N.B. Murphy and R. Pellé
International Livestock research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya. Email:n.murphy@cgnet. corn
References
Trypanosomosis is a major constraint to livestock productivity in sub-Saharan Africa and an important constraint in other developing regions of the world. Of the three main approaches to control animal trypanosomosis, only chemotherapy is applicable in all ecoregions. The extensive use of available drugs for the control of bovine trypanosomosis has resulted in the appearance of drug resistance in many parts of Africa (Peregrine, 1994). Furthermore, because of the close chemical relationships between trypanocides (Leach and Roberts, 1981), the development of resistance to individual compounds often appears to be associated with cross-resistance to others (Whiteside, 1961; Williamson, 1970). All chemotherapeutic compounds have been on the market for a minimum of 30 years, and the prospects of developing new agents is low, as the returns expected by commercial companies are too small to warrant investment. The optimum solution for controlling trypanosomosis would be the development of a cheap, simple vaccine with a broad protective spectrum. Although the mechanisms of antigenic variation in trypanosomes preclude a vaccine based on the major variant surface glycoproteins, the potential for a multicomponent vaccine based on surface receptor molecules and/or pathogenic molecules secreted by these parasites, still remains. To this end we have undertaken the analysis of trypanosome-host interactions through the identification of trypanosome genes involved in the establishment and maintenance of infection and cause of disease.
Biological processes, such as the cell-division cycle, differentiation and development, are driven by changes in gene expression. To gain a better understanding of the molecular mechanisms involved in the control of proliferation and differentiation in trypanosomes we have developed a differential display PCR method, randomly amplified differentially expressed sequences (RADES), for the rapid identification of differentially expressed trypanosome or leishmania genes (Murphy and Pellé, 1994). The RADES-PCR method differs from other differential display methods in that the template for the PCR fingerprinting step is amplified double-stranded cDNA which can be generated from very small quantities of starting material. Fingerprints are visualised by agarose gel electrophoresis, which simplifies the purification, cloning and analysis steps. The method exploits the fixed 5' and 3' sequences of all trypanosome mRNA sequences characterised to date (De Lange et al., 1984; Parsons et al., 1984) and therefore can be carried out with samples which are contaminated with host material, a feature which is unique to kinetoplastids. This feature is particularly important for intracellular and extracellular mammalian forms in which lengthy purification procedures can themselves alter gene expression. The method is applicable to all kinetoplastids.
Signals for growth control and differentiation are of both host and parasite origin. Parasites respond to signals from the host, signals from each other and in turn send signals to the host to establish and maintain an infection and cause disease. Infective but non-dividing metacyclic forms in tsetse flies that pass into a host begin to rapidly differentiate to actively-dividing bloodstream forms in response to a temperature change and signalling factors from the host. Signals from the infecting parasites prevent the host from mounting an appropriate immune response to combat establishment of the infection. When an infection is established, the actively-dividing trypanosomes are capable of undergoing a differentiation event to become non-dividing. At present, there is little information available on how growth and differentiation of the parasite is controlled, although a decrease in growth rate can allow the host to control and eliminate an infection.
We have applied the RADES-PCR method in two systems; one in which actively dividing Trypanosoma brucei brucei bloodstream forms differentiate to non-dividing forms, and the second in which non-dividing metacyclic forms of T. congolense differentiate to actively dividing bloodstream forms. Over 300 differentially expressed sequences have been identified and partially sequenced from T. b. brucei and T. congolense. Sequences have been checked for homologues in the current sequence databases using Basic Local Alignment Search Tool (BLAST) software. The types of genes not previously identified in trypanosomes that have been identified include receptor and transporter genes, genes encoding signal transduction molecules, molecules controlling the cell division cycle, molecules involved in endocytosis, secretion, sorting and targeting of proteins within the cell and mitochondrial transporter molecules. In addition, T. congolense homologues of ESAGs 2, 6 and 7 have been identified that would not have been isolated by classical hybridisation screening methods due to major differences in the primary sequences of these genes. This is not true for ESAG 4 which encodes adenylate cyclase. A comparative analysis between ESAGs 6 and 7 of T. brucei with the T. congolense homologue is contributing to the definitive identification of the lectin-binding domain of the encoded protein and of the regions that are under strong immunological pressure. These comparative studies are contributing important information towards the rational selection of anti-trypanosome vaccine candidate antigens.
