Vanessa Ferris

Elucidating the mechanism of genetic exchange is fundamental for understanding how genes for such traits as virulence, disease phenotype, and drug resistance are transferred between pathogen strains. Genetic exchange occurs in the parasitic protists Trypanosoma brucei, T. cruzi, and Leishmania major, but the precise cellular mechanisms are unknown, because the process has not been observed directly. Here we exploit the identification of homologs of meiotic genes in the T. brucei genome and demonstrate that three functionally distinct, meiosis-specific proteins are expressed in the nucleus of a single specific cell type, defining a previously undescribed developmental stage occurring within the tsetse fly salivary gland. Expression occurs in clonal and mixed infections, indicating that the meiotic program is an intrinsic but hitherto cryptic part of the developmental cycle of trypanosomes. In experimental crosses, expression of meiosis-specific proteins usually occurred before cell fusion. This is evidence of conventional meiotic division in an excavate protist, and the functional conservation of the meiotic machinery in these divergent organisms underlines the ubiquity and basal evolution of meiosis in eukaryotes.

The mechanisms underlying virulence in human African trypanosomiasis are poorly understood, although studies with experimental mice suggest that unregulated host inflammatory responses are associated with disease severity. We identified two trypanosomiasis foci with dramatically different disease virulence profiles. In Uganda, infections followed an acute profile with rapid progression to the late stage (meningoencephalitic infection) in the majority of patients (86.8%). In contrast, infections in Malawi were of a chronic nature, in which few patients progressed to the late stage (7.1%), despite infections of several months' duration. All infections were confirmed to be Trypanosoma brucei rhodesiense by testing for the presence of the serum resistance-associated (SRA) gene, but trypanosomes isolated from patients in Uganda or Malawi were distinguished by an SRA gene polymorphism. The two disease profiles were associated with markedly different levels of tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in plasma. In Uganda but not Malawi early-stage TNF-alpha was elevated, while in Malawi but not Uganda early-stage TGF-beta was elevated. Thus, rapid disease progression in Uganda is associated with TNF-alpha-mediated inflammatory pathology, whereas in the milder disease observed in Malawi this may be ameliorated by counterinflammatory cytokines. These differing host responses may result either from differing virulence phenotypes of northern and southern trypanosomes or from immune response polymorphisms in the different host populations.

BACKGROUND: Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy. RESULTS: To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP). Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial) DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones. CONCLUSION: The strategy of using production of yellow hybrids to indicate mating in trypanosomes provides a robust and unequivocal system for analysis of genetic exchange. Mating occurred with high frequency in these experimental crosses, limited only by the ability of both parental trypanosomes to invade the salivary glands. Yellow hybrids appeared as soon as trypanosomes invaded the salivary glands, implicating the short, unattached epimastigote as the sexual stage. The recovery of diploid, triploid and tetraploid hybrids in these crosses was surprising as genetic markers appeared to have been inherited according to Mendelian rules. As the polyploid hybrids could have been produced from fusion of unreduced gametes, there is no fundamental conflict with a model of genetic exchange involving meiosis.

BACKGROUND: The tsetse-transmitted African trypanosomes cause diseases of importance to the health of both humans and livestock. The life cycles of these trypanosomes in the fly were described in the last century, but comparatively few details are available for Trypanosoma (Nannomonas) congolense, despite the fact that it is probably the most prevalent and widespread pathogenic species for livestock in tropical Africa. When the fly takes up bloodstream form trypanosomes, the initial establishment of midgut infection and invasion of the proventriculus is much the same in T. congolense and T. brucei. However, the developmental pathways subsequently diverge, with production of infective metacyclics in the proboscis for T. congolense and in the salivary glands for T. brucei. Whereas events during migration from the proventriculus are understood for T. brucei, knowledge of the corresponding developmental pathway in T. congolense is rudimentary. The recent publication of the genome sequence makes it timely to re-investigate the life cycle of T. congolense. METHODS: Experimental tsetse flies were fed an initial bloodmeal containing T. congolense strain 1/148 and dissected 2 to 78 days later. Trypanosomes recovered from the midgut, proventriculus, proboscis and cibarium were fixed and stained for digital image analysis. Trypanosomes contained in spit samples from individually caged flies were analysed similarly. Mensural data from individual trypanosomes were subjected to principal components analysis. RESULTS: Flies were more susceptible to infection with T. congolense than T. brucei; a high proportion of flies infected with T. congolense established a midgut and subsequent proboscis infection, whereas many T. brucei infections were lost in the migration from foregut to salivary glands. In T. congolense, trypomastigotes ceased division in the proventriculus and became uniform in size. The trypanosomes retained trypomastigote morphology during migration via the foregut to the mouthparts and we confirmed that the trypomastigote-epimastigote transition occurred in the proboscis. We found no equivalent to the asymmetric division stage in T. brucei that mediates transition of proventricular trypomastigotes to epimastigotes. In T. congolense extremely long epimastigotes with remarkably elongated posterior ends were observed in both the proboscis and cibarium; no difference was found in the developmental stages in these two organs. Dividing trypomastigotes and epimastigotes were recovered from the proboscis, some of which were in transition from trypomastigote to epimastigote and vice versa. It remains uncertain whether these morphological transitions are mediated by cell division, since we also found non-dividing cells with a variously positioned, juxta-nuclear kinetoplast. CONCLUSIONS: We have presented a detailed description of the life cycle of T. congolense in its tsetse fly vector. During development in the fly T. congolense shares a common migratory pathway with its close relative T. brucei, culminating in the production of small metacyclic trypanosomes that can be inoculated with the saliva. Despite this outward similarity in life cycle, the transitional developmental stages in the foregut and mouthparts are remarkably different in the two trypanosome species.