Two U.S. companies, one biotech, the otherpharmaceutical, are about to reveal that they haveacquired licenses to a British invention for isolating andcloning virulence genes in a bacterium's genome.

A paper in last week's Science, dated July 21, describesthe patent-pending process under the title: "Simultaneousidentification of bacterial virulence genes by negativeselection." Its inventor is microbiologist David Holden ofHammersmith Hospital's Royal Postgraduate MedicalSchool in London.

"If one accepts that the coding capacity for a pathogenicbacterium might be 4,000 genes," Holden told BioWorldToday, "only a small proportion of these, we think, arespecifically required for facilitating infection and growthin the host that the pathogen infects."

The problem his invention solves, Holden explained, is:"How do you screen for those specific virulence genesagainst the background of the much larger number in thebacterial genome?"

The traditional genetic approach, he observed, "is tomutate the genome at random, inactivating one gene at atime, injecting it into an animal and monitoring whathappens: Does it cause disease or not?"

This method, Holden went on, "is very laborious, time-consuming and expensive. Having to screen manythousands of mutants individually would simply beprohibitive."

His approach to circumventing that genomic gridlock tagslarge numbers of random bacterial mutant sequences withbar code-like fragments of DNA. To generate thesemutants, he deploys transposon mutagenesis, inserting thetransposons at random into the bacterial genome. "There,in a proportion of cases, they will inactivate the genesthey've hit."

Each semi-random bar-code, or genetic signature, isabout 40 base pairs long, flanked by two 20-bp "arms."Together, they comprise "a pool of tags of enormouscomplexity." Ligating that pool into a plasmid harboringthe transposon, Holden said, "you can be virtually certainthat each transposon will carry a signature different fromany other." The short-arm flankers enable polymerasechain reaction (PCR) to amplify the tags for hybridizationanalysis.

To demonstrate the technique, as described in Science,Holden chose the pathogen, Salmonella typhimurium,which causes typhoid fever in mice. Starting out with 10pools of 96 different bar-coded S. typhimurium mutantseach, "we injected each pool into two mice.

"After the animals became sick, which took a few days,we recovered 10,000 bacterial cells from their infectedorgans. Extracting the bacterial DNA and amplifyingtheir tags revealed the equivalent of "the dog that didn'tbark."

That is, Holden explained, "if a transposon had gone intoa virulence gene, that mutant strain wouldn't be able togrow in the host. The tag, therefore, won't be present, sowe know that the gene carrying the mutant identified bythat tag is a virulence gene.

"Very rapidly, in just a few months," he related, "weended up identifying sequences of some 28 such genes _a fifth of the estimated total in the S. typhimuriumgenome."

Observing that "biotech companies are sequencing thegenomes of more and more bacteria," Holden continued,"what that gives them is a data base, but doesn't tell themanything about function. Once you have the sequences _and simultaneously you've generated a strain attenuatedin its virulence _ our approach could give you at leastthree places to go:

* "First, a better understanding of the pathogenesisprocess, from a strictly academic point of view. That'sclearly what we're interested in."

* "Second, it provides a potential drug target, either thegene or its product. " (Anent this implication, Holdenpoints out that "it's unlikely that such a drug would causeside effects in human cells, because virulence genesusually evolved specifically to deal with whateverimmune defenses the host organism presents.")

* "Third, it automatically generates a variety of strainsattenuated in virulence, hence, of potential use in avaccine or vaccine carrier."

Non-Exclusive Licenses, But Exclusive Fields Of Use

Cellular pathologist Jonathan Gee is managing director ofthe Royal Postgraduate Medical School's wholly ownedsubsidiary company for technology transfer.

"Holden's invention," Gee told BioWorld Today, "is anenabling technology, applicable in a number of differentfields _ human, animal and plant pathogens. Certainly inthe first two," he added, "the technology can be helpful indeveloping both drugs and vaccines, which is why we'vedecided on a non-exclusive licensing policy."

Gee explained: "The reason is that we want to benefit themaximum number of people, animals and plants, so wefeel the best way to do that is to have as many companiesas possible using the invention for finding a drug."

But he added a demurrer: "Within that over-all non-exclusive policy, we don't rule out exclusivearrangements covering defined fields."

The two U.S. licensees that have just signed on, Geeadded, "are in the process now of agreeing press releases.Meanwhile, we are continuing active efforts to grantfurther licenses. People who are interested shouldcertainly get in contact with us; we'd be happy to discusslicensing." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.