By David N. Leff
The commonest vector-borne disease in the U.S., Europe and parts of Asia made the cover of Nature this week, in its issue dated Dec. 11, 1997. The journal showcased the complete genomic sequence of Borrelia burgdorferi, the microbe that carries Lyme disease from tick to human.
It brings to an even dozen the number of total genomes published since 1995, when The Institute for Genomic Research (TIGR) launched the series with the 1.83-megabase sequence of Haemophilus influenzae in 1995.
Since then, the Rockville, Md., institute has sequenced another five of the remaining 11 genomes, culminating so far in B. burgdorferi.
First author of their paper in Nature is molecular biologist Claire Fraser, TIGR's vice president of research. The article is titled: "Genomic sequence of a Lime disease spirochaete, Borrelia burgdorferi." Including Fraser, it is signed by 37 co-authors, 32 from TIGR, three from the University of Utah, in Salt Lake City, and two at MedImmune Inc., in Gaithersburg, Md.
Weighing in at 1.44 megabases, B. burgdorferi's genome is far from the largest, or smallest, of the dozen thus far sequenced. But it would appear to be the most tantalizing. Scattered through the Nature article's seven pages, characterizing its linear central chromosome and "at least" 17 plasmids, are such phrases as "not clear," "not understood," "not yet known," "has not yet been determined."
Organism Has More Than 1,200 Genes
But what is already clear, understood, known and determined about B. burgdorferi's 533,000 base pairs, comprising 1,283 genes, should fuel "the tremendous interest of the Lyme disease community," Fraser told BioWorld Today.
That community, of course, is intently engaged in developing and testing vaccines to foil the shifty B. burgdorferi spirochete.
"In Lyme disease vaccine development," Fraser observed, "I think the strategy likely be taken is one that MedImmune has been using in trying to develop vaccines for other human pathogens. It uses computer programs to try to predict which of the genes in any genome might likely encode antigenic outer surface protein [OSP]."
She continued: "There look to be a fairly large number of OSPs in Borrelia that were not known before we had its entire genome sequence in front of us. So one strategy would be to try and understand whether these OSPs are differentially expressed in the tick vector vs. a mammalian host. There's prior evidence that some or a majority of the OSPs in Borrelia change when the spirochete transfers from the tick into a warm-blooded animal. (See BioWorld Today, Nov. 26, 1997, p. 5.)
"So if, from our genome sequence," Fraser went on, "we can get a better understanding of whether this is something unique to just a limited set of proteins, or a much bigger set of proteins, that would be important information. OSPs that are known to be expressed in humans and other warm-blooded animals could then be evaluated for their antigenicity. Those that elicit a strong immune response would become candidates for vaccine development."
"Another very important application of the genome sequence," the TIGR researcher pointed out, "would be in trying to understand what genes are involved in the ability of the organism to get from its first site of deposition in the skin to tissues throughout the body.
"What the DNA sequence provides," Fraser added, "is a starting point for identifying genes that may potentially be involved in different aspects of the biology of Borrelia. It's certainly much easier to design better experiments if you have the whole genetic blueprint in front of you than if you're just working with a limited knowledge of the genetic complement of the organism, and trying to come up with meaningful experiments — which in Borrelia in particular is much like a black box."
Countering bacterial antibiotic resistance, not just in Lyme disease but in general, is a broader bonus sought in genomic sequence analysis, Fraser suggested. She cited the case for Treponema pallidum, the 1.14 megabase syphilis pathogen, which she and her group have just finished sequencing and are preparing for publication.
"Fortunately," she pointed out, T. pallidum is still sensitive to antibiotics, but with the way microbes are evolving, certainly the specter of a drug-resistant form of syphilis is not unheard-of.
"In terms of medicine and human pathogens," Fraser said, "there is on tap a tremendous potential here in not only finding new targets for vaccines, but in cases where you're dealing with antibiotic-resistant organisms, perhaps uncovering entirely new types of targets that can be used in the design of antimicrobial agents.
"In the 21st century," she suggested, "these may not necessarily look like the antibiotics in use today. We may be targeting an entirely different set of protein pathways, based on having a complete genetic blueprint that, up until we showed that this sequencing technique could work, just wasn't possible."
How Does TIGR Do It?
Fraser summed up the technique that, over 18 months, from March 1996 to September 1997, enabled her team to sequence the genome of B. burgdorferi:
"We started with purified DNA from the organism. The first step was to use a mechanical technique, a nebulizer, to shear the DNA into small fragments. Then we selected the appropriate-sized fragments, on the order of 2 to 3 kilobases each, and cloned those into a plasmid that would allow us to replicate these various plasmid clones, each conditioning a unique piece of the Borrelia genome, in Escherichia coli.
"Next, we selected clones at random, grew up and isolated plasmid DNA, and used each individual plasmid as a template for DNA sequencing. We sequenced the Borrelia insert from both ends, collected the DNA sequence information from 19.078 sequencing reactions in our computer, and then, using algorithms — software that we'd developed here specifically for microbial genome work — reassembled the nearly 20,000 bits of the Borrelia sequence into a final single consensus sequence, for both the large chromosome and the plasmids."
Editor's note: The complete, annotated DNA sequence of Borrelia burgdorferi is available on the world wide web at http://www.tigr.org/tdb/mdb/bbdb/bbdb.html. *