Figure 1.
Effect of temperature and pH stress on general translation in Leishmania
. Polysome profile analysis of L. infantum promastigotes (no stress; 25°C and pH 7.3) and parasites exposed for different time periods to either elevated temperature (37°C) or low pH (5.5) or to a combined temperature and acidic pH (T°+pH) stress. Both elevated temperature and low pH are major signals triggering amastigote differentiation within the phagolysosome of the host macrophage [8], [10]. Leishmania lysates were fractionated by 15% to 45% sucrose density ultracentrifugation and absorbance (Abs) at 254 nm was continuously recorded. The 40S and 60S ribosomal subunits, 80S monosome and polyribosome peaks are indicated. Data displayed here represent one of 3–5 separate experiments.
Figure 2.
The effect of temperature and acidic pH stress on the de novo protein synthesis in Leishmania.
(A) Levels of [35S]-methionine incorporation (as an indicative of general translation rates) in L. infantum promastigotes subjected from 1 to 8 hours to either an elevated temperature (37°C) stress (A) or to a combined temperature and low pH (pH 5.5) stress (C). Thirty minutes before protein sample collection, 1 µCi/ml [35S]-methionine protein labeling mix was added to the culture media lacking methionine. Protein synthesis was measured as the incorporated radioactivity by a scintillation counter and expressed as cpm. Results are the mean of a minimum of six independent experiments. (B) [35S]-Met-labeled proteins resolved on 10% SDS-PAGE. Cells grown at 25°C/pH 7.3 (no stress) or exposed to elevated temperature (37°C) (T°) only or to a combined temperature and acidic pH (pH 5.5) stress for 1, 4 and 8 hours were labelled with [35S]-Met for 30 min. An autoradiograph of the SDS-PAGE analysis is shown here. Examples of upregulated or downregulated proteins under stress are indicated by arrows. (D) Pulse-chase assay to assess the stability of L. infantum proteins under stress. L. infantum promastigotes were incubated in a methionine-deprived medium supplemented with [35S]-Met for 1 hour at 25°C and then transferred to a non-radioactive methionine-containing medium at 37°C and pH 5.5 and grown for 24 hours. Aliquots were taken at 1, 3, 5 and 24 hours and the radioactivity (in cpm) was measured by a scintillation counter. A decrease in cpm numbers corresponds to protein degradation under stress. Results are the mean of three independent experiments. Significant differences between the various conditions in (C) are indicated (* p<0.05 and **p<0.01) using one-way ANOVA followed by the Tukey-Kramer test.
Figure 3.
Regulation of general translation throughout the Leishmania infantum life cycle.
Promastigote to amastigote differentiation (A). Polysome profile analysis of L. infantum promastigotes (Pro) and axenic amastigotes (Ama) grown in MAA 20 medium at 37°C and pH 5.5 for 4 days in average (1st passage). L. infantum differentiates into amastigote-like forms after ∼4 days of growth under these conditions as illustrated in (A). Amastigote to promastigote differentiation (B). Polysome profile analysis of L. infantum axenic amastigotes transferred from the MAA 20 medium at 37°C and pH 5.5 to a MAA medium at 25°C and pH 7.3 for up to 8 hours. Data displayed here represent one of three separate experiments. (C) The effect of L. infantum amastigote to promastigote differentiation on global protein synthesis as estimated by the incorporation levels of [35S]-Met into the Leishmania proteins (as described in Figure 2). Results are the mean of four independent experiments.
Figure 4.
The translation initiation factor 2-alpha subunit (eIF2α) is phosphorylated upon temperature and acidic pH stress and during promastigote to amastigote differentiation.
L. infantum promastigotes (Pro) (no stress) or promastigotes exposed to elevated temperature (37°C) or to a combined temperature and low pH (pH 5.5) stress for 4, 5 and 8 hours (A) or axenic amastigotes (Ama) and or amastigotes subjected to promastigote differentiation (B) were lysed and whole-cell lysates were used in immunoblots with a rabbit polyclonal anti-eIF2α [pS51] phosphospecific antibody (eIF2α-P) to detect eIF2α phosphorylation. Protein loading was monitored using an anti-Leishmania specific eIF2α antibody described in [27].
Figure 5.
Translation of Leishmania housekeeping genes under stress.
(A–B) Northern blot hybridization of L. infantum RNA samples isolated from sucrose gradient fractions (F: free mRNPs; M: monosomes; L: light polysomes; H: heavy polysomes) to determine the association of the constitutively expressed alpha-tubulin mRNA with translating polysomes in the absence of stress (no stress) or under temperature (T°) or acidic pH stresses (A) and or in the presence of the endoplasmic reticulum (ER)-inducing agents thapsigargin (Tg) or tunicamycin (TN) (B, upper panels). The ethidium bromide-stained gels used for northern blot analysis served as a loading control (rRNA is indicated). Polysome profiling analysis of L. infantum promastigotes untreated (no stress) or treated with 0.4 µM of thapsigargin (Tg) for 4 hours (B, lower panels). Data shown here are representative of at least four independent experiments with similar results.
Figure 6.
Translation of L. infantum developmentally regulated transcripts under temperature and acidic pH stresses.
(A) Northern blot hybridization of L. infantum RNA isolated from sucrose gradient fractions (F, M, L and H are as in Figure 5) to assess the association of the amastigote-specific A2 transcript with the ribosomes under temperature stress or acidic pH stress or a combination of both stresses. The ethidium bromide-stained gels used for northern blot analysis served as a loading control (rRNA is indicated). (B) Western blot analysis of L. infantum protein lysates using a monoclonal anti-A2 antibody to determine expression levels of A2 protein under different stresses, including high temperature, low pH and Tg-induced ER stresses. Controls using L. infantum promastigotes (Pro) (non stress) or axenic amastigotes (Ama) were included for both northern and western blots. (C) Northern blot hybridization of L. infantum total RNA to determine A2 mRNA expression during amastigote to promastigote differentiation for up to 48 hrs. (D) Northern blot hybridization of L. infantum RNA isolated from sucrose gradient fractions to determine the association of the A2 mRNA with monosomes or translating polysomes. (E) Western blot with an anti-A2 antibody of L. infantum amastigotes transferred into MAA-20 medium at 25°C and pH 7.3 for up to 5 hours to allow its differentiation into promastigote forms. The anti-alpha-tubulin antibody was used as loading control. Data shown here are representative of three independent experiments with similar results.