Busting the Gut Busters

It can take as few as 10 live bacteria in a undercooked patty of ground beef to spark an infection of the notorious hamburger bug, Escherichia coli O157:H7. Most strains of E. coli dwell peacefully within people, but O157:H7 explodes in the gut like a string of firecrackers. It sabotages the body, programming cells in the intestine to stick to it in an embrace that initiates a cascade of destruction. More than 60 people in the United States die this way each year, and at least 70,000 are sickened.

This fast-food demon, the most deadly food-borne E. coli in this country, has been lacing family meals since its first outbreak in the early 1980s. About a third of the cattle in the United States now harbor the strain. When a slaughterhouse inadvertently grinds some gut contents into the hamburger, the bug can make its way into people.

Primarily a North American microbe, O157:H7 is just one strain of E. coli that co-opts the human body for its own ends. Its microbial family, enterohemorrhagic E. coli, spans the globe. All members of the group spew lethal Shiga toxins, which cause intestinal bleeding and can hit a person’s other organs with deadly force.

In underdeveloped countries with crowded conditions and contaminated water, another E. coli family takes a toll in human lives. Enteropathogenic E. coli strains lack Shiga toxins, but they kill hundreds of thousands of infants each year.

With the intent of someday developing treatments to disarm these devastating microbes, scientists are discovering how these two virulent bacteria wreak havoc. Recent studies have shown how they shoot through the stomach into the intestine, spear holes in its cells, inject poisons, spew toxins, and latch toxins onto immune cells that then travel throughout the body.

The studies are also revealing what strategies virulent E. coli share with other killers, such as Cholera and dysentery-causing Shigella, and explaining how these bacteria exchange genetic material at a furious pace that ensures their constant evolution.

Common objectives

The two families of E. coli have common objectives: They must resist the stomach acid, recognize when they’ve reached the intestine, latch onto it for awhile, and get out again when it’s time to infect another host.

It’s that last process–diarrhea–that’s kept such destructive E. coli in circulation for centuries among people who get sick but survive their infections. “There is an advantage to an enteric organism to cause diarrhea because then it can then infect a new host,” says Thomas S. Whittam of Michigan State University in East Lansing.

One recent advance in E. coli research is the decoding and analysis of the O157:H7 genome by two different teams. The genome was published in the Jan. 25 Nature by Fred R. Blattner of the University of WisconsinMadison and his colleagues (SN: 2/3/01, p. 71) and also in the Feb. 28 DNA Research by Hideo Shinagawa of Osaka University in Japan and his colleagues.

To pinpoint genes involved in disease, the researchers compared the O157:H7 DNA sequence with that of a harmless E. coli strain. Genes found only in O157:H7 pop out as possible sources of virulence.

The sheer number of unique genes in O157:H7 was a “surprise,” says Whittam. Blattner’s group found 1,387 unique genes out of its total of 5,416. An even bigger surprise was the number of O157:H7’s genes that also appear in other disease-causing gut microbes, such as Salmonella and Shigella, Whittam adds.

The presence of these shared sequences sheds light on what scientists say is an ongoing, furious exchange of genetic material among bacteria, “a much more powerful force in evolution than thought originally,” says Blattner. Because of that exchange, new microbial varieties are constantly evolving, adding to the hundreds of virulent E. coli strains already in existence, he notes (SN: 7/22/00, p. 60; http://sciencenews.org/20000722/bob2.asp).

Widespread genes

One set of particularly widespread genes regulates a process central to infection, aptly dubbed attaching and effacing. These genes enable E. coli O157:H7and other gut-disrupting bacteria to transform an intestinal cell from an efficient nutrient-absorbing machine into a staging ground for microbes.

In the space within the intestine, the process starts with the production of what look like long needles extending from the bacteria. Studies on enteropathogenic E. coli suggest that each projection consists of a single type of protein spooled into a tube. That tube appears to pierce the membrane of an intestinal cell and injects proteins that subvert the cell’s own machinery.

“It’s a cleverly devised, evolved system to inject toxins right into the cell instead of into the gut where they can be wasted, diluted, or produce an immune response,” adds Whittam.

The injected proteins include the bacterium’s own docking platform–a molecule called tir–that sets up the next step in its invasion.

Research in the late 1990s on enteropathogenic E. coli showed that tir inside the host cell worms its way to the cell’s outer membrane. It then pokes through and straddles the membrane, one end anchored in the host cell and the other dangling outside. This free end locks onto a protein called intimin on the surface of the bacterium. The result of the tir-intimin interaction is a microbe firmly attached to the surface of an intestinal cell.

