By David N. Leff
Britain¿s Sir Vincent Wigglesworth (1899-1994) was one of the world¿s preeminent entomologists. Besides an imposing oeuvre of books and papers, his life work is memorialized by a small but vital bacterium that bears his name ¿ Wigglesworthia glossinidae.
¿I christened this bacterium in 1995,¿ recalled molecular entomologist Sarap Aksoy, ¿because in the early 1900s Sir Vincent very carefully described this microorganism. His descriptions are still the norm today. I felt I had to name this bacterium after him.¿
Aksoy herself, a faculty parasitologist at Yale University School of Medicine, is a much-published expert on the tsetse fly (Glossina glossinidae), which broadcasts sleeping sickness over much of equatorial Africa. The insect¿s deadly cargo is Trypanosoma brucei spp., which holds an estimated half-million men, women and children on death row from the disease. (See BioWorld Today, April 17, 2000, and Dec. 3, 1998.)
Among tsetse¿s guilty secrets are three bacteria, which symbiotically inhabit different tissues in the fly. They are Wigglesworthia ¿ which Aksoy named ¿ plus Sodalis glossinidae and Wolbachia.
¿Wigglesworthia is a very fastidious bacterium we knew nothing about,¿ Aksoy observed. ¿It¿s an obligate ¿ tsetse only ¿ microbe that the fly depends on for its nutrition and its fecundity. We had no handle on this bacterium, because we cannot culture it. It¿s an intracellular organism that lives within the insect. People have been working with this bacterium for 80 or 90 years, trying to understand what it provides to the flies that makes it obligate to them.
¿The second bacterium, Sodalis,¿ Aksoy went on, ¿has been extensively written up. Our group has genetically transformed that microorganism, and we¿re using it to investigate the possibility of expressing the anti-trypanosomal genes in tsetse flies.¿
Aksoy is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS) dated June 19, 2001. It¿s titled: ¿A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies.¿
Spying On E. Coli¿s Gene Store
¿This is the first heterologous application of gene arrays,¿ Aksoy told BioWorld Today. ¿The technique allowed us to gain a broad view of this bacterium¿s genome without necessarily going into the extensive analysis of genome sequencing. It¿s a very quick hybridization experiment that showed us, first of all, the rough genome¿s size, based on the number of genes we could detect, and the coding capacity of its very small ¿ 700-base pair ¿ genome. As more and more of these genomes are being sequenced, such arrays will be available, and can be used to understand a wide variety of organisms very quickly. They look extremely promising for future analysis.¿
Aksoy explained that glass DNA chips are called microarrays, whereas the five-by-eight-inch piece of nitrocellulose nylon filters her team used are gene macroarrays.
To identify the functions of Wigglesworthia¿s 700-base-pair genome, the Yale team matched that gene complement with the 4,000 base pairs of E. coli. This strategy assumed that before the now-slimmed-down bacterium took up symbiotic housekeeping in tsetse, it likely shared the evolution-designed genes of the still free-living microorganism.
¿On this gene array,¿ Aksoy narrated, ¿were spotted all of Escherichia coli¿s coding regions, based on its known sequence. So about 4,000 spots representing its genome. And we knew exactly which gene each spot corresponded to. So we hybridized the array overnight to radioactively labeled Wigglesworthia DNA. Then we exposed the array, like any Southern or Northern blot, and looked to see which spots hybridized. Finally, software told us what spots were positive. We had identified about 650 Wigglesworthia genes that hybridized to E. coli genes.
¿These symbiotic bacteria,¿ Aksoy pointed out, ¿live on the same single diet as their host organism. Tsetses always feed on blood, so the nutrition they get is rather restricted. Blood is low in certain vitamins that tsetse flies need. Hence it was assumed ¿ but not demonstrated ¿ that this Wigglesworthia bacterium could be supplementing the fly¿s diet with additional vitamins.
¿In fact, in our analysis we saw an unusually high number of genes in the B vitamin pathway. So we were really excited that the assumptions made by scientists for many years are true: a lot of B vitamin gene pathways appeared to be conserved, and Wigglesworthia synthesizes the vitamins for the fly. That¿s the nature of their symbiotic association. In return, the prokaryotic bacterium is missing genes for many amino acids, so we¿re assuming that it¿s obtaining those amino acids from the eukaryotic insect cells.
¿We began this work,¿ Aksoy observed, ¿because the symbionts hold the key to the fecundity of tsetse flies. So if somehow we were able to manipulate or damage these bacteria, the flies would simply stop reproducing. It provides a huge potential for controlling these insect vectors.
¿We work on all three bacteria in tsetse flies,¿ she went on. ¿We use them for different purposes. As we didn¿t have a way to culture Wigglesworthia, we were prevented from being able to develop a genetic transformation system, and manipulate its genome further. This is one avenue we can explore. Another is the basic evolutionary biology of these bacteria. Each of them had been associated with the fly from day one. If you look at different tsetse species, some newer, some older, the evolution of their bacteria reflects their age.¿
Fighting Back At Flies, Parasites, Bacteria
¿We¿re quite far ahead, actually, interfering with the viability of these flies ¿ especially with Sodalis, which is tsetse¿s midgut bacterium. We have been able to culture these bacteria in vitro; express foreign genes in bacteria and put these recombinant bacteria back into flies. In that scenario,¿ she continued, ¿we¿re looking for new candidate anti-trypanosomal genes.
¿Alternatively, we can express insect genes in these bacteria, which might be involved in killing parasites. Because insects have a natural immune response, naturally they can kill parasites, but somehow a few parasites seem to escape. It takes only one to cause disease.¿
Aksoy concluded: ¿If more of these gene arrays from related but different bacteria were commercially available, by doing this very simple hybridization we would have a more accurate map of the Wigglesworthia genome.¿