June 02, 2005
Gram-Negative Bacteria Shoot Their Way Into Cells
The bacteria that cause food poisoning, bubonic plague, and whooping
cough all deploy the same weapon to infect the body. A molecular
“syringe” sticks out of the bacteria, pokes a hole in a
nearby cell, and squirts in venomous proteins that hijack the cell's
machinery.
For the first time, Howard Hughes Medical Institute researchers have
captured a detailed picture of the large doughnut-shaped base of the
syringe barrel embedded in the bacterial membranes. The findings are
reported in a paper in the June 2, 2005, issue of the journal
Nature.
“We believe this ring forms the foundation upon which all of the other components assemble. This provides a real potential point of intervention.”
Natalie C.J. Strynadka
This first atomic picture of a major structural component of the
hazardous molecular hypodermic may help scientists develop a new kind
of drug that can disable the syringe and render disease-causing
bacteria harmless while sparing beneficial bacteria. Currently, doctors
must fight bacterial infections with antibiotics, which kill all
bacteria, good and bad. Furthermore, the researchers are optimistic
that drugs of this type might be effective against pathogens that are
resistant to existing antibiotics.
 | | | | |  |  | | | | |  |  | Schematic of the Type III secretion "syringe" in enteropathogenic E.coli
Twenty highly conserved proteins form an apparatus which spans the inner and outer membrane of the bacteria... more
| | | | | |  | | EscJ inner-membrane ring structure
A composite figure showing a surface rendering of the critical EscJ inner-membrane ring structure... more
Images: Natalie Strynadka
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"We believe this ring forms the foundation upon which all of the
other components assemble," said senior author Natalie Strynadka, a
HHMI international research scholar and associate professor of
biochemistry at the University of British Columbia in Vancouver,
Canada. "Without this assembly, there is no pathogenesis. This provides
a real potential point of intervention."
The syringe is used almost exclusively by pathogenic bacteria, such
as the Escherichia coli (E.coli) sometimes found in
uncooked hamburgers, the Pseudomonas that cause life-threatening
infections in the lungs of people with cystic fibrosis, and the plague
bacillus, Yersinia pestis, that causes so-called black death.
Many major plant pathogens also use the same piercing needle to
puncture plant cells.
The barrel of the syringe spans the inner and outer membranes of
gram-negative bacteria, a major category of microbes that have
multilayered cell walls. Two proteins at the tip of the needle drill
into other cells. Each species of gram-negative bacteria injects a
distinctive blend of proteins that do different things to the cell and
result in diverse diseases. The shared molecular machinery that injects
the customized protein is called a type III secretion system.
"This is a major leap forward in understanding how type III systems
work, because these things are so conserved among the rogues' gallery
of gram-negative bacteria," said co-author B. Brett Finlay, another
HHMI international research scholar and the Peter Wall Distinguished
Professor in the Michael Smith Laboratories at the University of
British Columbia. "It's been a black box. This gives us an inside
edge."
The new molecular model shows a circle of 24 identical interlocking
molecules that hint at how the other syringe components may fit
together. This large ring sits on top of the inner membrane of the
gram-negative bacterium, the portal where infectious proteins gather to
exit the cell.
The new model is based on experiments by first author Calvin Yip, a
graduate student in Strynadka's lab, using a technique called x-ray
crystallography. The number of molecules was confirmed by analysis of
whole bacteria in the lab of co-author Sam Miller at the University of
Washington. Together, these experiments revealed details that confirm
and enhance the blurry picture of the secretion system previously
reported by other researchers relying on an electron microscope.
The protein ring, called EscJ, is a large, fatty molecule that had
defied repeated attempts to grow it into the orderly crystals needed
for detailed structural studies. One of the problems was that the
ring's surface is glazed with positively charged atoms that push clones
of themselves away like magnets repelling each other.
The breakthrough in the lab came when Yip made a mutant version to
reduce some of the surface charge. That allowed the mutant EscJ
molecules to line up in orderly arrays in a crystal. Researchers in
Finlay's lab confirmed that the mutant protein could still form a
functional syringe in bacteria. Based on the way the EscJ molecule is
packed in the crystal, the scientists suspect that EscJ forms a ring
that functions as a molecular platform for the assembly of the
secretion system.
The greatest excitement about the new atomic structure comes from
the potential to selectively target a type of disease-causing bacteria,
the researchers said.
"Part of why this is so important is that antibiotics are becoming
resistant and are not working anymore," Finlay said. "All the major
pharmaceutical companies have pulled out. There are no new antibiotics
in the drug development pipeline. We need different ways of thinking
about going after bacteria."
Additionally, the crystal structure may illuminate more bacterial
functions than their injection apparatus, Yip said. Several dozen genes
clustered together on a region of the chromosome known as a
"pathogenicity island" produce the components of the syringe, Yip
explained. Interestingly, several of those genes closely resemble
another set of genes that make up the flagella, the beating hairs that
propel some bacteria.
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