Effect of acidic pH on heat shock gene expression in Leishmania

https://doi.org/10.1016/S0166-6851(99)00037-7Get rights and content

Abstract

Temperature and pH shifts trigger differential gene expression and stage transformation in Leishmania. The parasites encounter dramatic fluctuations in the extra-cellular pH between the mid-gut of the sand fly (pH>8) and the phagolysosomal vacuole of mammalian macrophages (pH<6). The authors examined the effect of pH shifts on heat shock gene expression in Leishmania amazonensis and Leishmania donovani promastigotes. Acidic pH resulted in preferential stability of the hsp83 transcripts at 26°C, but hsp transcripts were not preferentially translated as observed during heat shock. Pre-conditioning of promastigotes to acidic pH did not alter the temperature threshold for hsp synthesis but lead to an increase in hsp synthesis mainly in L. donovani at 37°C, and to a slight decrease in the arrest of tubulin synthesis in L. amazonensis. The stage specific morphological alterations that take place in vitro correlated with the arrest in tubulin synthesis and occurred at different temperatures in L. donovani and L. amazonensis.

Introduction

Throughout their life cycle, Leishmania parasites encounter a wide range of environmental conditions such as temperature, pH and osmotic pressure. Promastigotes proliferate at an average temperature of 26°C in a mildly basic environment (>8) in the intestinal tract of the sand fly vector. Amastigotes survive temperatures typical to mammalian hosts, and a pH range of 4.5–6.0 within the phagolysosomal vacuole of macrophages [1], [2]. Heat shock proteins are therefore believed to aid in overcoming the damages inflicted by the immediate environment of the parasites [3], [4], [5], [6]. The different Leishmania species vary in the range of temperatures which they resist. While cutaneous species grow at temperatures of mammalian skin, namely 32–35°C, and cannot adapt to 37°C, visceral species such as L. donovani grow at 37°C and are sensitive only to higher temperatures. Since these differences are inheritable and cannot be overcome by gradual adaptation, they could have a role in determining the target organs for infection by the different species [7]. Although parasites of cutaneous species are found in internal organs, the strictly limited range of temperature resistance could contribute to the virulence of these parasites within a given organ.

Stage specific gene expression in Leishmania can be mimicked in vitro by applying temperature and pH shifts similar to those which occur during the parasite life cycle. Exposing Leishmania amazonensis promastigotes to elevated temperatures results in the appearance of β-tubulin amastigote specific transcripts (2.8 and 3.6 kb) [5] and the elimination of a promastigote specific mRNA encoding for protein kinase A [8]. Incubation in acidic conditions results in the appearance of an amastigote specific antigenic epitope in promastigotes of Leishmania major [9] and in expression of the amastigote specific form of the gp63 metaloproteinase in L. amazonensis, exhibiting optimal activity at pH 5.5–6 [10]. The pH optimum for proline transport shifts from 7 to 5.5 between promastigotes and amastigotes of L. donovani, and exposure of promastigotes to pH 5.5 activates the amastigote specific proline transporter [11]. Similarly, expression of the cystein proteinase CPb in Leishmania mexicana is manifested when promastigotes are exposed to acidic pH, while temperature elevation is less effective in inducing CPb expression [12]. Accumulation of the amastigote specific transcripts encoding for the A2 protein requires both temperature elevation and reduced pH values [13], [14]. Thus, axenic cultivation of amastigotes is enabled for several Leishmania species upon adaptation to reduced pH and increased temperatures [15].

Exposure to temperatures typical to the mammalian host induces a stress response in Leishmania [3], [5]. The steady state level of heat-shock transcripts increases mainly due to their differential processing and stabilization, and translation of these transcripts is dramatically increased [16], [17], [18]. Since during the life cycle of Leishmania temperature elevation is combined with acidic pH, the authors examined the individual role of the pH shock in eliciting the stress response.

Section snippets

Parasites

L. amazonensis isolate MHOM/BR/77/LTB0016 and L. donovani LV9 were cultured in Schneider’s medium supplemented with 10% fetal calf serum (FCS), 4 mM l-glutamine and 25 μg ml−1 gentamycin. Parasites were also grown in RPMI supplemented with 10% FCS, 4 mM l-glutamine, 25 μg ml−1 gentamycin, biotin 0.0001%, hemin 0.0005%, biopterin 0.002 μg ml−1, HEPES 40 mM and adenine 0.1 mM. The RPMI media was titrated to pH 5.5 with 20 mM succinic acid.

RNA analysis

RNA was prepared using a commercial kit (TRI Reagent,

Exposure to acidic pH increases the stability of hsp83 transcripts

To determine whether exposure to acidic pH induces a stress response in Leishmania, the stability and accumulation of the hsp83 transcript in L. amazonensis was examined. Cells were incubated at pH 5.5 both at 26 and at 35°C, in the presence or in the absence of actinomycin d, a drug that arrests transcription. RNA was extracted from cell aliquots after 2, 4 and 6 h, and analyzed on Northern blots probed with hsp83. RNA loads were monitored by hybridization to rRNA. The hybridization data and

Discussion

The role of a pH stress on regulation of heat shock gene expression in Leishmania was examined. Similar to heat shock, exposure of L. amazonensis promastigotes to acidic pH resulted in increased levels of hsp83 mRNA due to its differential stability. However, transcripts encoding for hsps were not preferentially translated under these conditions, as observed during a temperature shock.

Regulation of intracellular pH is an important mechanism for parasite survival. The major mechanism involved in

Acknowledgements

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

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