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View from
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Lyme Disease
A Lucky
Catch
Artificial
Pancreas
People
About
Subscribe
Free
This issue...
News in
Brief
View from
the Inside
Lyme Disease
A Lucky
Catch
Artificial
Pancreas
People
About
Subscribe
Free
This issue...
News in
Brief
View from
the Inside
Lyme Disease
A Lucky
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Artificial
Pancreas
People
About
Subscribe
Free |
Untangling the Structure of Lyme
Disease
by Michaela Mann
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. |
"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 vaccine
failure, 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.
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.
 |
Computer-generated image of the
OspA structure found on the
B. burdgoferi bacterium.
OspA is supressed when the
bacterium moves from the tick
gut into mammalian blood
streams. |
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. burdgoferi
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.
Media contacts:
Diane Greenberg, BNL, (631)
344-2347,
greenb@bnl.gov
Mona S. Rowe, BNL, (631) 344-5056,
mrowe@bnl.gov
Research contacts:
John
Dunn, BNL, (631) 344-3012,
jdunn@bnl.gov
Subramanyam Swaminathan, BNL, (631)
344-3187,
swami@bnl.gov
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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