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. 2015 Mar-Apr;200(1-2):1-4.
doi: 10.1016/j.molbiopara.2015.04.001. Epub 2015 Apr 18.

Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei

Kiantra Ramey-Butler  1 , Elisabetta Ullu  2 , Nikolay G Kolev  3 , Christian Tschudi  4
Affiliations

Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei

Kiantra Ramey-Butler et al. Mol Biochem Parasitol. 2015 Mar-Apr.

Abstract

One distinctive feature of the Trypanosoma brucei life cycle is the presence of two discrete populations that are based on differential expression of variant surface glycoproteins (VSGs). Both are adapted to the environmental pressures they face and more importantly, both contribute directly to transmission. Metacyclics in the tsetse fly enable transmission to a new mammalian host, whereas bloodstream trypanosomes must avoid immune destruction to the extent that sufficient numbers are available for transmission, when the insect vector takes a blood meal. At present, there are few investigations on the molecular aspects of parasite biology in the tsetse vector and specifically about the activation of metacyclic VSG gene expression. Here we used an established in vitro differentiation system based on the overexpression of the RNA-binding protein 6 (RBP6), to monitor two metacyclic VSGs (VSG 397 and VSG 653) during development from procyclics to infectious metacyclic forms. We observed that activation of these two mVSGs was simultaneous both at the transcript and protein level, and manifested by the appearance of only one of the mVSGs in individual cells.

Keywords: Gene activation; Gene expression; Metacyclics; Surface coat; Trypanosoma brucei; VSG.

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Figures

Fig. 1
Fig. 1
Timeline of mVSG mRNA and protein expression and metacyclic cell proportion in cultures of T. brucei Lister 427 (29-13) induced to express RBP6. Cells were grown and induced with doxycycline as described previously [15] in the presence of 4.5 g/L glucose. (A) The percentage of metacyclic cells on each day of induced RBP6 expression was obtained as in [15] and cell samples were collected and processed for isolating total RNA or lysed with SDS-containing buffer. (B) Northern blots with equal amount of total RNA per sample (top) and probes specific for VSG 397 and VSG 653 were quantified with ImageQuantTL software (GE) (bottom). Large rRNAs stained with methylene blue are the loading controls. Day 0 of induction is set as 0% mVSG mRNA expression and the time point with highest value is set as 100% mVSG mRNA expression. (C) Western blots with equal cell number equivalents (top) processed with rabbit serum against the VSG 397 peptide CSDDAATYTSGSIAGTHALG (dilution 1:2,000) and rat serum against the VSG 653 peptide CRLHSGADNEGVVQNAGADN (dilution 1:1,000) were quantified with GeneTools software (Syngene) (bottom). A cross-reacting band served as a loading control. Day 0 of induction is set as 0% mVSG expression and the time point with highest value is set as 100% mVSG expression.
Fig. 2
Fig. 2
Coat formation by a single mVSG in metacyclics is followed by dispersal of the nucleolus. (A) Metacyclic cells were purified from culture also containing procyclics and epimastigotes on 0.1 mm diameter zirconia/silica beads column prepared in a Pasteur pipette in BBSG buffer (50 mM bicine, 50 mM NaCl, 5 mM KCl, 70 mM glucose). IF assay was performed with paraformaldehyde-fixed cells with the anti-VSG 397 rabbit serum (diluted 1:5,000) and anti-VSG 653 rat serum (diluted 1:500) and Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-rat secondary antibodies (Invitrogen), diluted 1:1,000. Note that some metacyclics are not recognized by either anti-VSG antibodies. (B) IF assay with affinity-purified anti-Pol I antibodies and L1C6 mouse monoclonal antibody was performed on PF, purified MF, and T.brucei Lister 427 single marker BF cells as described previously [3]. Arrow points to the ESB in BF. (C) Transmission EM of nucleus of a cell without a VSG coat (left) and a VSG-coated cell (right). The nucleolus is easily identifiable in the nonmetacyclic nucleus as an electron-dense sphere and it is not discernible in the metacyclic nucleus.
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