Bacterial communication,
toxin production tied to intriguing cell protein
BY KRISTIN WEIDENBACH
Stanford researchers have
found that a molecule once dubbed a "molecular
fossil" for its perceived irrelevance inside cells
may instead be involved in determining how dangerous
bacteria harm plants, humans and other animals.
Polyphosphate is a long,
chain-like molecule found in every living cell.
Scientists believe that in animals, one of its roles may
be to serve as a phosphate storage reservoir for the
production of ATP (adenosine triphosphate), which
provides the energy to power a cell. In bacteria,
polyphosphate helps these single-celled organisms adapt
to nutritional deficiencies and environmental stresses.
It also helps them survive the state of suspended
animation known as the stationary phase of growth.
In 1990, Arthur Kornberg,
MD, Nobel laureate and emeritus professor of
biochemistry, and a postdoctoral fellow in his lab
defined the enzyme that produces polyphosphate in the
bacterium Escherichia coli. The enzyme, polyphosphate
kinase (PPK), makes polyphosphate by stringing together
many phosphate residues. Kornberg has an enduring
interest in all kinds of enzymes and is particularly
interested in this one because its product is ubiquitous
in biology. And yet the function of this product
polyphosphate is relatively unknown.
"Everything in nature
is based on phosphate metabolism. Every cell in nature,
every compartment within every cell has polyphosphate.
It's found everywhere, and it's been conserved for
billions of years it must be doing something
important," Kornberg said. And yet, according to
Kornberg, very few researchers are studying polyphosphate
or the enzyme that makes it. Kornberg has made it the
focus of his research group.
Since finding and
characterizing the PPK gene, members of his lab have
learned that the PPK enzyme is similar in many bacteria,
including several that cause disease in humans. Organisms
such as yeast, plants, humans and other animals have a
different kind of enzyme for producing polyphosphate.
When the researchers made
mutant strains of bacteria lacking the gene for PPK, many
of the bacteria had difficulty moving around. Pseudomonas
aeruginosa, which causes dangerous infections in people
with a deficient immune system, were unable to use their
flagella to swim or swarm around, and they were unable to
use tiny cell extensions, called pili, for twitching
motility.
Now, the Kornberg team has
found that P. aeruginosa bacteria without the gene are
also unable to form microbial communities called
biofilms, and they are unable to communicate with each
other via a process called quorum sensing. Biofilms and
quorum sensing are phenomena that occur when the bacteria
encounter a new environment and must adapt to survive.
Quorum-sensing communication between bacteria involves
the release of small molecules that float among the
bacteria and deliver a chemical message. When the
bacteria determine, via quorum-sensing communication,
that a critical mass of organisms has assembled, they use
their flagella to slide toward each other and construct a
thick, slimy biofilm.
"These bugs talk to
each other. They sense when the population has become so
dense that they have to develop another way of life a
biofilm," Kornberg explained.
Within a biofilm they are
protected from harsh changes in the environment. The
tartar on your teeth and the scum on a bathroom sink are
both biofilms. They are the scourge of surgeons because
they form on catheters and pacemakers installed long-term
in patients. P. aeruginosa biofilms are the cause of
fatal pneumonia in people with cystic fibrosis. Once
sheltered within a biofilm, bacteria are relatively
immune to antibiotics and other drugs.
Besides biofilm
development, production of the proteins and toxins known
as virulence factors, which enable bacteria to cause
disease in their host, is also under quorum-sensing
control. Researchers in Kornberg's lab found that
bacteria without the PPK gene produced much lower amounts
of several toxins. Fourteen of 15 mice infected with
these mutant bacteria survived, whereas only one of 19
mice infected with normal bacteria carrying the PPK gene
survived.
Virulence factor
production, biofilm development, quorum-sensing
communication and flagella-initiated movement would seem
to be a disparate set of cellular processes to be
affected by the absence of a single enzyme, but Kornberg
believes that timing is what ties them all together.
"Many people focus on
growing cells. We discovered that even though the bugs
appeared to be healthy in their rapidly growing phase,
when they enter the stationary phase life in the slow
lane they die," he explained. "Several
hundred genes are needed for adaptations for survival to
ensure the longevity of those organisms. For lack of PPK
they don't express those genes, and they die."
Kornberg and Harun Rashid,
PhD, who conducted much of the current research while a
postdoctoral fellow in Kornberg's lab, admit that they do
not know exactly how all these diverse processes fall
under PPK's control, but the enzyme's position at the top
of a regulatory hierarchy within the cell makes it a
prime contender as the target for a new antibacterial
drug.
"It opens up the
possibility of using PPK as a novel antibiotic,"
said Rashid. "Inhibitors of this enzyme could be
used as an antibiotic to disrupt cell-to-cell
communication in bacteria."
Because the gene that
makes polyphosphate kinase is the same in many
disease-causing bacteria and yet different from the
enzyme that makes polyphosphate kinase in humans, a drug
that will seek out and disrupt the bacterial enzyme will
kill the harmful microbes and leave human cells intact.
Kornberg says that Stanford is already negotiating with
pharmaceutical companies to search for potential drugs.
Kornberg, Rashid and their
colleagues published the results of their study in the
August 15 issue of the Proceedings of the National
Academy of Sciences. Rashid is now a research scientist
at Genencor, Palo Alto. Researchers at Texas Tech
University Health Sciences Center, the University of
Rochester and Binghamton University contributed to the
study. Funding was provided by the National Institutes of
Health and the Cystic Fibrosis Foundation. SR
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