By David N. Leff
"Summertime, and the livin' is easy."
Those memorable, singable words from the opera "Porgy and Bess" apply not just to kids on vacation, but to various interconnected forms of backyard and woodsy wildlife. Now, with summer less than a season away, it's time to recall how these purveyors of Lyme disease line up:
¿ White-tailed deer - Odocoileus virginianus.
¿ White-footed field mouse - Peromyscus leucopus.
¿ Deer tick - Ixodes dammini.
¿ Lyme disease bacterium - Borrelia burgdorferi.
¿ Human victim - Homo sapiens.
Here, from the top down, is the daisy chain of warm-weather borreliosis (Lyme disease) infection:
The blood-sucking mature tick's preferred food is the deer's blood. Immature ticks imbibe their sanguinary fix from the mice. In exchange, the rodents pass the B. burgdorferi on to the tick, which then relays the bacterium to its ultimate human recipient.
If diagnosed in time, Lyme disease can be cured by common antibiotics. If it misses that window of opportunity, the tick bite may eventuate into a lifetime of painful, debilitating - but not fatal - symptoms that mimic rheumatoid arthritis and other serious neurological and cardiac ills.
Adding evasion to injury, B. burgdorferi cloaks its genetics in inscrutability, even three years after complete sequencing of its 1,283-gene genome. (See BioWorld Today, Dec.2, 1999, p. 1; and Dec. 12, 1997, p. 1.)
"To work with Borrelia is not easy," observed molecular biologist Felipe Cabello, at New York Medical College in Valhalla. "It's a very difficult bacterium to grow. With E. coli," he added, "you can do an experiment in two or three days. With B. burgdorferi, it takes you six weeks to two months."
Cabello is senior author of a paper in today's issue of The Proceedings of the National Academy of Sciences (PNAS), released April 18, 2000. It's titled: "Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi."
"At the present time," Cabello told BioWorld Today, "the study of Borellia burgdorferi is limited by the lack of cloning vectors. We set a goal to try to develop that. The purpose of cloning vectors," he explained, "is first, to complement the pathogen's null mutants, and, second, study gene expression in that microorganism."
Pathogen's Way With Mammals, Ticks
"Our purpose," Cabello continued, "was trying to isolate mutants that might tell us something about the functions of Borellia genes. We were looking to identify genes that are involved in the spirochete's ability to infect its mammalian host and its arthropod host - the ticks. Genetic approaches to the bacterium are very limited," he pointed out. "And I think this cloning vector we report in PNAS will expand that."
Besides its linear chromosome, B. burgdorferi's genome includes upwards of 17 plasmids (smaller free-standing DNA sequences), all totally refractory to laboratory analysis. To circumvent this genetic stonewalling, Cabello and his co-authors resorted to a nonpathogenic bacterium, Lactobacillus lactis. It's found in fermented dairy products, as well as in beer, wine, sewage, and flora of the human mouth, intestinal tract and vagina.
One clue - the similar 28.6 percent nucleotide content of cytosine and guanine in the DNA of C. lactis and B. burgdorferi - suggested to the co-authors a strategy aimed at unmasking Borellia's stubborn secrecy. Their "truth serum" was a well-known bacterial plasmid, pGK12, which contains replication functions of L. lactis, and is able to replicate in Staphylococcus, Streptococcus, Bacillus and E. coli.
"This pGK12 plasmid is very promiscuous," Cabello pointed out. "It can replicate in both gram-negative and gram-positive bacteria. That was the reason we thought we could use it as a cloning vector in B. burgdorferi - and it worked. The plasmid was able to propagate outside the chromosome in the bacterium's cytoplasm, where it replicated independently.
"Also," he recounted, "with the plasmid, we were able to introduce into B. burgdorferi enhanced green fluorescent protein, as a visual indicator of gene expression. What we did," Cabello observed, "may seem a little bit primitive, but given the stage of Borrelia burgdorferi genetics, I think it's an important finding.
"It could be applied, for example," he suggested, "to isolate knockout mutants of Borrelia, then complement those mutants and put them in for functional tests. If you inactivate the genes," Cabello explained, "you produce a null mutant. Nothing happens to the biology of Borrelia. It cannot infect; maybe it cannot grow very well. And if you then clone the wild-type gene into this pGK12 plasmid and introduce it back into the pathogen, you might see a recovery of the gene function because you are able to isolate mutants that lack the function."
Cabello explained this complementation process: You inactivate a gene in the Borrelia chromosome. That gene does not produce a protein. So let's assume it's not able to infect ticks very well. With this plasmid, you then put back the wild-type gene - the gene without any defects. Now, Borrelia can infect the tick. So you have a confirmation that the inactivated gene is involved in the ability of the pathogen to infect ticks."
From this feat, Cabello construed a range of practical consequences: "One that can arise from this finding," he surmised, "is that you can, for example, inactivate certain genes, allowing you to produce a less pathogenic Borrelia."
In The Long Run: Vaccines, Diagnostics, Antibiotics
"The relevance of this paper at the present time," he continued, "is that it would allow you to identify the functions of some Borrelia genes. And with that in the future, of course, you can develop vaccines, better diagnostic assays and antibiotics. But those are all a long way down the road, I think."
Cabello's paper was submitted to the Academy of Sciences by academician Edwin Kilbourne, research professor of microbiology and immunology at New York Medical College. "I think the importance of this paper," he commented to BioWorld Today, "is that until the work appeared, there apparently has not been an easy way of cloning the genes that cause Lyme disease - the Borrelia burgdorferi. This will greatly accelerate progress in the genetic analysis of that organism."