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Harvard Researchers Find Cholera Bacterium May Take Instruction From A Virus

Harvard Medical School

How the cholera bacterium got its virulence
June 27, 1996 Contact: Misia Landau 617-432-2342,
Keren McGinity 617-432-0441

Harvard researchers find cholera bacterium may take instruction from a virus

BOSTON-In 1993, as cholera swept through India, scientists were faced with a set of perplexing questions: What caused the deadly Bengal strain of cholera to reappear? Where did the deadly cholera pathogen come from in the first place?

Scientists have known that the cholera bacterium (Vibrio cholera) owes its virulence to two factors-the cholera toxin and another protein,TCP pili, which enables it to clump together and burrow into the intestines. But how the Vibrio cholera got those deadly factors has been a mystery.

Two Harvard Medical School scientist have found a partial answer to this puzzle. It appears that the cholera pathogen responsible for the Indian epidemic (Vibrio cholera 01) picked up one of its most lethal patches of DNA-the gene coding for the cholera toxin-from a virus, CTX phage.

"Here you have this dumb bacterium-Vibrio cholerae doesn't know how to become a pathogen. And the virus instructs it by introducing the cholera gene into the bacterial genome. The virus is the smart player in the interaction," says John Mekalanos, Higgins Professor of Microbiology and Molecular Genetics. He and Matthew K. Waldor, research fellow in medicine, announced their findings in the June 28 issue of Science.

The virus's first clever act is to select its students. It appears to introduce the gene for cholera toxin only into those bacteria that express the TCP pili protein.

Once inside the bacterium, the cholera toxin gene is activated by the same gene that turns the TCP pili gene on. This gene, known as Tox R, is designed to sense the intestinal environment. This ingenious arrangement is designed to bestow the cholera toxin gene on those bacteria located in an area where it can be used, namely the intestine.

"What this says is, 'Multiply in a sinister place. You have to bring something ot the party-TCP pilus-but I'll bring the band and we'll have a dance," says Mekalanos.

One reason the wily virus has eluded scientists' grasp for so long is that it belongs to a relatively rare class of viruses, the filamentous phages. Unlike other bacterial viruses, filamentous viruses do not kill the host cell though they may slow down growth, so there is no visual thinning of a bacterial lawn to mark an infection.

Even if filamentous viruses left a mark, researchers had little reason to suspect they were the culprits behind cholera. Filamentous viruses have never before been known to donate a fully functioning gene to a bacterium. Yet in the series of experiments reported in Science, Mekalanos and Waldron show that the CTX phage filamentous virus does move from a donor bacterium (Vibrio cholera 01) to a non-virulent recipient bacterium, bringing with it all of its genes. Some of these viral genes-including the gene for cholera toxin-may be expressed in the recipient bacterium.

In the first set of experiments, the researchers replaced the cholera toxin gene of Vibrio cholerae 01 with an antibiotic-resistant gene. They then mixed this antibiotic-resistant Vibrio with a recipient bacterium marked with a second antibiotic-resistant gene. The recipient bacterium was chosen for its ability to produce TCP pili under laboratory conditions. They found some recipient bacteria had acquired a resistant to both antibiotics, indicating they had, in fact, acquired an extra piece of DNA.

When they isolated and purified the particle of DNA, they found it had the long stringy shape characteristic of a filamentous virus. It was also single stranded, another hallmark of a filamentous virus.

To get a better sense of how the virus and bacteria actually interact, the researchers repeated the experiment with a recipient bacterium that does not produce TCP pili under laboratory conditions. Virtually no transfer of the virus occurred. But when they put the same mixture of recipient and donor into the gut of a mouse and then measured the transfer rate 24 hours later, they found stunning results. "The recipient strain is a million times better recipient of a virus in the intestine than it is under any laboratory conditions," says Mekalanos.

The discovery contradicts a long-standing and widely held notion about the emergence of infectious disease. Researchers traditionally have believed that bacteria pick up their virulence factors outside of the human body, in watery sewage systems or in stagnant coastal waters. However, it now seems that at least some pathogens originate inside the human body.

"From these data it sounds like the mixing pot may be our own intestines. And in a way we're constantly eating foods of various sorts. The body is its own little pressure cooker-cooking up, moving genes back and forth," says Mekalanos. "This phenomenon that we've uncovered may be the tip of the iceberg."

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