Immune System Molecule Weaves
Cobweb-Like Nanonets to Snag
Salmonella, Other Intestinal
Microbes
ScienceDaily (June 21, 2012) — A
team of researchers led by UC Davis
Health System has found that human
alpha-defensin 6 (HD6) -- a key
component of the body's innate
defense system -- binds to microbial
surfaces and forms "nanonets" that
surround, entangle and disable
microbes, preventing bacteria from
attaching to or invading intestinal
cells.
The research describes an entirely
new mechanism of action for
defensins, an important group of
molecules known to bolster the
defenses of circulating white blood
cells, protect cellular borders from
invasive pathogens and regulate
which "friendly" microbes can
colonize body surfaces. The discovery
provides important clues to
inflammatory bowel diseases,
especially Crohn's disease, which may
be caused, in part, by deficiencies in
HD6 levels or function.
A paper describing the work appears
in the June 22 issue of the journal
Science.
"During the past 25 years, researchers
have learned a lot about the biological
function of defensins, but the role of
HD6, a particular molecule that is
highly expressed in the intestines, was
a mystery," said Charles L. Bevins,
professor of microbiology and
immunology at UC Davis. "We now
know that HD6 has a very unique role
in the body's innate immune system.
Its ability to latch onto microbial
surfaces and self-assemble to cast a
fibrous net around bacteria, including
pathogens like Salmonella and
Yersinia, as well as fungi and
protozoan parasites, gives the
intestine, a critical part of the body, a
powerful and broad spectrum of
defense against potential threats."
Bevins is co-senior author of the
paper along with his UC Davis
colleague Professor Andreas Bäumler,
an expert in bacterial pathogenesis;
UCLA Emeritus Professor Robert I.
Lehrer, whose laboratory was the first
to discover defensins in the early
1980s; and Professor Wuyuan Lu, a
synthetic protein chemist from the
University of Maryland School of
Medicine whose work provided clues
to HD6's subtle and unique
properties. First author Hiutung Chu, a
graduate student in the Bevins lab
who is now a fellow at the California
Institute of Technology, was a driving
force on the nine-year quest to solve
the HD6 puzzle.
About the protein HD6
Defensins are a family of structurally
related, small peptides with antibiotic
activity found throughout nature in
plants and animals. Humans make six
different alpha-defensins. Two of
these, HD5 and HD6, are secreted by
Paneth cells, specialized secretory cells
located within the folds of the small
intestinal lining. HD5 has well-known
antibacterial properties while the
function of HD6 had been unknown.
The defensin-rich secretions of Paneth
cells work in conjunction with nearby
intestinal stem cells to maintain micro
flora balance and renew intestinal
cellular surfaces.
Chu's graduate work focused on
characterizing the biological activity of
HD6 in studies using cultured
intestinal epithelial cells and
transgenic mouse models. Although
Chu and Bevins anticipated HD6
activity would be very similar to other
alpha-defensins, which kill pathogens
by poking holes in the microbial
membrane, their early research
studies repeatedly showed that HD6
did not kill bacteria. Puzzled, they then
looked for other possible functions,
collaborating with UC Davis professors
Angela Gelli and Scott Dawson to see
if HD6 might kill only certain bacteria,
fungi or parasites. It did not.
After two years into the project and
feeling frustrated about the negative
results, Bevins and Chu carefully
reviewed the experimental data.
That's when they recognized two
crucial pieces of information. The first
was that whenever HD6 was added to
suspensions of either bacteria or
fungi, a white haze, or precipitate,
formed in the solution (see image
below). The second was that early
studies conducted in collaboration
with Bäumler had shown that while
HD6 did not kill the bacterial
pathogen Salmonella, it protected
transgenic mice from an otherwise
lethal infection.
"When we put these two results
together, we were able to
systematically show that HD6 was
inhibiting microbial invasion and
uncover HD6's unique structure and
function at multiple levels," said
Bevins.
On the road to discovery
The UC Davis team then collaborated
with Lehrer, whose research focuses
on the study of defensins and other
antimicrobial peptides that serve as
natural antibiotics. In his laboratory,
he had a surface plasmon resonance
instrument that measured molecular
binding in real time. This technique
captured the progressive assembly of
HD6 molecules, from binding to
bacterial proteins at the microbial cell
surface to the self-assembly to form
fibrils and the sequential addition of
fibrils (see images below).
