A major surface glycoprotein of trypanosoma brucei is expressed transiently during development and can be regulated post-transcriptionally by glycerol or hypoxia

Abstract

Differentiation is a means by which unicellular parasites adapt to different environments. In some cases, the developmental program may be modulated by interactions with the host, but the mechanisms are largely unknown. Trypanosoma brucei is transmitted between mammals by tsetse flies. The development of the procyclic form in the tsetse midgut is marked by the synthesis of a new glycoprotein coat, composed of EP and GPEET procyclins, that is important for survival. Here we demonstrate that the composition of the coat changes in response to extracellular signals in vitro and during development in vivo. EP and GPEET are coinduced when differentiation is initiated. Subsequently, EP expression is maintained, whereas GPEET is repressed after 7-9 days. The timepoint at which GPEET is repressed coincides with the appearance of parasites in a new compartment of the fly midgut. In culture, down-regulation of GPEET can be prevented by exogenous glycerol or accelerated by hypoxia. Regulation is post-transcriptional, and is conferred by the GPEET 3' untranslated region. The same sequence also regulates expression of a reporter gene in the fly. The finding that GPEET is expressed during a defined window during the establishment of infection suggests that it has a specific function in host-parasite interactions rather than a generalized role in shielding underlying membrane molecules.

Figure 1

Detection of GPEET and EP procyclins on the surface of trypanosomes by flow cytometry. The percentage of cells showing increased fluorescent staining (within the gray area of the histogram) relative to undifferentiated cells was determined. (A) Appearance of procyclins during synchronous differentiation of stumpy forms into procyclic forms. (B) GPEET surface expression depends on the culture medium. Procyclic forms derived from a stumpy population were cultured in SDM-79 or DTM for 12 days. (C) Constitutive expression of GPEET requires glycerol. Stumpy forms were transferred to normal SDM-79 medium (−glycerol) or SDM-79 supplemented with 10 mm glycerol (+glycerol) and triggered to differentiate by the addition of cis-aconitate. At day 11, those trypanosomes that had been previously cultured in the presence of glycerol were cultured for a further 6 days in the absence of glycerol and vice versa.

Figure 2

Growth properties of procyclic forms in different medium. Cultures were started from stumpy forms following triggered differentiation (day 0) and diluted at 2-day intervals. The population growth was calculated as cell density multiplied by the cumulative dilution factors. (●) DTM; (○) SDM-79 alone, or (♦) SDM-79 supplemented with 10 mm glycerol; (▿) 15% FCS; (×) 1.6 mm glutamate; (⋄) 11 mm glutamine; (+) 20 μm biotin, or (▵) 12 μm haemin. SDM-79 supplemented with all compounds from DTM except glycerol is indicated by an asterisk.

Figure 3

Glycerol-dependent regulation of GPEET expression. (A) Protein expression. Lysates from equal numbers of procyclic forms were analyzed by SDS-PAGE and immunoblotting using the mAb 5H3. An equivalent blot was incubated with an antiserum against the T. brucei paraflagellar rod (PFR) as a control for the sample loading. (B) Steady-state level of mRNA. Total RNA was hybridized with radiolabeled probes from the coding region of GPEET, PAG3, and β-tubulin or from the 3′ UTR of EP1 as indicated in the Materials and Methods section. Hybridization signals were quantified using a PhosphorImager and normalized to β-tubulin (β-tub) mRNA. (C) Nascent RNA expression. Run-on transcripts from isolated nuclei were hybridized to a DNA fragment consisting of the coding region of GPEET released from pBluescript (pBS) (left). The signals were normalized to the amount of RNA hybridized to EP1 (data not shown). (D) Targeting construct to replace the coding region of GPEET and downstream sequences with the hygromycin acetyltransferase gene (hyg). Arrows indicate the promoter, solid boxes the 5′ and 3′ UTRs, and broken lines the locus-specific sequences. (E) Northern blot analysis of the trypanosome mutant C-hyg. Total RNA was hybridized with radiolabeled sequences from hyg or probes as indicated in B. Procyclic forms of the wild-type (A,B,C) or clone C-hyg (E) were cultured in SDM-79 in the absence (−) or presence (+) of 10 mm glycerol.

Figure 4

Transient transfections of AnTat 1.1 procyclic forms cultured in SDM-79 in the presence of 10 mm glycerol (+) or absence of glycerol (−). Values are represented as the means (n = 3) ± s.d. of CAT activities relative to the activity obtained with the construct pA-CAT/EP1.

Figure 5

(A) Schematic depiction of the two polymorphic allelic loci EP/PAG1 and EP/PAG2, and the two allelic loci GPEET/PAG3 together with the constructs used to stably transform trypanosomes by homologous recombination. Locus-specific sequences upstream of the promoter region and within the 3′ end of the PAG genes are indicated by broken lines. Arrows indicate the promoter region and solid boxes the 5′ and 3′ UTRs. The 3′ UTR and intergenic region from EP1 are indicated by light gray boxes and the corresponding sequences from the GPEET gene are indicated by dark gray boxes. Mutants are named according to the locus that has been targeted (A stands for EP/PAG1, C for GPEET/PAG3) followed by the gene to be expressed and the origin of the 3′ UTR and intergenic sequences. (B–F) Immunofluorescent staining of trypanosomes isolated from the midgut of infected tsetse flies 3 days (B) or 14 days (C–F) after infection with procyclic forms. Wild-type trypanosomes of strain 427 (B,C) were labeled with anti-GPEET antibodies (K1 antiserum) and the mutant clones A-GARP/EP1 (D), C-GARP/EP1 (E), and C-GARP/GPEET (F) were labeled with a rabbit antiserum against GARP. (Left) The FITC image; (right) the corresponding DAPI image from the same field.

Figure 6

Down-regulation of GPEET mRNA under hypoxic conditions. Total RNA was extracted from procyclic forms cultured under hypoxic conditions (<1% O₂) with increased concentrations of CO₂ (10%–20%), or under atmospheric conditions in the presence (+) or absence (−) of glycerol. Northern blots were hybridized with radioactively labeled probes as indicated in the legend to Fig. 3. Hybridization signals were quantified using a PhosphorImager and normalized to β-tubulin (β-tub) mRNA.