After tir and intimin hook up, the intestinal cell no longer shapes its own destiny. Tir inside the intestinal cell initiates a cascade of events that effaces the nutrient-absorbing projections from the intestinal cell’s outer surface and forces the cell to rearrange itself into a bacterium-embracing pedestal.

The attachment apparently enables infectious bacteria to displace harmless E. coli. Virulent E. coli hang on to torment the intestine, while harmless E. coli get flushed out of the gut as diarrhea kicks in.

At first glance, sticking onto the inside of the intestine might seem at odds with one of E. coli‘s main goals: to infect a new host.

Researchers point out, though, that intestinal cells slough off every few days, likely taking huge rafts of virulent E. coli with them. Other virulent E. coli head down the intestinal tract after budding from an attached bacterium.

Scientists have recently discovered the three-dimensional structure of the tir and intimin proteins locked together. Natalie C.J. Strynadka of the University of British Columbia and her colleagues published the structure in the July 29, 2000 Nature.

Understanding that structure could eventually lead scientists to a means of prying virulent E. coli off the intestinal wall. “Now that we have a picture of how the two molecules interact, we could design drugs to disrupt that interaction,” says Michael Donnenberg of the University of Maryland School of Medicine in Baltimore.

Shigella, Salmonella, Cholera, Yersinia, and even some plant pathogens have genes for attaching to and effacing intestinal cells.

Although not as well studied as its enteropathogenic cousin, the bacterium O157:H7 also has such genes. The sequence of the genes in O157:H7 suggests that they were acquired from another bacterium, somewhere deep in the gut of a now-dead animal. The sequencing work has also uncovered O157:H7 genes for what could be a second attaching-and-effacing system, and scientists have just begun to explore that operation.

Dynamic interaction

Virulence is a tightly controlled process that keeps bacteria in tune with their surroundings. If a bug deploys its attaching-and-effacing system in a host’s stomach, the misguided bacterium will be attempting to make its home in a pool of acid. Researchers in the lab headed by James Kaper of the University of Maryland School of Medicine are on a quest to understand how virulent E. coli bacteria sense where they are in the body and respond with the appropriate behavior.

“The interaction between bacteria and the host cell is very dynamic,” says Donnenberg. “It’s not as simple as: Attach and cause disease.”

When virulent E. coli bacteria hit the stomach, they crank up systems to resist acid, the Maryland scientists have found. Sooan Shin of Kaper’s lab has identified a protein in enteropathogenic E. coli that responds to the conditions in the stomach in two ways. It activates genes that produce acid-resistance proteins, and it keeps a firm lock on the attaching and effacing genes that will go to work in the intestine. The research will be published in an upcoming issue of Molecular Microbiology.

The bacteria sense that they have reached the large intestine by picking up on a sense of community. In a healthy intestine, the billions of benign E. coli in the gut produce a molecule that says, “Here we are!” Normally, microbes use it to communicate with each other. Vanessa Sperandio in Kaper’s lab reports that O157:H7 picks up on this signal. “It’s kind of like a pheromone for bacteria,” she says.

The signaling molecule tells O157:H7 to activate its arsenal. About 10 percent of its genes kick on in response, Sperandio reports in an upcoming Journal of Bacteriology. Besides triggering attachment and effacement, these genes churn out a host of proteins that perforate, wound, and otherwise disable the intestine. Some of these proteins, speculate the Maryland researchers, may cause the infamous diarrhea.

Recent findings

Most of the recent findings about how virulent E. coli bacteria cause diarrhea have been from studies of enteropathogenic E. coli.

Recently, Donnenberg and Gail Hecht of the University of Illinois in Chicago published work identifying a protein from an enteropathogenic E. coli that nudges intestinal cells apart. The researchers reported in the March Journal of Clinical Investigation that the bacteria inject the protein into intestinal cells and that the substance, through unknown mechanisms, loosens the attachments that hold the cells together. The result is that body fluid from outside the intestine leaks in and contributes to diarrhea.

Donnenberg and several of his colleagues have shown that the same protein that loosens the connections between cells can eventually kill them. The protein initiates a process of cell suicide called apoptosis. This may further break down the intestinal walls, the researchers reported in the March Cell Microbiology.

Separately, Hecht recently showed that enteropathogenic E. coli can force intestinal cells to spew chloride ions into the intestinal interior. A counterbalancing rush of water from the cells follows the excess chloride, an event that also probably contributes to diarrhea.

With this knowledge, says Kaper, scientists may devise new therapies that stop diarrhea by reducing cells’ release of chlorine.

