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Function and Regulation of Surface Glycoproteins of African Trypanosomes
Summary: Isabel Roditi wants to learn more about surface proteins that govern survival and transmission of Trypanosoma brucei, the parasite that causes sleeping sickness, by its insect host, the tsetse fly. Using a fully transmissible strain of T. brucei enables her to monitor the parasite throughout its life cycle in the fly. The long-range goal of such studies is to find ways to interrupt transmission of the disease.
Some of the deadliest parasites afflicting humans and their domestic animals are transmitted by insects. These include various sub-species of Trypanosoma brucei, which cause sleeping sickness in humans and Nagana in ruminants in large areas of sub-Saharan Africa. These diseases would be even more widespread were it not for the fact that T. brucei is strictly reliant on the tsetse fly for its transmission between mammals. The insect is not a mere "flying syringe" that mechanically transfers parasites from one host to the next as it takes a blood meal. Instead, there is a complex interaction between the parasite and the tsetse as the trypanosomes differentiate, proliferate, and migrate through different tissues before being transmitted to a new host.
Our laboratory is interested in how trypanosomes sense their environment, how they regulate gene expression in order to adapt to different hosts, and in particular what surface proteins are important for successful transmission. In the long term, this information might be exploited to control infection, either by inducing premature differentiation and the subsequent destruction of trypanosomes in the mammalian host, or by preventing differentiation and completion of the life cycle in the insect host. With the advent of transfection systems that allow trypanosomes to be genetically manipulated, we have found that a surprising number of parasite surface molecules are not required in culture. Indeed, their functions become apparent only when they are studied in the fly. Learning more about the function of these surface proteins might also enable us to interfere with parasite transmission. In carrying out these studies, we are fortunate to have a long-standing collaboration with Professor Reto Brun (Swiss Tropical Institute, Basel), who runs one of the few tsetse laboratories in the world.
After the discovery of abundant surface glycoproteins known as procyclins about 20 years ago, a simple "two coat" model of the trypanosome life cycle was developed. The metacyclic form that initiates an infection in the mammalian host and the bloodstream forms that sustain the infection have a coat of variant surface glycoproteins (VSG); periodic switching of the active VSG enables bloodstream forms to evade an adaptive immune response. Until recently, all other life-cycle stages (procyclic and mesocyclic forms in the tsetse midgut and epimastigote forms in the salivary glands) were believed to have an invariant coat of procyclins. This part of the model has now been radically overhauled: we have shown that midgut forms of the parasite express procyclins, but that the composition of the coat varies significantly during the course of an infection. Moreover, the proteins do not form the coat of epimastigotes and, most unexpectedly, are not essential for transmission, although they clearly confer a competitive advantage.
Using a combination of database mining and differential screening of micro-and macroarrays, we identified several candidate genes that were stage-regulated and were likely to encode surface proteins. In the past year, we have shown that epimastigote forms in the salivary glands express a family of glycosylphosphatidylinositol-anchored proteins (brucei alanine-rich proteins, BARP) are expressed by epimastigote forms in the salivary glands; these proteins represent the first molecular marker for this stage of the life cycle. A BARP 3′-untranslated region (UTR) is sufficient to activate expression in epimastigote forms and prevent expression in bloodstream and procyclic forms. Another stage-regulated surface protein that is currently being investigated is the transmembrane protein PSSA-2, which is required for efficient colonization of the salivary glands.
An unusual feature of trypanosomes and related organisms is that transcription is polycistronic, with monocistronic mRNAs subsequently being generated by coupled trans-splicing and polyadenylation reactions. Adjacent genes in a transcription unit can give rise to very different levels of steady-state mRNAs, and the presence of mRNA is not an automatic guarantee that the protein is synthesized. In addition to BARP, elements in the 3′ UTRs of VSG, procyclins, and several other stage-specific mRNAs have been shown to control RNA stability and/or translation. We have shown that at least one element in a procyclin mRNA, known as the glycerol-responsive element (GRE), acts as a sensor of environmental factors and metabolites. Although the first regulatory elements in trypanosome mRNAs were identified well over a decade ago, the cognate RNA-binding proteins are still unknown. We are using several approaches, including biochemical purification of RNA-binding complexes and genome-wide screens with RNAi libraries, and have recently identified the first protein to interact with the GRE and to influence the steady-state levels of a procyclin transcript.
Cyclical transmission of T. brucei by tsetse is a surprisingly inefficient process; flies can eradicate an infection from the midgut or stall the progress of the trypanosomes to the salivary glands. Even in a laboratory set-up, where the conditions for transmission are optimized, only about 10 percent of the flies that take an infective blood meal will support the entire life cycle. Migration to the salivary glands occurs during a defined period, after which only a few migrating forms can be detected.
We have no idea of the degree of diversity of trypanosomes in a mature infection, but hypothetically a single cell might be enough to colonize a gland. The extent to which a population is restricted during transmission has important implications for the acquisition and maintenance of traits such as human-serum resistance and drug resistance. To investigate population dynamics during transmission, we recently initiated a project in which trypanosomes were tagged with sequences integrated into a ribosomal DNA spacer. Within this sequence are 10 variable base pairs, giving each trypanosome a unique signature. The experimental procedure we use is to infect flies with a mixture of tagged trypanosomes. Parasites are subsequently isolated from the midgut and salivary glands of individual flies at various time points after infection, and the repertoire of tags is analyzed by polymerase chain reaction and sequencing. By comparing data obtained from individual flies, it should be possible to assess the magnitude of the bottlenecks during the establishment of midgut and salivary gland infections, respectively, and also to see if certain tags are consistently enriched during transmission.
Genomewide screens can be performed with the procyclic form of trypanosomes, but they have yet to be applied to some of the most important issues in trypanosome biology. These include antigenic variation, the phenomenon of human-serum resistance by T. brucei gambiense and T. brucei rhodesiense, and the acquisition of drug resistance, which all need to be studied in bloodstream forms of the parasite. Unfortunately, it has not been possible to produce representative RNAi libraries, or to use libraries for complementation, because of the extremely low rate of stable transformation in this stage of the life cycle. We recently achieved a 1,000-fold improvement in the rate of stable transformation. The transformation rate (about 1 in 10,000 of the starting population) is comparable to that obtained for procyclic forms, bringing high-throughput analyses and genomewide screens of bloodstream forms within reach.
In addition to support from HHMI, work in our laboratory is financed by the Swiss National Science Foundation and the Canton of Berne. Collaborative projects with the Swiss Tropical Institute are also supported by the Roche Research Foundation and the Novartis Foundation.
Last updated August 2008
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