Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library

Abstract

RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.

Fig. 1. Clone 1 is resistant to conA killing and has reduced affinity for conA following RNAi induction. (A) Trypanosome strain 29-13, uninduced clone 1 and tetracycline-induced clone 1 (for 14 days) were grown for 24 h in the presence of increasing concentrations of conA. Cell densities were determined using a Coulter counter (model Z1) and cell numbers plotted as a percentage of control untreated cells. The Coulter counter was set to exclude cell aggregates larger than 7.65 µm. This method may not discriminate between living and some dead cells, leading to a conservative estimation of the EC₅₀. The data presented here are representative of four independent experiments in which the EC₅₀ for induced cells was consistently 4- to 6-fold greater than that of parental 29-13 cells. (B) Living trypanosomes were incubated with 10 µg/ml fluorescein-conjugated conA for 15 min at room temperature in cytoM and then analyzed by flow cytometry (10 000 cells/assay). Cell line 29-13, uninduced clone 1 and induced clone 1 (14 days) were analyzed. The two peaks seen in both 29-13 and uninduced clone 1 could be due to the presence of both normal cells and large cells about to divide. Laser intensity was adjusted to yield autofluorescence from unstained cells of ∼10 fluorescence intensity units. (C) 29-13 cells, uninduced clone 1 and tetracycline-induced (14 days) clone 1 were incubated with 5 µg/ml conA for 24 h and then visualized by phase microscopy.

Fig. 2. The hexokinase 1 gene is silenced by RNAi using a construct targeting either the 3′ UTR or the open reading frame. (A) The hexokinase 1 and 2 genomic locus showing DNA fragments (HKORF, HK1 3′ and HK2 3′) used to silence hexokinase expression by RNAi and as probes. (B) Northern analysis of total RNA from uninduced (–) and tetracycline-induced (+) (for 40 h) cells containing the constructs pZJM(HK1 3′) or pZJM(HKORF). Total RNA was purified from 5 × 10⁷ parasites and electrophoresed on a formaldehyde–1.5% agarose gel. rRNA levels were estimated by ethidium bromide staining to ensure equal loading of RNA. The asterisk indicates dsRNA expressed from the pZJM insert.

Fig. 3. Cells expressing dsRNA corresponding to the open reading frame of hexokinase have reduced ConA–fluorescein affinity. Trypanosomes (clone 2) were grown in the presence of tetracycline and analyzed by flow cytometry as described in Figure 1B. The inset in the day 14 panel shows cells harboring pZJM(UMSBP) induced for 14 days. The day 21 panel also shows cells induced for 14 days and then washed free of tetracycline for 7 days (Clone 2 +/– tet).

Fig. 4. Trypanosomes with silenced hexokinase express GPEET- procyclin. Procyclins were purified and prepared for MALDI-TOF MS. An internal standard (insulin, 5733 Da, indicated by an asterisk) was added to some samples. Identical spectra were obtained when insulin was omitted. [M-H]^– ions were identified using the assignments in Acosta-Serrano et al. (1999). EP1-1, EP1-2 and EP3 had m/z of 11 531, 10 430 and 9723, respectively. Full-length GPEET and phosphorylated GPEET (+P) had m/z of 6142 and 6222, respectively. Species indicated as (-10) lack 10 amino acids from the N-terminus, a cleavage occurring during HF treatment (Acosta-Serrano et al., 1999). GPEET species identified as (-4) lack the four N-terminal amino acids. GPEET can also be phosphorylated, with the predominant form being phosphorylated once (+P). Other species correspond to multiply phosphorylated forms (Mehlert et al., 1999). The ions at m/z 8504 and 10 987 (indicated with a hash) are not procyclins; the latter is probably KMP-11 (Acosta-Serrano et al., 1999). (A) Parental 29-13 cells. (B) Cells containing pZJM(HK1 3′) induced for 21 days. (C) Cells containing pZJM (HKORF) induced for 21 days. (D) Cells containing pZJM (HKORF) induced for 14 days and then washed free of tetracycline for 7 days. Prior to washing out tetracycline, cells harboring pZJM(HKORF) (and induced for 14 days) were virtually indistinguishable from cells induced for 21 days, with the exception being that EP1-1(-10) and EP3(-10) could be detected at very low levels. These procyclins are from the cells examined by flow cytometry in Figure 3.

Fig. 5. Effect of silencing PKs on cell growth and conA binding. (A) Trypanosomes grown in triplicate in normal SDM-79 medium (upper panel) or low glucose SDM-79 (lower panel) were induced to express PK dsRNA as indicated. Cell densities were determined with a Coulter counter. Cells were diluted 10-fold when the density reached 5 × 10⁶/ml, and densities shown in the graph are the products of cell density and the dilution factor. Error bars are too small to be visible. (B) Trypanosomes were grown in low glucose SDM-79 and then incubated with 10 µg/ml fluorescein-conjugated conA and analyzed by flow cytometry as described in Figure 1. Cells were induced for 5 days prior to analysis.

Fig. 6. Growth in glucose-depleted medium leads to loss of conA binding in parental 29-13 cells. Trypanosomes (29-13) grown in normal SDM-79 were seeded into low glucose medium, grown for 4 days and then passed (1:10) into glucose-depleted medium when they reached a density of ∼5 × 10⁶ cell/ml. After 5 days growth in glucose-depleted medium, glucose (5 mM) was added back to a portion of the culture (29-13 –/+ glucose). Cells were analyzed by flow cytometry on day 7 using conA–FITC as described in Figure 1. As a control, cells harboring pZJM(HK1 3′) were induced for 10 days.

Fig. 7. Trypanosoma brucei grown in glucose-depleted medium upregulates GPEET-procyclin. 29-13 cells adapted to low glucose medium were grown for 4 days in glucose-depleted medium prior to procyclin isolation and analysis as described in Figure 4. EP1-2(-N-term), the ion at m/z 8089, probably results from non-specific cleavage of 24 residues from the N-terminus of EP1-2 (Acosta-Serrano, 1999). See Figure 4 for discussion of ions at m/z 8504 and 10 987 (indicated with a hash). (A) Parental 29-13 cells cultured in SDM-79 medium. (B) 29-13 cells grown for 4 days in glucose-depleted medium.