Despite these findings, Donnenberg cautions that a complete understanding of the mechanisms by which enteropathogenic and enterohemorrhagic E. coli cause diarrhea remains elusive.

Beyond the intestine

Many scientists study how O157:H7 reaches beyond the intestine. Damage to the kidneys and other organs by Shiga toxin is the leading cause of death from the infection. Furthermore, the toxin’s peculiar biology sets up the main barrier to fighting the microbe effectively with antibiotics.

Antibiotics can take days to decimate an E. coli infection, Kaper notes, and in the case of O157:H7 they may actually cause harm.

Phil Tarr of Children’s Hospital in Seattle reported last year that antibiotics appear to exacerbate an O157:H7-caused illness called hemolytic uremic syndrome, the main cause of kidney failure in children in the United States. “I’m still taking a charitable view [of doctors] who want to prescribe antibiotics [for O157:H7]. I personally don’t recommend it,” says Tarr.

Why don’t antibiotics seem to work? In experiments in test tubes and animals, several research groups have discovered that antibiotics cause O157:H7 to increase its spewing of Shiga toxins.

The toxin is actually produced by viral DNA that resides in the bacterium’s DNA. Antibiotics stress the bacterium and nudge the DNA from the toxin-encoding virus out of the bacterial DNA. Then, the virus replicates madly inside the bacterium, cranking out toxin all the while. Eventually, the bacterium bursts and the toxin makes its way to the kidneys and other organs.

Since the toxin hasn’t been detected in fluid from circulating blood, scientists have wondered how Shiga toxin travels through the body. Recently, researchers have gathered evidence that the bacterium uses the immune system to deliver the toxin to various organs.

A Dutch team led by Leo A.H. Monnens of University Hospital in Nijmegen reported in the March Journal of Infection and Immunity that Shiga toxin latches onto immune system cells called polymorphonuclear leukocytes. These cells then travel through the body, taking toxin with them.

The data from Monnens’ group suggests that the toxin moves from the immune cells into local cells in the kidneys and other organs. Once there, the toxin interferes with protein synthesis, a process essential to cell survival.

Patchwork of new genes

Whittam calls a cow’s intestines a “giant mixing bowl” for genetic material. When E. coli moves into a new cow herd, it picks up loose bits of DNA from the herd’s resident viruses and bacteria, he says. Within as few as 100 generations–just a matter of days, for a bacterium–a virulent E. coli can pick up a patchwork of new genes.

Right now, the next killer E. coli strain is perhaps being born deep in the bowels of a cow or a person, says Whittam.

Alison O’Brien’s group at the Uniformed Services University of the Health Sciences in Bethesda, Md., is doing its best to keep that from happening. The researcher are generating vaccines against Enterohemorrhagic E. coli. In collaboration with Evelyn A. Dean-Nystrom of the National Animal Disease Center in Ames, Iowa, the researchers injected animals with intimin, the protein that gives E. coli its sticking power. The idea is that the immune system will produce antibodies against intimin and later prevent any invading bacteria from clinging to the intestine.

O’Brien says that the results so far are promising. The researchers have yet to test vaccines in cattle, but immunized pigs show marked resistance to O157:H7 infection. “That means you can block adherence,” notes O’Brien.

O’Brien’s goal is to engineer the intimin gene into the corn that goes into cattle feed. Such feed, O’Brien reasons, could be an effective vaccine against O157:H7 and related enterohemorrhagic E. coli. It may even stop any new strains that are set to bust out of the intestinal mixing bowl.

Even now, a new strain might be preparing to leap into people. “I think it would be foolish to say that is not a possibility, now that we have the experience of O157:H7,” says Kaper.

No one knows why it took O157:H7 until the 1980s to hit the human population. Recent trends of raising cattle in close quarters and changes in how meat is processed might have fostered O157:H7’s emergence.

“With thousands of cattle on hundreds of farms going to tens of slaughter houses to one hamburger plant, where everything is mixed together to allow cross contamination . . . it’s no surprise that O157:H7 emerged,” says Kaper.

Whittam and his colleagues are surveying people hit in outbreaks of virulent E. coli to monitor for the appearance of any especially damaging strain, “the next O157:H7,” he says. Recently, the researchers identified a new strain of virulent E. coli whose prevalence seems to be on the upswing.

Whether it’s as nasty as O157:H7 remains to be seen. If so, scientists are better prepared to fight it than they were to battle O157:H7, says Whittam. “I think the next one, people will learn about even more quickly,” he predicts.