By David N. Leff and

Lisa Seachrist

A press conference Wednesday morning at the National Press Club in Washington, D.C., presented to the media the 26th bacterial genome to be totally sequenced.

That poster-boy bug is Vibrio cholerae, one of the deadliest pathogens on the planet - where cholera is pandemic in underdeveloped countries, from Asia to the Middle East, Africa and Latin America - not sparing the U.S. Gulf Coast.

Since cholera first devastated the human race - starting in Asia 2,000 years ago - it has launched seven pandemics "The seventh one, now ongoing," observed cholera-directed microbiologist Matthew Waldor, at Tufts University Department of Medicine, in Boston, "began in 1961 on the Indonesian Island of Sulawesi. It now has spread to most of the continents of the world, including the Americas in 1991."

The media event heralded the publication today of Nature, dated Aug. 3, 2000, announcing Vibrio's debut as a duly sequenced member of the microbial genome club. The journal's article is titled: "DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae." Of its 32 co-authors, the two officiating at the media event were John Heidelberg, the lead author, and Claire Fraser, senior author. Both represented TIGR - The Institute for Genomic Research, in Rockville, Md., which carried out the actual DNA sequencing operations. A third co-author, John Mekalanos, Harvard Medical School, is a leading cholera vaccine developer.

In his brief presentation, Mekalanos told the reporters "how Vibrio cholerae goes from being an environmental organism to a human pathogen by acquisition of 'accessory genetic elements.'" He then cited "a new interesting virulence gene identified by the genome sequencing project, [which] has improved our ability to make safer, live, oral cholera vaccines. . . . . [and] antibiotic targets."

"The genomic sequence of V. cholerae," the Nature paper noted, "confirmed the presence of a large integron island (a gene capture system) located on chromosome II. "Several lines of evidence," it added, "suggest that chromosome II was originally a megaplasmid captured by an ancestral Vibrio species, [which] presumably acquired genes from diverse bacterial species before being captured."

Vibrio's Surf 'n' Turf Lifestyle

The cholera pathogen's three-faced lifestyle lends credence to this scenario. In between visiting pandemics on its human victims' guts, the bacterium populates oceans, coastal waters and brackish estuaries, where it wreaks havoc on seafood fish and mammals.

When V. cholerae assaults Homo sapiens, it disports itself along the entire length of the small intestine. To get there in the first place, it must be ingested by fecally contaminated food or drink. That's why tourists in the tropics are urged to wash their fruits and vegetables, and boil their water.

An attack of the infection usually starts painlessly with a flood-tide of profuse, watery diarrhea. Then come vomiting, muscle cramps, dehydration (urine pretty much dries up), intense thirst, sunken eyes, wrinkled skin on fingers, collapse of the circulatory system, stupor and death. If left untreated - as cholera often is in impoverished areas of the earth - 52 percent will die. Prompt, modern treatment cuts this mortality to one percent.

The intestinal colonization factor of V. cholerae is a Velcro-like coating of submicroscopic grappling hooks called type IV pili. These colonize the mucosal inner surface of the gut, then let loose their cholera toxin. The genome sequence confirmed that the genes involved in assembling these pili reside on chromosome I, as part of a 'pathogenicity island.' Among that large chromsome's other discerned genes is the cholera toxin itself.

In contrast, the much smaller chromosome II packs a larger fraction (59 percent) of genes encoding proteins of unknown function, and hypothetical proteins. One of the genes it carries was apparently acquired from an archaea - the third form of life.

Race For A Vaccine Goes On

Immunization is biotechnology's not-so-secret weapon against cholera. Its vaccines are being developed along two tracks - field trials in endemic areas of the world, and testing of healthy human volunteers, first vaccinated, then challenged with the bacteria. In the U.S., Maryland's Center for Vaccine Development, in Baltimore, and Harvard's John Mekalanos are among the leading U.S. cholera vaccinolgists.

The genome sequence confirmed the discovery by Waldor four years ago (then Mekalanos' post-doc) that the cholera toxin genes are encoded in a filamentous phage in the large V. cholerae chromosome I. (See BioWorld Today, July 5, 1996, p. 1.)

He told BioWorld Today where the vaccine work stands: "Two types are in some state of clinical trials now," Waldor said. "One is a live, attenuated bacterium, genetically engineered to be detoxified. One of these is from Maryland, and one from Mekalanos' work. The University of Maryland's version is farther along in clinical trials, and it's up to the FDA, I think, to approve it for travelers, because they have very good data in volunteer studies. The Harvard vaccine is also in volunteer studies; it is not as far along, but looks promising.

"The Maryland vaccine," Waldor went on, "was tried in a field study in Indonesia, and failed. I don't know what Mekalanos' plans are for field studies of his vaccine, It's crucial to test vaccines for efficacy in endemic areas. However, volunteer studies are probably useful for testing travelers' vaccines.

"The other kind of immunization," Waldor continued, "is killed, whole-cell oral vaccine. That has shown some efficacy in a field trial, but it doesn't confer long-lived immunity. So right now," he summed up, "we don't yet have in hand - at least that's been tested - an ideal cholera vaccine for developing countries.

"As for Vibrio's genomic sequencing - hype aside - just like the human genome, this is not the end of disease by any means," Waldor observed. "It's really just a vast, dense data set for scientists to have a new beginning to study this organism, to be clever and persistent - and to make new vaccines.

"So for instance," Waldor suggested, "you could imagine deleting certain sequences that could be potential virulence genes, constructing live vaccines, or expressing certain sequences, and using their proteins as subunit vaccines."

Waldor wrote the "News & Views" editorial accompanying the Nature article. He titled it: "Treasure trove for cholera research."

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