Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1

Abstract

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3'-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.

Fig 1. Sb(III) uptake and efflux by different species of Leishmania.

A. Promastigotes of different species of Leishmania were exposed to 10 μM potassium antimony tartrate [Sb(III)]. Cells were harvested at different time points and antimony accumulation was estimated using ICP-MS. B. Everted plasma membrane enriched vesicles were prepared from promastiogtes of different species and transport assayed with 0.1 mM Sb(TS)₂ and 10 mM ATP as energy source. The values at each time point were corrected for non-specific binding by subtraction of the values obtained with 10 mM AMP. Data were expressed as mean ± SE of three independent experiments in triplicate.-●- L. donovani,-○- L. infantum,-▼- L. major,-△- L. tropica,-■- L. braziliensis,-□- L. panamensis.

Fig 2. Common factors of antimonial resistance in different species of Leishmania.

A. Levels of MRPA mRNA: Total RNA was isolated from promastigotes of different species and MRPA mRNA expression levels were estimated using qPCR. Relative (with respect to L. donovani) MRPA expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate. B. Levels of total non-protein thiols: Promastigotes from different species of Leishmania were harvested and proteins were precipitated using tricloroacetic acid. Total non-protein thiols were estimated using dithionitrobenzoic acid. Data were expressed as mean ± SE of three independent experiments in triplicate. C. Levels of AQP1 mRNA: Total RNA was isolated from promastigotes of different species and AQP1 mRNA expression levels were estimated using qPCR. Relative (with respect to L. donovani) AQP1 expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate.

Fig 3. Volume regulation of promastigotes of different species of Leishmania.

Promastigotes of different species of Leishmania were subjected to a hypo-osmotic shock, and the relative changes in cell volume were measured by monitoring the absorbance at 550 nm. Data were expressed as mean ± SE of three independent experiments in triplicate.-●- L. donovani,-○- L. infantum,-▼- L. major,-△- L. tropica,-■- L. braziliensis,-□- L. panamensis.

Fig 4. Stability of AQP1 mRNA in promastigotes of different species of Leishmania.

Promastigotes of different species of Leishmania were exposed to sinefungin followed by actinomycin D treatment. Cells were harvested just before the exposure of actinomycin D (0 minute) and at different time points after the exposure to actinomycin D. Total RNA was isolated and AQP1 mRNA levels were estimated using qPCR. Relative (with respect to 0 minute) AQP1 mRNA levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate.-●- L. donovani,-○- L. infantum,-△- L. major,-▼- L. tropica,-■- L. braziliensis,-□- L. panamensis.

Fig 5. Effect of AQP1 3’-UTR from different species of Leishmania on levels of LUC mRNA, protein expression and activity when transfected in promastigotes of L. donovani.

A. Chimeric constructs with the luciferase (LUC) reporter gene placed under the control of the 3’-UTR of the AQP1 mRNA from different Leishmania species were prepared to assess the role of these sequences in the species-specific regulation process. The neomycin resistance gene (NEO) and LUC transcripts in pSPYNEOαLUC vector were processed either by a 92 synthetic polypyrimidine stretch (Y90AG) (YNEO) or by the intergenic region of the α-tubulin gene (α-IR), respectively. The 3’-UTR (approximately 1.8 kb) followed by a 200 bp intergenic sequence of the AQP1 3’-UTR (IR) from different species were PCR amplified from the genome of different species of Leishmania and introduced at the 3’ end of the LUC gene. The LUC-expressing constructs were transfected into different species of Leishmania, and the effects of the AQP1 3’-UTR on LUC mRNA and protein expression and activity were measured. B. LUC mRNA levels: Total RNA was isolated from promastigotes of L. donovani expressing different chimeric constructs of LUC, and LUC mRNA expression levels were estimated using qPCR. Relative (with respect to LUC) LUC mRNA expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate. C. LUC activity and expression: Estimation of LUC activity (□) was carried out using whole cell lysates. Percent LUC activity was calculated keeping vector control at 100%. Data were expressed as mean ± SE of three independent experiments in triplicate. D. Representative Western blot analysis of transfected promastigotes: Whole cell (1 x 10⁶/lane) lysates of different transfectants were fractionated on SDS-PAGE and blotted onto nitrocellulose membrane. Levels of LUC expressions were detected using an anti-luciferase antibody. α-tubuline was used as loading control. Lanes: 1. pLUC, 2. pLUC-Ld, 3. pLUC-Li, 4. pLUC-Lm, 5. pLUC-Lt, 6. pLUC-Lb, and 7. pLUC-Lp. Amount of luciferase expression (■) relative to cells transfected with pSPYNEOαLUC was estimated by densitometric analysis using ImageJ software followed by normalization against the amount of α-tubulin of the respective cells. Error bars were calculated from the mean ± SE of two independent experiments. Ld- L. donovani, Li- L. infantum, Lm- L. major, Lt- L. tropica, Lb- L. braziliensis, Lp- L. panamensis.