Through the expertise of Lu, a
synthetic protein chemist and expert
in defensin structure and function
relationships, the team obtained
sufficient quantities of the highest-
grade HD6 peptide and subtle
molecular variants of HD6 to test their
hypotheses experimentally. Lu was
able to identify critical structural
components of HD6 that enabled it to
self-assemble into fibrils. One feature
unique to HD6 is the manner in which
four HD6 molecules combine to form
a building block whose further
assembly creates both fibers and nets.
The researchers also found that
changing just one of the 32 amino-
acid residues of the HD6 molecule --
histidine-27 -- impaired HD6's ability
to form a tetramer in the x-ray crystal
structure. As a result, HD6 lost the
special binding that Lehrer found in
his real-time experiments, blocked the
ability of HD6 to form nanonets and
abrogated its ability to inhibit bacterial
invasion.
The Bäumler laboratory created vital
bacterial mutants affecting the
molecules that HD6 initially binds to
on the surface of the microbe. When
those molecules were knocked out in
the transgenic mouse model, HD6 did
not form the fibrils on the bacterial
surface.
"This series of experiments provided
the vital 'glue' to bind the many facets
of the story together, and to convince
ourselves and our peers that we had
finally solved the mechanism of HD6
action," commented Bevins.
Clues to innate immunity and
inflammatory bowel diseases
The UC Davis research describes how
HD6 contributes to the body's innate
immunity, which protects from
microbes that the immune system
might not have any experience in
managing.
"The innate immune system has to be
able to deal with diverse microbes that
might have all kinds of tricks that
cause infection," said Bevins. "After
we've been exposed to a microbe or
an infection the first time and survive
it, the adaptive immune system can
recognize and remember specific
pathogens to generate immunity and
to mount stronger defenses each time
the pathogen is encountered. HD6 is
a major player in helping the body
prevent potentially dangerous
pathogens from coming into close
physical contact with intestinal
epithelial cells, as well as the stem
cells that continuously renew the
epithelial cell surface."
Previously published studies from the
Bevins lab have linked alpha-defensins
and Crohn's disease, a chronic
inflammatory bowel disease that
investigators associated with HD5 and
HD6 deficiencies. The secretions of
these defensins typically occur at the
base of the out pouches (so-called
crypts) of the small intestinal surface,
where they are ready to fend off
bacteria that become dangerously
close to the intestinal lining.
Individuals with Crohn's disease,
however, tend to accumulate invasive
bacteria in this same area, developing
a chronic inflammation that is self-
perpetuating.
"With less of these important defense
molecules, microbes that would
normally exist in the gut, can irritate
the intestinal surface and cause the
chronic inflammation that
characterizes Crohn's disease," said
Bevins. "We know a lot about HD5's
antimicrobial activities, so it makes
sense why reduced HD5 levels might
contribute or allow this condition to
progress. Now we have a clue how
HD6 levels play a role."
Future studies on Crohn's disease by
this team aim to better understand
exactly why alpha-defensin-
expression is reduced in individuals
with Crohn's disease, and perhaps
devise strategies to boost the body's
production of these vital molecules.
"The multidisciplinary approach that
we used to 'crack' the obscure and
complex action of HD6 exemplifies
the power of team science," Bevins
said. "Not to be underestimated,
however, is the courage and
tenaciousness of graduate student
Hiutung Chu in leading the
experimental investigations. Many
blind alleys were visited as we
investigated this molecule, and those
frustrating diversions can erode
confidence and morale. Hiutung
deserves tremendous credit for
persevering through those setbacks."
Other authors on the paper include
Marzena Pazgier from the University
of Maryland School of Medicine, Grace
Jung from the David Geffen School of
Medicine at UCLA, Bo Shen at the
Cleveland Clinic Foundation, Nita H.
Salzman at the Medical College of
Wisconsin and Mark A. Underwood,
Glenn M.Young, Sean-Paul Nuccio,
Patricia A. Castillo, Maarten F. deJong,
Maria G. Winter, Sebastian E. Winter
and Jan Wehkamp, all from the
University of California, Davis.
This research was supported by
grants from the National Institutes of
Health.
http://www.sciencedaily.com/releases/2012/06/120621151512.htm