effect of acidic ph on heat shock gene expression in leishmania

Molecular and Biochemical Parasitology 100 (1999) 95–101

Effect of acidic pH on heat shock gene expression inLeishmania

Srinivas Garlapati, Edit Dahan, Michal Shapira *

Department of Life Sciences, Ben-Gurion Uni6ersity, PO Box 653, Beer-She6a 84105, Israel

Received 5 October 1998; accepted 9 February 1999

Abstract

Temperature and pH shifts trigger differential gene expression and stage transformation in Leishmania. Theparasites encounter dramatic fluctuations in the extra-cellular pH between the mid-gut of the sand fly (pH\8) andthe phagolysosomal vacuole of mammalian macrophages (pHB6). The authors examined the effect of pH shifts onheat shock gene expression in Leishmania amazonensis and Leishmania dono6ani promastigotes. Acidic pH resulted inpreferential stability of the hsp83 transcripts at 26°C, but hsp transcripts were not preferentially translated asobserved during heat shock. Pre-conditioning of promastigotes to acidic pH did not alter the temperature thresholdfor hsp synthesis but lead to an increase in hsp synthesis mainly in L. dono6ani at 37°C, and to a slight decrease inthe arrest of tubulin synthesis in L. amazonensis. The stage specific morphological alterations that take place in vitrocorrelated with the arrest in tubulin synthesis and occurred at different temperatures in L. dono6ani and L.amazonensis. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Leishmania amazonensis ; Leishmania dono6ani ; pH shock; Differential gene expression

1. Introduction

Throughout their life cycle, Leishmania para-sites encounter a wide range of environmentalconditions such as temperature, pH and osmoticpressure. Promastigotes proliferate at an average

temperature of 26°C in a mildly basic environ-ment (\8) in the intestinal tract of the sand flyvector. Amastigotes survive temperatures typicalto mammalian hosts, and a pH range of 4.5–6.0within the phagolysosomal vacuole ofmacrophages [1,2]. Heat shock proteins are there-fore believed to aid in overcoming the damagesinflicted by the immediate environment of theparasites [3–6]. The different Leishmania speciesvary in the range of temperatures which theyresist. While cutaneous species grow at tempera-

* Corresponding author. Tel.: +972-7-6472663; fax: +972-7-6472890.

E-mail address: [email protected] (M. Shapira)

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S. Garlapati et al. / Molecular and Biochemical Parasitology 100 (1999) 95–10196

tures of mammalian skin, namely 32–35°C, andcannot adapt to 37°C, visceral species such as L.dono6ani grow at 37°C and are sensitive only tohigher temperatures. Since these differences areinheritable and cannot be overcome by gradualadaptation, they could have a role in determiningthe target organs for infection by the differentspecies [7]. Although parasites of cutaneous spe-cies are found in internal organs, the strictlylimited range of temperature resistance could con-tribute to the virulence of these parasites within agiven organ.

Stage specific gene expression in Leishmania canbe mimicked in vitro by applying temperature andpH shifts similar to those which occur during theparasite life cycle. Exposing Leishmania ama-zonensis promastigotes to elevated temperaturesresults in the appearance of b-tubulin amastigotespecific transcripts (2.8 and 3.6 kb) [5] and theelimination of a promastigote specific mRNA en-coding for protein kinase A [8]. Incubation inacidic conditions results in the appearance of anamastigote specific antigenic epitope in promastig-otes of Leishmania major [9] and in expression ofthe amastigote specific form of the gp63 metalo-proteinase in L. amazonensis, exhibiting optimalactivity at pH 5.5–6 [10]. The pH optimum forproline transport shifts from 7 to 5.5 betweenpromastigotes and amastigotes of L. dono6ani,and exposure of promastigotes to pH 5.5 activatesthe amastigote specific proline transporter [11].Similarly, expression of the cystein proteinaseCPb in Leishmania mexicana is manifested whenpromastigotes are exposed to acidic pH, whiletemperature elevation is less effective in inducingCPb expression [12]. Accumulation of theamastigote specific transcripts encoding for theA2 protein requires both temperature elevationand reduced pH values [13,14]. Thus, axenic culti-vation of amastigotes is enabled for several Leish-mania species upon adaptation to reduced pH andincreased temperatures [15].

Exposure to temperatures typical to the mam-malian host induces a stress response in Leishma-nia [3,5]. The steady state level of heat-shocktranscripts increases mainly due to their differen-tial processing and stabilization, and translationof these transcripts is dramatically increased [16–

18]. Since during the life cycle of Leishmaniatemperature elevation is combined with acidic pH,the authors examined the individual role of thepH shock in eliciting the stress response.