Fig 6. Effect of AQP1 3’-UTR from different species of Leishmania on levels of LUC mRNA, protein expression and activity when transfected in promastigotes of L. major.

A. LUC mRNA levels: Total RNA was isolated from promastigotes of L. major expressing different chimeric constructs of LUC, and LUC mRNA expression levels were estimated using qPCR. Relative (with respect to LUC) LUC mRNA expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate. B. LUC activity and expression: Estimation of LUC activity (□) was carried out using whole cell lysates. Percent LUC activity was calculated keeping vector control at 100%. Data were expressed as mean ± SE of three independent experiments in triplicate. C. Representative Western blot analysis of transfected promastigotes: Whole cell (1 x 10⁶/lane) lysates of different transfectants were fractionated on SDS-PAGE and blotted onto nitrocellulose membrane. Levels of LUC expressions were detected using an anti-luciferase antibody. α-tubulin was used as loading control. Lanes: 1. pLUC, 2. pLUC-Ld, 3. pLUC-Li, 4. pLUC-Lm, 5. pLUC-Lt, 6. pLUC-Lb, and 7. pLUC-Lp. Amount of luciferase expression (■) relative to cells transfected with pSPYNEOαLUC was estimated by densitometric analysis using ImageJ software followed by normalization against the amount of α-tubulin of the respective cells. Error bars were calculated from the mean ± SE of two independent experiments. Ld- L. donovani, Li- L. infantum, Lm- L. major, Lt- L. tropica, Lb- L. braziliensis, Lp- L. panamensis.

Fig 7. Effect of AQP1 3’-UTR from different species of Leishmania on levels of LUC mRNA, protein expression and activity when transfected in promastigotes of L. braziliensis.

A. LUC mRNA levels: Total RNA was isolated from promastigotes of L. braziliensis expressing different chimeric constructs of LUC and LUC mRNA expression levels were estimated using qPCR. Relative (with respect to LUC) LUC mRNA expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate. B. LUC activity and expression: Estimation of LUC activity (□) was carried out using whole cell lysates. Percent LUC activity was calculated keeping vector control at 100%. Data were expressed as mean ± SE of three independent experiments in triplicate. C. Representative Western blot analysis of transfected promastigotes: Whole cell (1 x 10⁶/lane) lysates of different transfectants were fractionated on SDS-PAGE and blotted onto nitrocellulose membrane. Levels of LUC expressions were detected using an anti-luciferase antibody. α-tubulin was used as loading control. Lanes: 1. pLUC, 2. pLUC-Ld, 3. pLUC-Li, 4. pLUC-Lm, 5. pLUC-Lt, 6. pLUC-Lb, and 7. pLUC-Lp. Amount of luciferase expression (■) relative to cells transfected with pSPYNEOαLUC was estimated by densitometric analysis using ImageJ software followed by normalization against the amount of α-tubulin of the respective cells. Error bars were calculated from the mean ± SE of two independent experiments. Ld- L. donovani, Li- L. infantum, Lm- L. major, Lt- L. tropica, Lb- L. braziliensis, Lp- L. panamensis.

Fig 8. Role of the 3’-UTR in species-specific functionality of AQP1.

A. Sb(III) sensitivity of L. donovani promastigotes overexpressing different constructs of AQP1-3’-UTR. B. Sb(V) sensitivity of intracellular amastigotes of L. donovani overexpressing different constructs of AQP1-3’-UTR. C. Sb(III) uptake by L. donovani promastigotes overexpressing different constructs of AQP1-3’-UTR. D. Volume regulation of L. donovani promastigotes overexpressing different constructs of AQP1-3’-UTR;-●- Vector alone,-○- LdAQP1-Ld-3’-UTR (pLd-Ld),-▼-LdAQP1-Lm-3’-UTR (pLd-Lm),-△-LmAQP1-Ld-3’-UTR (pLm-Ld), -■-LmAQP1-Lm-3’-UTR (pLm-Lm), Data were expressed as mean ± SE of three independent experiments in triplicate.