RESUMO
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.
Assuntos
Estágios do Ciclo de Vida , Meiose , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologia , Animais , Fusão Celular , Células Clonais , Cruzamentos Genéticos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Haploidia , Masculino , Meiose/genética , Modelos Biológicos , Homologia de Sequência do Ácido Nucleico , Transfecção , Trypanosoma brucei brucei/genéticaRESUMO
Like yeast, Trypanosoma brucei is a model organism and has a published genome sequence. Although T. b. brucei strain 427 is used for studies of trypanosome molecular biology, particularly antigenic variation, in many labs worldwide, this strain was not selected for the genome sequencing project as it is monomorphic and unable to complete development in the insect vector. Instead, the fly transmissible, mating competent strain TREU 927 was used for the genome project, but is not as easily grown or genetically manipulable as strain 427; furthermore, recent findings have spread concern on the potential human infectivity of TREU 927. Here we show that a 40-year-old cryopreserved line of strain 427, Variant 3, is fly transmissible and also able to undergo genetic exchange with another strain of T. b. brucei. Comparison of Variant 3 with lab isolates of 427 shows that all have variant surface glycoprotein genes 117, 121 and 221, and identical alleles for 3 microsatellite loci. Therefore, despite some differences in molecular karyotype, there is no doubt that Variant 3 is an ancestral line of present day 427 lab isolates. Since Variant 3 grows fast both as bloodstream forms and procyclics and is readily genetically manipulable, it may prove useful where a fly transmissible version of 427 is required.
Assuntos
Dípteros/parasitologia , Recombinação Genética , Trypanosoma brucei brucei/fisiologia , Animais , Southern Blotting , Impressões Digitais de DNA , Repetições de Microssatélites , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/classificaçãoRESUMO
BACKGROUND: Genetic exchange occurs between Trypanosoma brucei strains during the complex developmental cycle in the tsetse vector, probably within the salivary glands. Successful mating will depend on the dynamics of co-infection with multiple strains, particularly if intraspecific competition occurs. We have previously used T. brucei expressing green fluorescent protein to study parasite development in the vector, enabling even one trypanosome to be visualized. Here we have used two different trypanosome strains transfected with either green or red fluorescent proteins to study the dynamics of co-infection directly in the tsetse fly. RESULTS: The majority of infected flies had both trypanosome strains present in the midgut, but the relative proportion of red and green trypanosome strains varied considerably between flies and between different sections of the midgut in individual flies. Colonization of the paired salivary glands revealed greater variability than for midguts, as each gland could be infected with red and/or green trypanosome strains in variable proportions. Salivary glands with a mixed infection appeared to have a higher density of trypanosomes than glands containing a single strain. Comparison of the numbers of red and green trypanosomes in the proventriculus, salivary exudate and glands from individual flies showed no correlation between the composition of the trypanosome population of the proventriculus and foregut and that of the salivary glands. For each compartment examined (midgut, foregut, salivary glands), there was a significantly higher proportion of mixed infections than expected, assuming the null hypothesis that the development of each trypanosome strain is independent. CONCLUSION: Both the trypanosome strains used were fully capable of infecting tsetse, but the probabilities of infection with each strain were not independent, there being a significantly higher proportion of mixed infections than expected in each of three compartments examined: midgut, proventriculus and salivary glands. Hence there was no evidence of competition between trypanosome strains, but instead co-infection was frequent. Infection rates in co-infected flies were no different to those found routinely in flies infected with a single strain, ruling out the possibility that one strain enhanced infection with the other. We infer that each fly is either permissive or non-permissive of trypanosome infection with at least 3 sequential checkpoints imposed by the midgut, proventriculus and salivary glands. Salivary glands containing both trypanosome strains appeared to contain more trypanosomes than singly-infected glands, suggesting that lack of competition enhances the likelihood of genetic exchange.