2. Materials and methods

2.1. Parasites

L. amazonensis isolate MHOM/BR/77/LTB0016 and L. dono6ani LV9 were cultured inSchneider’s medium supplemented with 10% fetalcalf serum (FCS), 4 mM L-glutamine and 25 mgml−1 gentamycin. Parasites were also grown inRPMI supplemented with 10% FCS, 4 mM L-glu-tamine, 25 mg ml−1 gentamycin, biotin 0.0001%,hemin 0.0005%, biopterin 0.002 mg ml−1, HEPES40 mM and adenine 0.1 mM. The RPMI mediawas titrated to pH 5.5 with 20 mM succinic acid.

2.2. RNA analysis

RNA was prepared using a commercial kit(TRI Reagent, Scientific Research Laboratories),and separated over 1.2% denaturing agarose gelsthat were further blotted and hybridized withspecific probes. The intensity of hybridization wasdetermined by analysis in a Molecular DynamicsPhosphorImager. The DNA probes used for hy-bridization were the 3.6 kb SalI genomic repeatunit of hsp83 from L. amazonensis [19] and arDNA probe of L. major, (Jaffe C.L. unpublisheddata).

2.2.1. Metabolic labelingLabeling experiments were performed in RPMI.

Parasites (3×107 cells) were preincubated at dif-ferent temperature and pH conditions in completemedia and then labeled with 20 mCi[35S]methionine for 30 min at the same conditions.Following labeling, the cells were harvested at4°C, washed once with cold phosphate bufferedsaline (PBS) and lysed in SDS-PAGE samplebuffer [20]. Incorporation of [35S]methionine wasmeasured by precipitation with trichloroaceticacid (TCA). Metabolically labeled proteins con-taining the same amounts of incorporated radio

S. Garlapati et al. / Molecular and Biochemical Parasitology 100 (1999) 95–101 97

label corresponded to similar cell counts [21] andwere separated by SDS-PAGE over 9% gels thatwere further processed for fluorography.

3. Results

3.1. Exposure to acidic pH increases the stabilityof hsp83 transcripts

To determine whether exposure to acidic pHinduces a stress response in Leishmania, the stabil-ity and accumulation of the hsp83 transcript in L.amazonensis was examined. Cells were incubatedat pH 5.5 both at 26 and at 35°C, in the presenceor in the absence of actinomycin D, a drug thatarrests transcription. RNA was extracted fromcell aliquots after 2, 4 and 6 h, and analyzed onNorthern blots probed with hsp83. RNA loadswere monitored by hybridization to rRNA. Thehybridization data and their densitometric evalua-tion indicate that the stability of the hsp83 tran-script at pH 5.5 increased in response toextracellular acidification of the culture media(2.590.5-fold; Fig. 1 lanes g and h, top panel)resulting in a two-fold increase (2.090.3-fold) inthe steady state levels after 4 h (lanes e and f, toppanel). A similar effect on stability of the hsp83transcript was observed upon heat shock. Elevat-ing the temperature from 26 to 35°C resulted indifferential stabilization of hsp83 RNA at 35°C(1.790.1-fold; Fig. 1 lanes g and h, bottompanel) and an increase in its steady state level atthe higher temperature (3.790.1-fold; Fig. 1lanes e and f, bottom panel and [16]). A predomi-nant band that hybridizes above the hsp83 tran-script was observed, degrading with similarkinetics. This band most probably represents anon-processed dimer resulting from polycistronictranscription [22]. Although the temperature in-crease had a stronger effect, acidification of theextracellular environment led to a typical stressinduced stabilization of the hsp83 transcript.Hsp83 transcripts in L. dono6ani were also stabi-lized by two-fold in response to a pH shock (datanot shown).

3.2. Extracellular acidic pH does not inducepreferential synthesis of hsp70 and hsp83

Since acidification of the extra cellular environ-ment increased the stability of the hsp83 tran-script, the authors examined whether the hsptranscripts were preferentially translated in re-sponse to a pH shock. L. amazonensis cells weremetabolically labeled at 26°C after exposure tonormal and to acidic pH during a time range of1–4 h, to avoid the potential bypassing of anarrow window of induction. No change was

Fig. 1. pH shock leads to preferential stabilization of the hsp83transcript in L. amazonensis. Top panel: parallel cultures wereincubated at 26°C during 2, 4 and 6 h in the absence (lanes a,b, e, f, i and j) or presence (lanes c, d, g, h, k, and l) ofactinomycin D, at pH 7.4 and at pH 5.5. Bottom Panel:parallel cultures were incubated at 26 and at 35°C during 2, 4and 6 h, in the absence (lanes a, b, e, f, i and j) or in thepresence (lanes c, d, g, h, k, and l) of actinomycin D. Samplesof extracted RNA (10 mg) were analyzed on Northern blotswhich were hybridized with the hsp83 coding gene, and with arRNA probe.


