In a critical scene in the film remake of the classic 1960s TV series "The Fugitive," actor Harrison Ford sheds his coat and replaces it with another. This simple deception allows him to escape detection by the swarm of police officers trailing him.
The African trypanosome, a blood parasite that causes African sleeping sickness, is, like Ford's character in the film, a fugitive that changes its "coat" each time the human immune system is about to nab it. Woven of 10 million copies of a single sugar-coated molecule called a glycoprotein, the trypanosome's surface changes every few days by virtue of a switch that activates a new gene.
The ability of a species to create numerous, successive surfaces as a survival tactic is known as antigenic variation. An exhausted host immune system can no longer quell infection after repeated rounds of futile antibody response.
Rockefeller's George Cross, head of the Laboratory of Molecular Parasitology, is one of a handful of pre-eminent scientists investigating the African trypanosome. In 1975 he showed that antigenic variation is based upon the amino acid arrangement of successive surface proteins of a single trypanosome lineage. Since then, he and his colleagues have identified many characteristics of the genes and proteins that are responsible for antigenic variation. Though the picture is not complete, the Cross lab has unearthed clues to the mechanisms that regulate variable-surface genes so precisely during infection.
That's why Cross, the Andrι and Bella Meyer Professor, is betting on a molecular genetics approach to combat two diseases caused by trypanosomes: nagana, a livestock illness, and African sleeping sickness. "The parasite's antigenic variation renders vaccines unlikely to work, and older therapies such as pentamidine, suramin and melarsoprol are nearly as poisonous to humans as they are to trypanosomes."
Though other micro-organisms vary their surface antigens to counter the host's immune system, trypanosomes do so at an unrivaled rate. Also intriguing to scientists such as Cross is that the protozoan parasite is ancient, and anciently diverged, with some striking differences from multicellular beings.
The middle against both ends
Trypanosome's outlaw status sets it apart, but does not deter interest. "I've always been drawn to oddball organisms. I believe unusual adaptations can provide new insights to standard biology problems," says Shuba Gopal, a fifth-year graduate fellow in Associate Professor Terry Gaasterland's Laboratory of Computational Genomics at Rockefeller.
Seventy-five percent of trypanosomes' genes share no similarities with multicellular organisms. According to Gaasterland, the proteins made by this organism, or the ways in which they are made, are so different from ours and all other known "higher" organisms', they're provocative.
As a window to these genetic differences, Gopal studies the parasite's ability to generate messenger RNA (or mRNA) through an unusual process called "trans-splicing."
Most organisms convert their own genetic information into a usable form, called mRNA, by a process called "cis-splicing." "Cis" refers to a process of splicing together informational cassettes ("exons") that are separated by non-coding "introns" on the same piece of RNA, to make a readable transcript. This process works like a tightly safeguarded library: the cassettes' master versions always stay in the library. Copies circulate via mRNA.
Trypanosome genetic material is different in content and in form. Trypanosome genes contain no "introns" and therefore do not need to cis-splice their RNA to decode its information. "The process is closer to what we observe in bacterial transcription," says Gopal. But trypanosome genes all lack a small piece at their start, which is apparently needed for mRNA to be translated into protein, and this "mini-exon" has to be added to the RNA in a process called "trans-splicing."
When attempting to decode a DNA sequence, computers use relatively simplistic methods that cannot say, for certain, what is a real gene and what stretch of sequence just coincidentally looks like a gene. If trans-splicing is required for mRNA in trypanosomes, identifying DNA signals that specify trans-splicing sites can help discriminate real genes from others that are predicted, by the computer, to be possible genes. To this end, Gopal developed a system that classifies different kinds of splice signal data. Her goal was to learn how to predict trans-splicing signals. This has involved her in cycles of computer prediction and experimental confirmation. Each cycle improves the veracity of the predictive algorithm.
Gopal, in her bioinformatics-to-bench research, focuses on the genetic material at the "middle" of T. brucei's many chromosomes (11 essential, plus hundreds of mini-chromosomes). Another trypanosome researcher at Rockefeller primarily is concerned with what occurs at the "ends" of these chromosomes.
Bibo Li, a new research assistant professor in Cross's lab, who completed a postdoctoral fellowship with Titia de Lange in her Laboratory of Cell Biology and Genetics, studies the ends of trypanosome chromosomes, called telomeres.
Telomeres in trypanosomes turn out to have the closest biochemical lettering - that is, A,C,T and G organization of nucleotides found in DNA - to our own human telomeres.
As a rich source of insight on chromosomal aging and instabilities leading to cancer, telomeres have been an important basic research topic. Rockefeller's de Lange, a leader in the telomere field, conducted her own graduate work on telomeres in T. brucei.
Li's tenure in the Cross lab has only begun, but the research she undertakes will illuminate more than one set of biomedical problems. By identifying proteins that act on telomeres, she will potentially contribute to further understanding of cancer onset and its possible treatments.
She can conduct endogenous gene experiments in T. brucei that cannot be done in humans, and reap the benefit of the parasite's rapid reproduction cycle. In addition, learning more about trypanosome telomere function could reveal a vulnerability in the parasite's system. It has been suggested that silencing T. brucei's telomere might reduce its capacity for antigenic variance. This knowledge could translate into a long-awaited cure for sleeping sickness.
"Li is by far the most qualified person in the world to be working on trypanosome telomeres," says Cross. "She is familiar with all the existing systems - including yeast, mouse and human - and knows how to exploit the T. brucei model."
The molecular genetics of trypanosomes, whether at the "middle" or the "ends," promises new insights, the dimensions of which we can only estimate.
Orphaned but not abandoned
The National Institutes of Health define orphan diseases as those that afflict fewer than 200,000 people annually in the United States. The fatal sleeping sickness caused by African trypanosomes strikes between 200,000 to 300,000 people annually in equatorial Africa, the habitat of the tsetse fly, whose bite spreads trypanosomes among people and livestock.
In U.S. public health terms, African sleeping sickness is not a candidate for major research funding, nor costly drug development that would lead to cures for the disease. The AIDS and tuberculosis epidemics in developing nations eclipse the distant drama of African sleeping sickness. The trypanosomes-caused human disease is therefore an orphan. Yet, the NIH funds Cross's lab due to the intrinsic value of the organism, not to mention Cross's strong legacy of discoveries and accomplished laboratory alumni. Championing orphan diseases is hard work. Cross travels the globe to lobby for sustained efforts to combat the diseases caused by African trypanosomes.
"If we do get new epidemics of African sleeping sickness in areas of civil strife, it's going to be very difficult to control them. We could easily begin the 21st century as we began the 20th - with significant loss of life and deep impoverishment due to the parasites."
Gaasterland would be gratified to see basic research conducted in her lab reverberate on the public health scene, too.
"The genome of T.brucei is computationally challenging to annotate," says Gaasterland. "However, the very same properties that make it hard to annotate make it powerful in comparative genomics. By virtue of its remote position in the phylogenetic tree of life, the parasite provides essential data for understanding protein families."
Gaasterland explains further: "This cuts both ways. Human, mouse, fly and worm all help to identify important sites in trypanosome proteins. With such remote evolution, anything that has remained the same is likely to be essential in some way. It is possible that our study of the T.brucei genome could further unravel the mechanisms the parasite uses during infection."
Orphaned and on the run, the biological outlaw known as trypanosome could someday be reformed. It is already contributing to the intellectual life of science.
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