RESUMO
Blood smears and blood lysate samples from freshwater fishes captured in the Okavango Delta, Botswana, were examined to determine whether their trypanosomes were all Trypanosoma mukasai, a species of supposed broad host specificity and widespread existence across Africa. Trypanosomes and/or babesiosomes occurred in 20/32 blood smears, and morphometric analysis of trypanosomes from 13/32 smears showed features suggestive of T. mukasai, including nuclear indices consistently >1. In 16/32 blood lysate samples from which DNA was extracted, trypanosome DNA was detected in 12/16 by PCR (polymerase chain reaction), using trypanosome-specific ssu rRNA gene primers. Two samples positive for trypanosomes in blood smears yielded no amplifiable trypanosome DNA, but 4 samples with no detectable infection in blood smears were positive for trypanosome DNA, suggesting an overall trypanosome prevalence rate of 17/32 (53%) among fishes and demonstrating the value of PCR in trypanosome recognition. Cloning and sequencing of the 12 amplified fragments revealed 2 genotypic groups among these fish trypanosomes. Group 1 trypanosomes were from cichlids and 3 families of catfishes, Group 2 from 2 types of catfishes. Sequence comparison showed that the consensus Group 1 sequence was most similar to that of Trypanosoma cobitis, representing European fish trypanosomes of the carassii type, while the consensus Group 2 sequence showed similarity with a trypanosome sequence from another African catfish, Clarias angolensis. It was concluded that the identification of T. mukasai remains a problem, but at least 2 genotypic groups of trypanosomes occur in Okavango Delta fishes, and catfishes in this region appear to contain both types.
Assuntos
Peixes/parasitologia , Trypanosoma/citologia , Trypanosoma/genética , Animais , Sequência de Bases , Botsuana , Primers do DNA , Genótipo , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.
Assuntos
Transferência Genética Horizontal , Variação Genética , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , Recombinação Genética/genética , Trypanosoma brucei rhodesiense/genética , Tripanossomíase Africana/epidemiologia , Tripanossomíase Africana/parasitologia , África Oriental/epidemiologia , Animais , Cruzamentos Genéticos , Fluorescência , Humanos , Especificidade da EspécieRESUMO
The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.
Assuntos
Primers do DNA/genética , Reação em Cadeia da Polimerase/métodos , Trypanosoma/genética , Moscas Tsé-Tsé/parasitologia , Animais , Dados de Sequência Molecular , Filogenia , RNA Ribossômico/genética , Tanzânia , Trypanosoma/isolamento & purificação , Tripanossomíase/parasitologiaRESUMO
BACKGROUND: Genetic exchange has been described in several kinetoplastid parasites, but the most well-studied mating system is that of Trypanosoma brucei, the causative organism of African sleeping sickness. Sexual reproduction takes place in the salivary glands (SG) of the tsetse vector and involves meiosis and production of haploid gametes. Few genetic crosses have been carried out to date and consequently there is little information about the mating compatibility of different trypanosomes. In other single-celled eukaryotes, mating compatibility is typically determined by a system of two or more mating types (MT). Here we investigated the MT system in T. brucei. METHODS: We analysed a large series of F1, F2 and back crosses by pairwise co-transmission of red and green fluorescent cloned cell lines through experimental tsetse flies. To analyse each cross, trypanosomes were cloned from fly SG containing a mixture of both parents, and genotyped by microsatellites and molecular karyotype. To investigate mating compatibility at the level of individual cells, we directly observed the behaviour of SG-derived gametes in intra- or interclonal mixtures of red and green fluorescent trypanosomes ex vivo. RESULTS: Hybrid progeny were found in all F1 and F2 crosses and most of the back crosses. The success of individual crosses was highly variable as judged by the number of hybrid clones produced, suggesting a range of mating compatibilities among F1 progeny. As well as hybrids, large numbers of recombinant genotypes resulting from intraclonal mating (selfers) were found in some crosses. In ex vivo mixtures, red and green fluorescent trypanosome gametes were observed to pair up and interact via their flagella in both inter- and intraclonal combinations. While yellow hybrid trypanosomes were frequently observed in interclonal mixtures, such evidence of cytoplasmic exchange was rare in the intraclonal mixtures. CONCLUSIONS: The outcomes of individual crosses, particularly back crosses, were variable in numbers of both hybrid and selfer clones produced, and do not readily fit a simple two MT model. From comparison of the behaviour of trypanosome gametes in inter- and intraclonal mixtures, we infer that mating compatibility is controlled at the level of gamete fusion.
Assuntos
Trypanosoma brucei brucei/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Quimera , Cruzamentos Genéticos , DNA de Protozoário/análise , DNA de Protozoário/genética , Feminino , Genes Reporter , Genótipo , Cariótipo , Masculino , Recombinação Genética , Reprodução , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/genéticaRESUMO
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.