Fig. 2. A pH shock of different lengths does not change thetranslational pattern at 26°C in L. amazonensis cells. Cellsgrown at 26°C pH 7.4 were incubated at 26°C pH 5.5 fordifferent time periods ranging from 1 to 4 h, and then labeledduring 30 min with [35S]methionine at the corresponding con-ditions. Total cell extracts containing equal incorporated radiolabel, which also corresponded to an equal number of cellswere separated on 9% polyacrylamide gels and subjected tofluorography.

3.3. Preconditioning at acidic pH slightly reducesthe temperature threshold which causestranslational arrest of tubulin during heat shock

Since a pH shock by itself was not sufficient toinduce preferential synthesis of hsps, its potentialindirect effects were searched for. Thus, the au-thors examined whether pre-incubation of L.amazonensis cells in acidified medium could alterthe temperature threshold that was required forpreferential synthesis of hsps. Cells from parallelcultures were spun down and resuspended in cul-ture media at pH 7.4 and pH 5.5 and furthergrown for 18 h at 26°C. Aliquots of 1 ml fromeach culture were then incubated for 1 h in aseries of temperatures that varied by incrementsof 1°C, ranging between 26 and 35°C. The cellswere then labeled during 30 min by the additionof [35S]methionine under similar conditions oftemperature and pH. The labeled proteins wereextracted and separated by SDS-PAGE, and theresults are depicted in Fig. 3. Preferential synthe-sis of hsp70 and hsp83 initiated at 30°C, howeverpre-exposure to acidic pH did not change thetemperature threshold for this induction. Synthe-sis of hsp83 seemed stronger at 35°C in the cellsthat were preincubated in acidic pH. Hardly anyincorporation could be observed when L. ama-zonensis cells were labeled at 37°C, indicating thatthe metabolic state of the cells at this temperature

observed in the pattern of protein synthesis inresponse to acidic pH, either at 26 or at 35°C(Fig. 2). A longer exposure to acidic conditions(18 h) also did not change the pattern ofmetabolic labeling (Fig. 3), and neither was thesynthesis of tubulin and non-heat shock proteinsarrested, unlike during a temperature stress (Fig.2, 35°C panel).

Fig. 3. Effect of a pH shock cells combined with gradually increasing temperatures on L. amazonensis. Cells incubated at 26°C atpH 7.4 and at pH 5.5 for 18 h. Samples from each of the two cultures were then incubated for 30 min at a series of increasingtemperatures ranging from 26 to 35°C. The cells were labeled during 30 min with [35S]methionine at the corresponding conditions.Total cell extracts were analyzed as described in the legend of Fig. 2.


Table 1Effect of preincubation in acidic pH on synthesis of hsps andtubulina

(°C) A/N ratio

hsp83hsp70Tubulins

1.03 0.93 1.00290.99 1.0331 1.03

0.97 0.921.04331.081.1834 1.19

0.96 1.335 1.19

a The numbers represent the ratio between the intensity oflabelin at pH 5.5 (A) and at pH 7.4 (N), for the specifiedproteins.

4). However, preconditioning at acidic pH had anindirect effect, resulting in a two-fold increase inhsp synthesis at 37°C. The difference in hsp syn-thesis between cells pre-incubated at normal andacidic pH was not observed at 39°C.

Different Leishmania species vary in their rangeof sensitivity to elevated temperatures. Visceralspecies, such as those related to the L. dono6anicomplex tolerate higher temperatures than cuta-neous species, such as L. amazonensis [7,23], andthese differences are reflected in the pattern ofprotein synthesis (Fig. 4). While in L. dono6anihsp synthesis was induced at 37°C, in L. ama-zonensis this occurred already at 30°C. Similarly,tubulin synthesis was inhibited in L. dono6ani at39°C and in L. amazonensis at 34°C. In bothspecies, the reduction in tubulin synthesis corre-sponds with the arrest of other proteins in the cell,which takes place during severe heat shock. Theinduction of hsp synthesis occurs at even lowertemperatures, when the stress is marginal, due tothe sensitive signaling system of the cells. Induc-tion of hsp synthesis at the lower range of heatshock could confer protection on the translationalmachinery, though this is yet to be shown.

4. Discussion

The role of a pH stress on regulation of heatshock gene expression in Leishmania was exam-ined. Similar to heat shock, exposure of L. ama-zonensis promastigotes to acidic pH resulted inincreased levels of hsp83 mRNA due to its differ-ential stability. However, transcripts encoding forhsps were not preferentially translated under theseconditions, as observed during a temperatureshock.

