New Strategy For Fighting Infection Proves Successful In Mice

April 25, 1997

St. Louis, April 25, 1997 -- Researchers in Maryland and Missouri have found a sticky solution to the problem of how to ward off infection in an era of antibiotic resistance.

The study, reported in today's issue of the journal Science, describes a strategy to prevent bacteria from getting a toehold on tissues and therefore from causing infection. The researchers developed a vaccine to prevent urinary tract infections caused by E. coli. The vaccine is a protein called an adhesin.

"Antibiotic resistance is becoming more and more of a problem - even simple infections like cystitis are getting harder to treat," says Scott J. Hultgren, Ph.D., associate professor of molecular microbiology at Washington University School of Medicine in St. Louis. "So it's becoming increasingly important to develop novel ways to fight infection. Vaccine development is one avenue to take."

Unlike antibiotics, which usually are given after bacteria have set up shop, the vaccine prevents E. coli from getting a foot in the door. "This is a very basic principle that should be applicable to a wide range of bacterial infections, including middle-ear infections, pneumonia, meningitis, kidney infections and gonorrhea," Hultgren says.

The technology for producing the vaccine was developed by Hultgren's team at Washington University School of Medicine in St. Louis. The product was tested by Solomon Langermann, Ph.D., and colleagues at MedImmune Inc., a biotechnology company in Gaithersburg, Md.

Bladder infection or cystitis affects mostly women, whose anatomy allows bacteria from feces to be swept into the urinary tract. At least half of American women have this itchy and painful problem once during their lives, and up to 10 percent suffer three to five infections per year. The disorder accounts for 7 to 8 million visits to physicians and hospitals each year at a cost of more than $1 billion.

E. coli - like many bacteria - is covered with fine hairs called pili, which have sticky tips. Hultgren's group has shown that one of the proteins at the tip, an adhesin, fits into a receptor on the bladder lining. By locking its pili into these receptors, a bacterium can cling to the bladder like Velcro. Even a flood of urine fails to sweep it away.

Adhesin-based vaccines offer a novel approach to ward off infectious disease. "The idea is very attractive because such a vaccine would give bacteria a double whammy - antibodies against the protein would both block attachment and mark bacteria for destruction by the immune system," Hultgren says.

Langermann and colleagues analyzed bacterial samples from cystitis patients across the United States. They found that FimH, the adhesin produced by the bladder-loving E. coli, hardly varies from strain to strain. Antibodies to FimH also cross-reacted with more than 95 percent of the strains and prevented binding to bladder cells. Therefore a single vaccine might prevent recurrent bladder infection, even if a succession of strains was involved.

By removing the FimH gene from E. coli, the researchers also proved that a bacterium without sticky pili is as useless as a Post-it Note without gum. "So the binding event is absolutely critical to the bacterium's ability to cause disease," Hultgren says.

Painstaking studies of pilus assembly allowed Hultgren's team to produce correctly folded adhesin that could be used as vaccine. "This is a nice example of how basic science can promote advances in clinical care," Hultgren says.

Over the past decade, his group has shown that the pilus assembly line begins when adhesin molecules and other components are shipped to the periplasmic space - the region between the two membranes that surround E. coli. Boomerang-shaped proteins called chaperones latch onto these building blocks and also mold adhesin into shape. When the chaperone-adhesin complexes reach the outer membrane, the chaperones release their cargo to tunnel-shaped proteins called ushers - mini spaghetti machines that assemble and extrude pili to the bacterial surface.

To obtain FimH for purification, the researchers stepped up adhesin production and derailed the assembly process. They attached "on switches" to chaperone and adhesin genes, inserted the resulting complexes into E. coli, and deleted the usher gene. The product is a bacterium that overproduces chaperones and correctly folded adhesin molecules. These complexes accumulate in the periplasm, staying soluble instead of gluing themselves onto pilus tips. "So by getting rid of the ushers, you end up with chaperone-adhesin complexes that are easy to purify in large amounts," Hultgren says.

He supplied purified FimH to Langermann, who showed that the adhesin triggers a strong, long-lasting immune response when injected into mice. The MedImmune researchers also applied the resulting antibodies to cultured cells from the bladder lining, finding that they prevent E. coli from binding to the cells. To see if the vaccine can have the same effect in live animals, they inoculated E. coli into the urinary tract of mice. The animals developed full-blown bladder infections - unless the E. coli lacked the gene for FimH. But vaccination with FimH allowed normal mice to resist infection - the linings of their bladders had 100 to 1,000 times fewer bacteria than those of unvaccinated mice. The anti-adhesin antibodies also prevented bladder infection when they were introduced into the bloodstream via the abdominal cavity.

"This is the first demonstration that antibodies targeted to a bacterial adhesin offer protection," Langermann says. "It also suggests that, by targeting proteins such as adhesins, one might be able to induce protection through systemic immunization."

The FimH vaccine is being tested on monkeys at the Karolinska Institute in Sweden by Staffan J. Normark, M.D., Ph.D., who identified adhesins as minor components of pili in 1987. If the monkey trials are successful, the vaccine will be tested in humans. The human bladder contains the same receptor as that of mice.

Because Hultgren's group has shown that almost 30 different kinds of adhesins are assembled via the chaperone-usher pathway, the researchers hope to develop additional products to fight infectious disease. "Dissecting out the molecular details of these pathways has allowed us to create advanced technologies for developing agents that block the assembly of adhesive pili," Hultgren says. "These would represent a whole new class of antibiotics - one of the first in 20-to-30 years."
A news release from MedImmune also is available (301-417-0770 x 295). For graphics ideas, contact Linda Sage (314-286-0119).

Langermann S, Palaszynski S, Barnhart M, Auguste G, Pinkner JS, Burlein J, Barren P, Koenig S, Leath S, Jones CH, Hultgren SJ. (1997), Science, 276, 607-611.

This study was funded by the National Institute of Diabetes and Digestive and Kidney Diseases and by MedImmune.

Washington University School of Medicine

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