Antigenic variation in clones of animal-infective <i>Trypanosoma brucei</i> derived and maintained <i>in vitro</i>

Doyle, J.J.; Hirumi, H.; Hirumi, K.; Lupton, E. N.; Cross, George A.M.

doi:10.1017/s0031182000000810

Cited by 99 publications

(58 citation statements)

References 21 publications

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“…As revealed by an indirect immunofluorescence assay using variant-specific antibodies, variants did not switch antigenic types during the adaptation period. However, consistent with the results of others (21,22), over the course of several months, switching occurred at low levels in cloned populations.…”

Section: Resultssupporting

confidence: 79%

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

Carruthers

Cross

1992

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

113

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This report describes a method for growing both bloodstream-and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HM-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 106 cells at maturity. Tanzania (19) and was cloned as a metacyclic taken directly from an infected tsetse salivary gland (kindly provided by L. Jenni, Swiss Tropical Institute, Basel). It has since been propagated through a total of eight animals (mice or rats) with intermittent cryopreservation. Prior to use in this study, all of these cell lines were recloned by limiting dilution in vitro and checked by immunofluorescence using variant-specific antibodies to ensure expression of the correct variant surface glycoprotein (VSG; except STIB 366E, for which variant-specific antibodies are unavailable).Adaptation of Bloodstream Forms to Liquid Culture. Trypanosomes were grown in either mice or rats to subsaturation density (1-5 x 108 cells per ml of blood) and adapted to grow in HMI-9 medium essentially as described (12). Briefly, 1 ml of infected blood [collected with 0.2 vol of citrate glucose anticoagulant (0.1 M sodium citrate/0.04 M glucose, pH 7.7)] was diluted in 9 ml of HMI-9 medium and then centrifuged at 200 x g for 5 min at room temperature. The supernatant (containing trypanosomes) was transferred to a T-25 culture flask and incubated upright at 370C for 2 hr to allow the majority ofthe remaining blood cells to settle. Trypanosomes in the supernatant were then used to initiate 5-ml cultures in HMI-9 at a density of 1.2 x 106 cells per ml. Cultures were fed daily by adding 2.5 ml of fresh medium after removing an equal volume. In the initial stages, many cells died. Once the trypanosomes began to divide rapidly (when densities recovered to 106 cells per ml), cultures were routinely diluted 1:10 to 1:20 daily to maintain densities in the range of 105-106 cells per ml.Preparation of Agarose Plates and Growth of Colonies.Agarose plates were prepared using 0.65% agarose as suggested by Lee and Van der Ploeg (17). To achieve the desired final concentration ofingredients, it was necessary to prepare and then mix 2x solutions ofmedium and agarose. 2x HMI-9 medium was prepared by dissolving 5.31 g of powdered Iscove's modified Dulbecco's medium (IMDM, GIBCO/ BRL) and 0.907 g of sodium bicarbonate in 92 ml of lowconductivity water followed by the addition of 40 ml of Abbreviation: VSG, variant surface glycoprotein.

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Section: Resultssupporting

confidence: 79%

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

Carruthers

Cross

1992

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

113

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“…Factors thought not to impact on the generation of ordered antigenic variation include dispersed anatomical location of trypanosomes within the host (39), infection-associated immunosuppression (40), and putative, direct-host involvement in the switching mechanism (17,41). Ordered antigenic variation occurs in the bloodstream without complication from parasites in other anatomical niches.…”

Section: T Rypanosomes Switch Antigens Primarily By Duplication Ofmentioning

confidence: 99%

Parasite-intrinsic factors can explain ordered progression of trypanosome antigenic variation

Lythgoe

Morrison

Read

et al. 2007

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

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Pathogens often persist during infection because of antigenic variation in which they evade immunity by switching between distinct surface antigen variants. A central question is how ordered appearance of variants, an important determinant of chronicity, is achieved. Theories suggest that it results directly from a complex pattern of transition connectivity between variants or indirectly from effects such as immune cross-reactivity or differential variant growth rates. Using a mathematical model based only on known infection variables, we show that order in trypanosome infections can be explained more parsimoniously by a simpler combination of two key parasite-intrinsic factors: differential activation rates of parasite variant surface glycoprotein (VSG) genes and densitydependent parasite differentiation. The model outcomes concur with empirical evidence that several variants are expressed simultaneously and that parasitaemia peaks correlate with VSG genes within distinct activation probability groups. Our findings provide a possible explanation for the enormity of the recently sequenced VSG silent archive and have important implications for field transmission.mathematical model ͉ Typanosoma brucei ͉ variant surface glycoprotein ͉ switching ͉ hierarchy

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“…The 'single marker' cell line, a derivative of T. brucei Lister 427, antigenic-type MITat 1.2, clone 221a (Doyle et al, 1980), expresses T7 RNA polymerase and the tet repressor, allowing inducible expression of ectopic genes under control of the T7 promoter and tet operator . All cell lines used in this study were derived from the 'single marker' cell line.…”

Section: Trypanosome Culturementioning

confidence: 99%

Histone H2AZ dimerizes with a novel variant H2B and is enriched at repetitive DNA inTrypanosoma brucei

Lowell

Kaiser

Janzen

et al. 2005

Journal of Cell Science

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H2AZ is a widely conserved histone variant that is implicated in protecting euchromatin from the spread of heterochromatin. H2AZ is incorporated into nucleosomes as a heterodimer with H2B, by the SWR1 ATP-dependent chromatin-remodeling complex. We have identified a homolog of H2AZ in the protozoan parasite Trypanosoma brucei, along with a novel variant of histone H2B (H2BV) that shares ∼38% sequence identity with major H2B. Both H2AZ and H2BV are essential for viability. H2AZ localizes within the nucleus in a pattern that is distinct from canonical H2A and is largely absent from sites of transcription visualized by incorporation of 5-bromo-UTP (BrUTP). H2AZ and H2BV colocalize throughout the cell cycle and exhibit nearly identical genomic distribution patterns, as assessed by chromatin immunoprecipitation. H2AZ co-immunoprecipitates with H2BV but not with histones H2B or H2A nor with the variant H3V. These data strongly suggest that H2AZ and H2BV function together within a single nucleosome, marking the first time an H2AZ has been shown to associate with a non-canonical histone H2B.

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Antigenic variation in clones of animal-infective Trypanosoma brucei derived and maintained in vitro

Cited by 99 publications

References 21 publications

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

Parasite-intrinsic factors can explain ordered progression of trypanosome antigenic variation

Histone H2AZ dimerizes with a novel variant H2B and is enriched at repetitive DNA inTrypanosoma brucei

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