Mysterious "Hairs" Help E. coli Cause Diarrhea.

STANFORD, Calif.--(BW HealthWire)--June 25, 1998--A stoic bunch of volunteers willingly swallowed diarrhea-causing organisms and then endured the consequences, helping Stanford scientists uncover an unusual strategy by which E. coli bacteria make people sick.

The researchers, led by professor of medicine Dr. Gary Schoolnik and senior research scientist David Bieber, describe the randomized, double-blind study in the June 26 issue of the journal Science.

The study involved 60 people who volunteered to spend three days in the NIH-funded General Clinical Research Center at Stanford University Medical Center. At the outset, each volunteer drank a liquid containing a disease-causing strain of Escherichia coli bacteria. Diarrhea from this particular strain poses a deadly threat to children in developing countries.

Many strains of E. coli live harmlessly in the intestines. However, a subgroup known as the enteropathogenic E. coli (EPEC) can cause diarrhea. No one knows exactly what enables EPEC to make people sick.

First Clue

A chance observation in 1991 by a postdoctoral research fellow in Schoolnik's laboratory provided some forward momentum in the burgeoning field of EPEC research. When the diarrhea-causing E. coli strain grew on blood agar instead of the usual growth medium, electron microscopy revealed mysterious hair-like appendages extending from each bacterium.

The researchers named these appendages bundle-forming pili, and the Schoolnik lab has been studying them ever since.

Each bundle-forming pilus is a thin, flexible protein filament that extends from the surface of the bacterial cell. The pili condense into rope-like structures that link adjacent bacteria into large clumps.

Several other pathogenic bacteria, including those responsible for gonorrhea, meningitis and cholera, display similar pili. A variety of circumstantial evidence suggested that the odd structures played a role in causing disease, Schoolnik said. He and his colleagues wondered if pili helped bacteria attach to host cells -- in this case, the epithelial cells lining the human gastrointestinal tract.

Compelling Evidence

To explore the possible role of pili in EPEC-related diarrhea, Schoolnik's team first identified the genes responsible for the presence of pili in these bacteria. Then they created mutant strains that lacked surface pili.

Schoolnik and Bieber administered three doses of each of the mutant bacteria to healthy volunteers. Another group of volunteers received a comparable dose of the non-mutant, or "wild-type," bacteria.

All stool specimens were collected and analyzed during each participant's three-day stay at the General Clinical Research Center.

The first round of clinical experiments produced dramatic results. Eleven of the 13 volunteers who received the wild-type strain developed diarrhea, and the highest dose had to be discontinued for the patients' welfare. In stark contrast, very few volunteers (five out of 30) became ill from the mutant strain lacking the pili.

This offered compelling evidence that bundle-forming pili play an integral part in causing disease.

Genetic Revelation

Next, to learn more about the structure and function of the unusual appendages, Schoolnik and his colleagues delved further into pilus genetics. Mutation of an alternative gene (called F) involved in pili production led to an intriguing result: it appeared that it's not the mere presence or absence of the pili, but rather the pili-dependent behavior of the bacteria, that is important for their ability to cause diarrhea.

At first glance the so-called F mutants seemed to behave exactly like the wild-type EPEC strain: they had pili, they attached to human epithelial cells and they clumped together in a similar fashion. But closer inspection with the video microscope revealed subtle differences in the behavior of the bacterial clusters. Wild-type bacteria formed aggregates that dispersed over time, whereas F mutant bacteria remained permanently clumped together in an agglutinated mass.

It is this pili-dependent aggregation and dispersal process that seems to be essential for the bacteria's virulence.

"We had a bet in the lab at that point," said Schoolnik. "Some bet that (the F mutant) would have increased virulence; some bet that it would have no virulence."

So it was back to the General Clinical Research Center.

Final Round

The final round of volunteer inoculations showed that the diarrheal response to the F mutant was greatly reduced at the highest dose given (10(11), or 100 billion, bacteria). "This is a dose at which you would expect an enormous amount of diarrhea if it was wild-type," Schoolnik said.

He and Bieber concluded that the F mutant bacteria retain the ability to infect and colonize the human gastrointestinal tract but fail to competently aggregate and disperse, and that this severely reduces their power to cause diarrhea.

Wild-type bacteria form "a primitive community," Schoolnik said. "The pili are somehow required for mediating dispersal of the community."

Members of his lab are now studying the basic biology of the pili, such as how they are shed or retracted when it is time for the bacteria to disperse.

Stanford co-authors on the Science paper include Sandra Ramer, Cheng-Yen Wu and Rosemary Fernandez, also from the Department of Medicine's Division of Infectious Diseases.

Funding for the research came from the National Institutes of Health (NIH).

Note to Editors: A feature article chronicling a volunteer's experience in the diarrhea study appeared in the Spring 1998 issue of Stanford Medicine magazine. For a copy, please e-mail Rosanne Spector at [e-mail] or call her at 650/723-6911.

    CONTACT:  Stanford University Medical Center
               Rosanne Spector, 650/723-6911
               [e-mail])
               Ruthann Richter, 650/723-6911
               [e-mail])
               Dr. Gary Schoolnik, 650/723-8158 (for comment)
               David Bieber, 650/723-7026 (for comment)