Assuntos
Vetores de Doenças , Estágios do Ciclo de Vida , Trypanosoma congolense/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologia , Estruturas Animais/parasitologia , Animais , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Microscopia , Saliva/parasitologia , Fatores de Tempo , Trypanosoma congolense/citologiaRESUMO
Unlike other dipteran disease vectors, tsetse flies of both sexes feed on blood and transmit pathogenic African trypanosomes. During transmission, Trypanosoma brucei undergoes a complex cycle of proliferation and development inside the tsetse vector, culminating in production of infective forms in the saliva. The insect manifests robust immune defences throughout the alimentary tract, which eliminate many trypanosome infections. Previous work has shown that fly sex influences susceptibility to trypanosome infection as males show higher rates of salivary gland (SG) infection with T. brucei than females. To investigate sex-linked differences in the progression of infection, we compared midgut (MG), proventriculus, foregut and SG infections in male and female Glossina morsitans morsitans. Initially, infections developed in the same way in both sexes: no difference was observed in numbers of MG or proventriculus infections, or in the number and type of developmental forms produced. Female flies tended to produce foregut migratory forms later than males, but this had no detectable impact on the number of SG infections. The sex difference was not apparent until the final stage of SG invasion and colonisation, showing that the SG environment differs between male and female flies. Comparison of G. m. morsitans with G. pallidipes showed a similar, though less pronounced, sex difference in susceptibility, but additionally revealed very different levels of trypanosome resistance in the MG and SG. While G. pallidipes was more refractory to MG infection, a very high proportion of MG infections led to SG infection in both sexes. It appears that the two fly species use different strategies to block trypanosome infection: G. pallidipes heavily defends against initial establishment in the MG, while G. m. morsitans has additional measures to prevent trypanosomes colonising the SG, particularly in female flies. We conclude that the tsetse-trypanosome interface works differently in G. m. morsitans and G. pallidipes.
Assuntos
Trypanosoma brucei brucei/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologia , Animais , Feminino , Trato Gastrointestinal/parasitologia , Humanos , Masculino , Fatores SexuaisAssuntos
Genoma de Protozoário , Glicoproteínas de Membrana/genética , Proteínas de Protozoários , Trypanosoma brucei rhodesiense/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Ordem dos Genes , Variação Genética , Dados de Sequência Molecular , Testes de Sensibilidade Parasitária , Mapeamento por RestriçãoRESUMO
BACKGROUND: Mating in Trypanosoma brucei is a non-obligatory event, triggered by the co-occurrence of different strains in the salivary glands of the vector. Recombinants that result from intra- rather than interclonal mating have been detected, but only in crosses of two different trypanosome strains. This has led to the hypothesis that when trypanosomes recognize a different strain, they release a diffusible factor or pheromone that triggers mating in any cell in the vicinity whether it is of the same or a different strain. This idea assumes that the trypanosome can recognize self and non-self, although there is as yet no evidence for the existence of mating types in T. brucei. RESULTS: We investigated intraclonal mating in T. b. brucei by crossing red and green fluorescent lines of a single strain, so that recombinant progeny can be detected in the fly by yellow fluorescence. For strain 1738, seven flies had both red and green trypanosomes in the salivary glands and, in three, yellow trypanosomes were also observed, although they could not be recovered for subsequent analysis. Nonetheless, both red and non-fluorescent clones from these flies had recombinant genotypes as judged by microsatellite and karyotype analyses, and some also had raised DNA contents, suggesting recombination or genome duplication. Strain J10 produced similar results indicative of intraclonal mating. In contrast, trypanosome clones recovered from other flies showed that genotypes can be transmitted with fidelity. When a yellow hybrid clone expressing both red and green fluorescent protein genes was transmitted, the salivary glands contained a mixture of fluorescent-coloured trypanosomes, but only yellow and red clones were recovered. While loss of the GFP gene in the red clones could have resulted from gene conversion, some of these clones showed loss of heterozygosity and raised DNA contents as in the other single strain transmissions. Our observations suggest that many recombinants are non-viable after intraclonal mating. CONCLUSION: We have demonstrated intraclonal mating during fly transmission of T. b. brucei, contrary to previous findings that recombination occurs only when another strain is present. It is thus no longer possible to assume that T. b. brucei remains genetically unaltered after fly transmission.
RESUMO
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.
RESUMO
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.