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Stanford Report, September 6, 2000

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