By David N. Leff

Out in California, late last year, 60 healthy (in mind as well as body) adult human volunteers swallowed cultures of intestinal bacteria isolated from the stools of patients with severe diarrhea. Other test subjects gulped down mutations of these pathogens, designed to understand and influence their virulence.

The bacterium in question is a variant of Escherichia coli called enteropathogenic E. coli (EPEC). "E. coli is an important cause of diarrhea and death among young children in poor countries," observed microbiologist Gary Schoolnik, at Stanford University, in Palo Alto, Calif.

In 1991, the World Health Organization estimated E. coli kills annually 3.2 million youngsters before their fifth birthday.

That happened to be the year in which Schoolnik discovered a feature of EPEC's weaponry linked to the microbe's diarrheal virulence. "A chance observation by a post-doc in my lab," he recalled, "that when we grew the EPEC strain on blood agar instead of the usual growth medium, electron microscopy revealed mysterious hairlike appendages extending from each bacterium.

"We determined that these flexible protein filaments, or pili, help EPEC bacteria grapple onto the smooth epithelial cells that line the intestines, and there clump into aggregates. Several other pathogenic bacteria," he added, "including those responsible for gonorrhea, meningitis and cholera, display similar pili.

"But then we wondered," Schoolnik told BioWorld Today, "what role, if any, do those pili play in actually bringing on diarrhea?"

Schoolnik reports his team's experiments seeking answers to that key question in the current issue of Science, dated June 26, 1998. Its title: "Type IV pili, transient bacterial aggregates, and virulence of Enteropathogenic Escherichia coli."

Altered Bacteria Don't Cause Diarrhea

For starters, he and his co-authors engineered various mutations in some of the 14 EPEC genes that encode pili proteins. "And at that point," he went on, "we invited the science-dedicated volunteers, ages 18 to 48, to drink a cocktail spiked with either the normal wild-type form of EPEC or one or another mutant, at various doses."

Bacteria made bald of pili by knocking out their filament-forming genes proved to be wimps at causing diarrhea in the volunteers.

"But microbes altered to express more of the microfilaments — by encoding the major repeating subunits of the pilus fiber." Schoolnik narrated, "did a better job than wild-type EPECs, of sticking to epithelial cells in vitro. They also failed to show the non-flagellar, twitching motility-dispersal movement by which the pathogens transit the gut."

Among those human guinea pigs (each of whom received a fee of $300) was a Ph.D. molecular biologist from Boston, named Kristin Weidenback, who received the highest dose of the pili-augmenting mutant. In an editorial titled "A tangled tale of E. coli virulence," accompanying Schoolnik's Science paper, Weidenback described the crucial experiment:

"The pili bundle together into ropelike filaments that interweave among bacteria, binding them into large aggregates," she wrote. "But the tests also suggest that another key to EPEC's virulence is the ability of the pili to disentangle themselves so the bacteria can go on to infect new intestinal cells."

Her article continued: "At first glance, the [high-pilation] mutant seemed to be an overachiever. Schoolnik . . . showed that these bacteria aggregate into clusters and stick to epithelial cells in greater number than the wild-type EPEC. . . .The wild-type bacteria form aggregates that disperse, but [these] mutants remained clumped together in a mass. . . . So they recruited more volunteers. . .

"Would these extra-pili EPECs prove more virulent, or not?" Schoolnik then asked. "We had a bet in the lab," he recalled. "Some bet that the mutant would have increased virulence; others that it would have none."

Those volunteers gave the answer, in a randomized, double-blinded experiment.

Of 16 subjects who ingested 20 billion or 100 billion colony-forming units of mutant bacteria, only four developed clinical diarrhea, as measured in dose-dependant stool volume. Of six who took the highest dose, none suffered the gut-wrenching ailment.

But in another cohort of 13 subjects, who swallowed 500 million to 20 billion cultures of wild-type EPEC, all but two members succumbed.

"We attribute this counter-intuitive result," Schoolnik concluded, "to the apparent fact that the high-pili mutants can infect and replicate in the human gut, but fail to disperse along the length of the intestinal tract, thus curtailing diarrheal virulence about 200-fold."

Microbiologist Michael Donnenberg, at the University of Maryland, in Baltimore, is pursuing the same line of research as Schoolnik. Citing the enigmatic ambiguities in investigating EPEC mechanisms, he commented, "The volunteer is a big black box. We put bugs in one end and measure diarrhea out the other end. As to what happens inside we really have no clue." *