Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

Abstract

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.

Introduction

Leishmania are a group of trypanosomatid parasites responsible for a spectrum of diseases ranging from mild cutaneous lesions to often lethal visceral leishmaniasis (Herwaldt, 1999, Handman, 2001). These parasites reside and multiply as extracellular promastigote forms in the gut of sandfly insect vectors and as obligatory intracellular amastigotes within the phagolysosomal system of macrophages in vertebrate hosts (Handman, 2001). Both developmental stages have evolved mechanisms to survive within their respective hostile hydrolytic environments. Although still poorly understood, these mechanisms include resistance to the host immune system and the means of salvaging essential nutrients from their hosts. It is likely that molecules present on the cell surface or secreted by Leishmania are involved in such mechanisms and are important for the parasite survival within its hosts. Some of the Leishmania cell surface molecules have been identified and characterised. They include lipophosphoglycans and glycoproteins such as enzymes or transporters (Schneider et al., 1992, Ferguson, 1997). Some of these molecules have been shown to help the parasite to evade killing by the immune system (Sorensen et al., 1994, Desjardins and Descoteaux, 1997). A few secreted proteins have also been characterised, e.g. secretory acid phosphatase, chitinase or thiol-specific antioxidant (Gottlieb and Dwyer, 1982, Shakarian and Dwyer, 1998, Webb et al., 1998). The secretory acid phosphatases represent the major secreted glycoproteins by Leishmania and are thought to represent important parasite virulence factors (Gottlieb and Dwyer, 1982, Bates and Dwyer, 1987). In addition, their secretion through the secretory pathway of Leishmania parasite has been studied extensively (Bates and Dwyer, 1987, Bates et al., 1989, Bates et al., 1990). Further, it is believed that membrane bound and secreted proteins of Leishmania follow a typical eukaryotic secretory pathway. They travel from the endoplasmic reticulum to the Golgi apparatus to the cell surface, or are secreted, via vesicular trafficking through a flagellar reservoir which is the only site of exocytosis in these parasites (Overath et al., 1997, Landfear and Ignatushchenko, 2001). Therefore, the endoplasmic reticulum quality control of these proteins must be essential in Leishmania to ensure proper folding and to maintain their critical functions in parasite survival.

In higher eukaryotes, the quality control of protein folding in the endoplasmic reticulum is regulated by a series of resident proteins including molecular chaperones, glucosidases and glucosyltransferase [reviewed in (Ellgaard et al., 1999, Parodi, 2000, Helenius and Aebi, 2001)]. The endoplasmic reticulum chaperone calreticulin plays an important role in such quality control process (Zhang et al., 1997, Trombetta and Helenius, 1998). Calreticulin promotes the folding and assembly of newly synthesised glycoproteins and helps to retain in the endoplasmic reticulum misfolded or incompletely assembled proteins that are targeted to proteasome degradation (Hurtley et al., 1989, Klausner and Sitia, 1990, Zhang et al., 1997, Ellgaard et al., 1999). The central proline rich domain (P-domain) of calreticulin plays a critical role in the chaperone function of the molecule since it possesses the lectin-like binding site that interacts with glycans of nascent glycoproteins in the endoplasmic reticulum (Tatu and Helenius, 1997, Vassilakos et al., 1998, Ellgaard et al., 1999). In addition, the P-domain binds calcium (Ca²⁺) with high affinity and low capacity (Baksh and Michalak, 1991) and interacts with other endoplasmic reticulum chaperones such as protein disulfide isomerase (Baksh et al., 1995, Corbett et al., 1999). Calreticulin also binds calcium Ca²⁺ with high capacity and low affinity via its carboxy terminal domain (C-domain) thereby contributes to the Ca²⁺ storage capacity of the endoplasmic reticulum (Nakamura et al., 2001). The N-terminal amino acid sequence of calreticulin (N-domain) is conserved amongst calreticulins from various organisms (Michalak et al., 1999) and also interacts with other endoplasmic reticulum chaperones such as protein disulfide isomerase and endoplasmic reticulum protein 57 (ERP57) (Baksh et al., 1995, Corbett et al., 1999). Calreticulin regulates the chaperone activity of protein disulfide isomerase and ERP57 by modifying its interaction with these proteins in response to fluctuating Ca²⁺ levels in the lumen of the endoplasmic reticulum (Baksh et al., 1995, Corbett et al., 1999).

The putative three-domain structure (N-, P-, and C-domains) has been conserved in all the calreticulin proteins identified to date (Michalak et al., 1999), ranging from human to primitive protozoan parasites such as Leishmania donovani and Trypanosoma cruzi (Joshi et al., 1996, Labriola et al., 1999). However, the role played by calreticulin in the secretory pathway of protozoan parasites remains to be elucidated. In this report, we used a leishmanial episomal expression system to express either the full length or the P-domain of the L. donovani calreticulin in L. donovani. We assessed the effect of such expression on the parasite secretory pathway by measuring the secretion of the parasite secretory acid phosphatases. Since the secretory acid phosphatases and other secreted and membrane bound proteins that traffic through the parasite secretory pathway represent potential virulence factors (Dwyer and Gottlieb, 1983), the survival of these transfected parasites inside human macrophages was assessed in vitro.