Regulation of intracellular pH is an importantmechanism for parasite survival. The major mech-anism involved in the intracellular pH regulationin Leishmania promastigotes has been previouslyassumed to be associated with the function of aprotein-translocating Na+/H+ATPase [24]. Fur-ther studies indicated that Cl− dependent trans-port processes are involved, possibly acting inparallel to the H+ATPase in maintaining a stableintracellular pH [25–27]. Although the intracellu-

was severely damaged (data not shown). Synthesisof tubulin and non-heat shock proteins was ar-rested above 34°C but pre-exposure to acidic pHslightly decreased the arrest, maintaining synthesiswhich was 1.2-fold higher at acidic pH as com-pared to pH 7.4. In addition, hsp83 translationwas slightly higher at 35°C (by 1.3-fold) afterpreconditioning the cells to acidic pH (Table 1).These results were observed in two independentexperiments.

The effect of acidic pH on protein synthesis wasexamined also in L. dono6ani. Only a marginalinduction in synthesis of hsp70 and hsp83 wasobserved in response to acidic pH at 26°C (Fig.

Fig. 4. L. dono6ani and L. amazonensis differ in the tempera-ture threshold required to arrest translation of tubulin. L.dono6ani cells were incubated during 18 h at 26°C pH 7.4 andat 26°C at pH 5.5. Samples of both cultures were incubated at26, 37 and 39°C for 1 h and then labeled with [35S]methionineat the corresponding conditions of temperature and pH.


lar pH is maintained neutral throughout the lifecycle, exposure to extracellular acidic pH inducessignaling in L. pifanoi by tyrosine phosphoryla-tion of several specific proteins, indicating thatsignaling occurs. Alternatively, despite thehomeostatic maintenance of the internal pH, mi-nor fluctuations may occur due to changes in theextracellular pH [28], and these fluctuations couldtrigger stress signaling sufficient for stabilizing thehsp83 transcript, but not for its preferentialtranslation.

A temperature shock in L. amazonensis led tothe induction of hsp synthesis, and to an arrest innormal protein synthesis, such as tubulin. Expo-sure to acidic pH does not trigger preferentialtranslation of hsps, but it preconditions the cellsto resist a temperature stress, by mildly alteringthe temperature threshold for the arrest of tubulinsynthesis in L. amazonensis. Pre-incubation inacidic pH caused a stronger hsp synthesis in L.dono6ani at 37°C (by two-fold) and in L. ama-zonensis at 35°C (by 20%). A preconditioningeffect for extracellular acidic pH was reported fortranscriptional activation of hsp70 in HeLa cells.Incubation with anti-inflammatory drugs such assodium salicylate activated the DNA binding ac-tivity of the heat shock factor (HSF) to the corre-sponding heat shock elements (HSE), but failed toactivate transcription of hsp70. Moreover, if thecells were exposed to an extracellular pH of 6.8,binding of the HSF occurred at lower concentra-tions of salicylate, as compared to cells incubatedat pH 7.6 [29]. Thus incubation at lower pHpreconditions the eukaryotic cell to reduce itsthreshold for HSF–HSE binding, which is thefirst step in transcriptional activation of heatshock genes. Exposure of Leishmania cells toacidic pH lead to a stronger induction of hsptranslation, and a delay in the arrest of tubulinsynthesis. Unlike in HeLa cells, expression of heatshock genes in Leishmania is controlled exclu-sively by post transcriptional processes [16,18]thus acidic pH affects gene regulation at the levelof RNA stability and translation.

Different Leishmania species vary in their sensi-tivity to increased temperatures. Cutaneous para-sites such as L. amazonensis cannot resisttemperatures that exceed 33°C, while the visceral

species can adapt to temperatures that exceed37°C [7,23]. Exposure of promastigotes to temper-atures higher than 32–33°C for L. amazonensis,37°C for L. dono6ani and 29°C for L. braziliensis,prevented their in-vitro transformation, and re-sulted in cell death [30]. In the author’s experi-ments these temperature differences were reflectedin the pattern of protein synthesis in the variousLeishmania species. While translational arrest oftubulin and non-heat shock proteins occurred inL. amazonensis already at 34°C, this arrest wasapparent in L. dono6ani only above 37°C. Thebiological basis for this intrinsic difference be-tween the species is yet unknown, and could notbe overcome by gradual adaptation processes. Inconclusion, the observations suggest that acidicpH by itself cannot induce a typical stress re-sponse in Leishmania. However, it could have aprotective role against damage caused by elevatedtemperatures during stage transformation, in vitroand in vivo.

Acknowledgements

The authors thank Prof. R. Morimoto from theNorthwest University for valuable discussions.This work was supported by Grant No. 93-200administered by the US–Israel Binational ScienceFoundation (BSF), Grant No. I-350-062-2/94from the German–Israel Binational Foundation(GIF) and Grant No. 215/98 from the IsraelScience Foundation.

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effect of acidic ph on heat shock gene expression in leishmania

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