A novel GFP approach for the analysis of genetic exchange in trypanosomes allowing the in situ detection of mating events

Bingle, Lewis; Eastlake, J. L.; Bailey, Mick; Gibson, Wendy

doi:10.1099/00221287-147-12-3231

Cited by 53 publications

(42 citation statements)

References 37 publications

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“…5). To search for haploid cells, we used K11, a trypanosome cell line expressing GFP under control of the Tet repressor (35). Segregation of the chromosomes carrying the GFP reporter and repressor loci during meiosis should yield fluorescent haploid trypanosomes.…”

Section: Resultsmentioning

confidence: 99%

“…The 1738 mRFP carried a transgene for monomeric red fluorescent protein (6,41). Strain K11 is derived from T. b. gambiense group 2 [MHOM/CI/ 78/TH2 (78E)] (42) and contains a GFP gene driven by a procyclin promoter under control of the tet repressor (35). Procyclic form (PF) trypanosomes were grown in Cunningham's medium (CM) (43) supplemented with 10% vol/vol heat-inactivated FCS, 5 μg/mL of hemin, and 10 μg/mL of gentamycin at 27°C.…”

Section: Methodsmentioning

confidence: 99%

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Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly

Peacock

Ferris

Sharma

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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126

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly

Peacock

Ferris

Sharma

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

126

156

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show abstract

“…AnTat1.1 procyclic trypanosomes nucleofected with the GFP-expressing construct pHD67E (Bingle et al, 2001) were also used to monitor parasite attachment and development within tissues. Cultures of bloodstream and procyclic forms were performed as described previously (Rotureau et al, 2011).…”

Section: Trypanosome Strains and Culturesmentioning

confidence: 99%

A new asymmetric division contributes to the continuous production of infective trypanosomes in the tsetse fly

et al. 2012

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SUMMARYAfrican trypanosomes are flagellated protozoan parasites that cause sleeping sickness and are transmitted by the bite of the tsetse fly. To complete their life cycle in the insect, trypanosomes reach the salivary glands and transform into the metacyclic infective form. The latter are expelled with the saliva at each blood meal during the whole life of the insect. Here, we reveal a means by which the continuous production of infective parasites could be ensured. Dividing trypanosomes present in the salivary glands of infected tsetse flies were monitored by live video-microscopy and by quantitative immunofluorescence analysis using molecular markers for the cytoskeleton and for surface antigens. This revealed the existence of two distinct modes of trypanosome proliferation occurring simultaneously in the salivary glands. The first cycle produces two equivalent cells that are not competent for infection and are attached to the epithelium. This mode of proliferation is predominant at the early steps of infection, ensuring a rapid colonization of the glands. The second mode is more frequent at later stages of infection and involves an asymmetric division. It produces a daughter cell that matures into the infective metacyclic form that is released in the saliva, as demonstrated by the expression of specific molecular markers -the calflagins. The levels of these calcium-binding proteins increase exclusively in the new flagellum during the asymmetric division, showing the commitment of the future daughter cell to differentiation. The coordination of these two alternative cell cycles contributes to the continuous production of infective parasites, turning the tsetse fly into an efficient and long-lasting vector for African trypanosomes.

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“…New therapies are clearly needed. The parasite has two major life cycle forms as follows: insect forms with asexual and sexual stages, present in the tse-tse fly vector, and mammalian host-specific asexual bloodstream forms (4,5). Kinetoplastids are phylogenetically ancient eukaryotes that have unusual DNA in both the nucleus and mitochondrion.…”

mentioning

confidence: 99%

Distinct Genes Encode Type II Topoisomerases for the Nucleus and Mitochondrion in the Protozoan Parasite Trypanosoma brucei

Kulikowicz

Shapiro

2006

Journal of Biological Chemistry

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Topoisomerases are essential for orderly nucleic acid metabolism and cell survival and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically intricate mitochondrial DNA (kinetoplast or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. brucei brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. brucei brucei, termed TbTOP2␣ and TbTOP2␤. Phylogenetically, they share a common node with other nuclear topoisomerases, clearly distinct from a clade that includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned ϳ1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes, but only a single message is detected in Leishmania or Crithidia. TbTOP2␣ encodes an ATP-dependent topoisomerase that appears as a single ϳ170-kDa band on immunoblots and localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2␤ is unclear. Although transcribed in trypanosomes, TbTOP2␤ is not detected by ␤-specific antiserum, and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2␣ as a promising target for the development of much-needed new therapies.Trypanosoma brucei sp. are hemoflagellates from the order Kinetoplastida that cause African trypanosomiasis (also known as sleeping sickness) in humans and a related disease in cattle (1, 2). These diseases are fatal if untreated, and current therapies are antiquated, toxic, and limited by the emergence of drug-resistant parasites (3). New therapies are clearly needed. The parasite has two major life cycle forms as follows: insect forms with asexual and sexual stages, present in the tse-tse fly vector, and mammalian host-specific asexual bloodstream forms (4, 5). Kinetoplastids are phylogenetically ancient eukaryotes that have unusual DNA in both the nucleus and mitochondrion. The total nuclear DNA content of T. brucei brucei is ϳ2.6 ϫ 10 7 bp per haploid genome. Chromosomes, which do not condense during mitosis, are divided into the following three classes: megabase chromosomes (11 pairs), intermediate chromosomes (1-5, ploidy uncertain), and minichromosomes (ϳ100, unpaired) each carrying a single variable surface glycoprotein gene (6). Sequence and analysis of the megabase chromosomes of T. brucei brucei were reported very recently (7). The mitochondrial DNA, termed kinetoplast DNA (kDNA), 2 is a topologically complex structure that consists of thousands of minicircles and tens of maxicircles interlocked in one massive disk-shaped network. Maxic...

show abstract

A novel GFP approach for the analysis of genetic exchange in trypanosomes allowing the in situ detection of mating events

Cited by 53 publications

References 37 publications

Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly

Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly

A new asymmetric division contributes to the continuous production of infective trypanosomes in the tsetse fly

Distinct Genes Encode Type II Topoisomerases for the Nucleus and Mitochondrion in the Protozoan Parasite Trypanosoma brucei

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