An aspect of these studies is the identification of parasite genes encoding potential immunomodulatory products. Evidence is accumulating which suggests that African trypanosomes can modulate the immune system of their mammalian hosts by secreting molecules with the capacity to mimic or interfere with host cytokines. During the analysis of RADES-PCR products we isolated a 600 bp T. b. brucei product which has a four fold higher level of expression in the actively-dividing long slender (LS) compared to the non-dividing short stumpy (SS) BSF. This product was sequenced and identified as a trypanosome cyclophilin homologue. Cyclophilins (Cyps) constitute a highly conserved family of proteins found in a wide variety of organisms and play an important role in the maturation and correct folding of receptor molecules. Recently it has been reported that macrophages secrete Cyps in response to stimulation by lipopolysaccharides and that the cytokine, IL8, is likely a Cyp variant. Cyps are also the major receptor for the cyclic undecapeptide immunosuppressant drug, cyclosporin A, which complexes with Cyps to block IL2-stimulated transcription and prevent T-cell proliferation. It is tempting to propose that Cyps might be used by trypanosomes for several functions. One possibility may be that Cyps are secreted by trypanosomes during the course of an infection and contribute to the modulation of immune responses of the mammalian host.
Further progress with the RADES-PCR technique has had two other important outcomes: (1) we have found that there are subtle differences in gene expression between in vivo and in vitro generated parasites which can be rapidly identified by this method and (2) the technique works on mixtures of parasite and host cells, even when the number of host cells is far in excess of the parasite cells. This has been achieved by exploitation of unique properties of trypanosome transcripts resulting in the selective amplification of trypanosome-specific sequences. This could be applied to experiments to examine differences in trypanosome gene expression early in infections of tolerant and susceptible animals.
The recent findings that apoptosis occurs in a number of parasitic kinetoplastids, Trypanosoma cruzi (Ameisen et al., 1995), T. b. rhodesiense (Welburn et al., 1996) and Leishmania amazonenis, has important implications for understanding the origins of programmed cell death (PCD) processes in eukaryotic cell survival and signalling in these species and offers new opportunities in controlling the diseases these organisms cause. To examine whether lectin-induced parasite death in T. b. rhodesiense is a programmed process and is under genetic control and to dissect the molecular mechanisms involved in the control of cell death we have examined RNA fidelity and applied the RADES-PCR differential display method, to determine whether genes are differentially expressed during the death process. The method has successfully identified differentially expressed sequences that establish that programmed cell death is an active process in these unicellular organisms and that key genes involved in the process can be rapidly identified (Murphy and Welburn, unpublished).
The work reported here is predominantly based on African trypanosomes, but we believe that the key processes and mechanisms of cell signalling and differentiation can be applied to all. We further believe that this approach to the understanding of these processes can lead to the final grasping of the "holy grail" of an effective vaccine which will incorporate anti-parasite and anti-disease components to overcome this debilitating disease. We would like to know whether others can envisage this or whether we are just clutching at straws.
Ameisen, J.C., Idziorek, T., Billaut-Mulot, O., Loyens, M., Tissier, J.P., Potentier, A. and Ouaissi, A., 1995. Apoptosis in a unicellular eukaryote (Trypanosoma cruzi): implications for the evolutionary origin and role of programmed cell death in the control of cell proliferation, differentiation and survival. Cell Death Diff., 2: 285-300.
De Lange, T., Berkvens, T.M., Veerman, H.J., Frasch, A.C., Barry, J.D. and Borst, P., 1984. Comparison of the genes coding for the common 5' terminal sequence of messenger RNAs in three trypanosome species. Nucleic Acids Res., 12: 4431-4443.
Leach, T.M. and Roberts, C.J., 1981. Present Status of Chemotherapy and Chemoprophylaxis of Animal Trypanosomiasis in the Eastern Hemisphere. Pharmacology and Therapeutics, 13: 91-147.
Murphy, N.B. and Pellé, R., 1994. The use of arbitrary primers and the RADES method for the rapid identification of developmentally regulated genes in trypanosomes. Gene, 141: 53-61.
Parsons, M., Nelson, R.G., Watkins, K.P. and Agabian, N., 1984. Trypanosome mRNAs share a common 5' spliced leader sequence. Cell, 38: 309-316.
Peregrine, A., 1994. Chemotherapy and delivery systems: haemoparasites. Veterinary Parasitology Special Issue, 54: 223-248.
Welburn, S.C. et al., 1996. Apoptosis in procyclic T. b. rhodesiense in vitro. Cell Death Diff., 3: 229-236.
Whiteside, E.F., 1961. Recent work in Kenya on the control of drug-resistant cattle trypanosomiasis. In: 8th Meeting International Scientific Committee for Trypanosomiasis Research. Publication 62, Commission for Technical Co-operation South of the Sahara, London, pp. 141-154.
Williamson, J., 1970. Review of chemotherapeutic and chemoprophylactic agents. In: Mulligan, H.W., ed. The Africa Trypanosomiasis. Allen and Unwin, London, pp. 125-221.
