoct fragile organic matter, such as protein
chains, into crystals that can withstand the powerful radiation
bombardment of the NSLS and yet retain their original structure. To do
this, the Brookhaven team drew upon available nuclear magnetic
resonance (NMR) information to identify the
least stable areas of the OspC protein-the C and N termini. They
truncated the protein to remove these termini and improve their chances
of crystallizing portions of the protein into a stable, viewable form.
They then expressed and purified the protein to ensure homogeneity, and
grew them as crystals.

These crystals were frozen to liquid nitrogen temperature and then
illuminated with the NSLS beams. By varying the wavelength of the light
beams and by using a technique called multiple wavelength anomalous
diffraction (MAD), the researchers generated more than 120,000
different "reflections" (diffraction patterns).

Using computer-assisted analysis and visual imaging techniques,
researchers resolved the diffraction patterns into vivid 3-D views of
both the shape and surface characteristics (such as the charge) of that
portion of the OspC protein. Once the basic shape of the protein-how it
"folds"-was determined for two of the invasive OspC groups, the
researchers used computer modeling techniques to infer the structure of
the remaining 17 groups, considerably speeding the investigative
process. The technique and findings are discussed by Swaminathan and
fellow researchers in the March 2001 edition of Acta Crytallographica,
D57. This information about structure and the techniques used to derive
it are expected to prove significant in understanding the behavior of
other disease-causing bacteria.

The next step, developing an OspC vaccine, is not a simple task.
However, says Dunn, having the structure of the OspC protein is a major
step forward.-Michaela Mann Media contacts: Diane Greenberg, BNL, (631)
344-2347, greenb@...
Mona S. Rowe, BNL, (631) 344-5056, mrowe@...

Research contacts: John Dunn, BNL, (631) 344-3012, jdunn@...
Subramanyam Swaminathan, BNL, (631) 344-3187, swami@...


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Related web links from DOE's Virtual Resource Library:

"Crystal structure of outer surface protein C (OspC) from the Lyme
disease spirochete, Borrelia burgdorferi," D. Kumaran, S.
Eswaramoorthy, B.J. Luft, S. Koide, J.J. Dunn, C.L. Lawson1, and S.
Swaminathan1, The EMBO Journal, Vol. 20, No. 5 pp. 971-978, 2001
(European Molecular Biology Organization)

"Borrelia burgdorferi outer surface protein C (OspC)," Kumaran, D.,
Eswaramoorthy, S., Dunn, J. J. & Swaminathan, S. (2001).
Crystallization and preliminary X-ray analysis of Acta Cryst. D57,
298-300. (requires subscription - Foundations of Crystallography
Online)

National Synchrotron Light Source website

Lyme Disease Network

Borrelia burgdorferi sensu lato Molecular Genetics Server

Protein Crystallization (steps involved in protein production,
purification, and crystallization)

Protein Crystallography

Interactive tutorial about diffraction

X-ray Anomalous Scattering (for crystallographers considering MAD
(multiple-wavelength anomalous diffraction)

-------------------------------------------------------------------Funding:
The National Synchrotron Light Source, at Brookhaven National
Laboratory in New York, is a national user research facility funded by
the U.S. Department of Energy's Office of Science, Division of Basic
Energy Science. The NSLS operates two electron storage rings: an X-Ray
Ring and a Vacuum Ultra Violet (VUV) Ring which provide intense focused
light spanning the
electromagnetic spectrum from the infrared through x-rays.

Laboratory: Brookhaven National Laboratory creates and operates major
facilities available to university, industrial and government personnel
for basic and applied research in the physical, biomedical and
environmental sciences, and in
selected energy technologies. The Laboratory is operated by Brookhaven
Science Associates, a not-for-profit research management company, under
contract with the U.S. Department of Energy.

Author: Michaela Mann is a science writer and electronic communications
specialist at Pacific Northwest National Laboratory in Richland,
Washington. She was formerly the managing editor and original website
developer of Energy Science News, an award-winning online newsletter
for DOE's Office of Science.
Ms. Mann is also a gifted licensed, practicing massage therapist.

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