Roles of free GPIs in amastigotes of Leishmania

doi:10.1016/S0166-6851(99)00003-1

Molecular and Biochemical Parasitology

Volume 99, Issue 1, 5 March 1999, Pages 103-116

https://doi.org/10.1016/S0166-6851(99)00003-1 Get rights and content

Abstract

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.

Introduction

Human leishmaniasis is transmitted by a sand fly that injects promastigotes, the insect vector stage of Leishmania, into a vertebrate during a blood meal. Promastigotes infect mononuclear phagocytes of the reticuloendothelial system and differentiate into amastigotes. This intracellular stage of Leishmania resides in a membrane-bound parasitophorous vacuole. Following many rounds of division amastigotes lyse the macrophages and the released parasites infect new host cells. Little is known about the requirements of amastigotes for survival and replication.

Free glycosylphosphatidylinositols (GPIs) are abundant on the plasma membranes of mammalian cells and the Leishmania [1], [2], [3]. L. major promastigotes contain 1–5×10⁶ molecules cell⁻¹ of lipophosphoglycan (LPG), a GPI-anchored polysaccharide, and 5×10⁵ molecules of gp63, a GPI-anchored protease [4], [5]. Two groups of ethanolamine (EtN)-containing GPIs (EtN-GPIs) are found in L. mexicana: protein-GPIs containing a ‘conserved core’ of EtN-phospho-Manα1-2Manα1-6Manα1-4GlcN-PI, and glycoinositolphospholipids (GIPLs) (∼10⁷ cell⁻¹), e.g. EPiM₃, EtN-phospho-Manα1-6(Manα1-3)Manα1-4GlcN-PI [6]. The functions of these GPIs in the Leishmania have remained elusive because null mutants in the glycan (i.e. Manα1-4GlcN-Ins) that is common to all three GPIs have not been reported (reviewed in [1], [7], [8], [9], [10]).

With the aim of investigating the effects of a GPI deficiency on intracellular parasitism, GPI-deficient L. amazonensis were generated by stable transfection with a gene from Trypanosoma brucei encoding a GPI-specific phospholipase C (GPI-PLC) [11], [12], [13], [14], [15]. GPI-PLC is an integral membrane protein which appears to co-localize with free GPIs on the cytoplasmic side of the endoplasmic reticulum (ER) membranes [16], [17], [18], [19]. There, it may cleave GPI intermediates (reviewed in [1], [9]), especially those occurring after Man₁GlcN-PI [18], [19], [20]. In this work, the engineered strain was shown to be deficient in EtN-GPIs. Amastigotes of this strain were more sensitive than corresponding promastigotes to the loss of EtN-GPIs; their growth was inhibited, and virulence of the parasites was reduced. GPIs may, therefore, be essential for amastigote replication. Based on these observations a hypothetical explanation for the apparent absence of a GPI-PLC in Leishmania is advanced.

Section snippets

Cell culture and transfection

Parasites were derived from the virulent stock 12 of L. amazonensis LV78 [21]. Promastigotes were transfected with pX63NEO.GPI-PLC [18] or with pX63NEO [22]. Transfectants were selected with 50 μg ml⁻¹ G418 and adapted to grow in medium with 200–800 μg ml⁻¹ G418. Amastigote lines were obtained by culturing transfected promastigotes [23] at pH 5.3, 33°C [24]. These axenic amastigotes were maintained in medium containing G418.

Cell lysis, partial fractionation and GPI-PLC assay

Parasite cells were lysed hypotonically, and a detergent fraction

Partial structures of amastigote GPIs

TLC profiles of polar glycolipids obtained from [³H]EtN-labeled promastigotes and amastigotes are depicted in Fig. 1, Fig. 2. Two glycolipids AmGPI-Z and AmGPI-Y were found predominantly in amastigotes. Both were cleaved by T. brucei GPI-PLC in vitro (Fig. 1), indicating that they are GPIs whose inositol moieties are not acylated [32], [33]. [³H]EtN-labeled AmGPI-Y and AmGPI-Z were resistant to digestion by α-mannosidase (JBAM) (Fig. 2), suggesting the absence of unsubstituted mannosyl residues

Deficiency of two amastigote GPIs is associated with cytostasis

Genes which may be used for targeted mutagenesis of the ‘EtN-GPI core’ (EtN-Man₃-GlcN-Ins-1-phospho-lipid) biosynthetic pathway have not been isolated from any protozoan parasite as yet. Consequently, mutants in the ‘EtN-GPI core’ have not been described in the Leishmania. Given our interest in studying the effects of a GPI deficiency on intracellular parasitism, we generated phenotypic GPI mutants by transfecting Leishmania with a GPI-PLC gene from T. brucei. GPI-PLC cleaves GPI intermediates

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^☆: Note: Dedicated to Professor Moises Agosin (University of Georgia).

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Roles of free GPIs in amastigotes of Leishmania☆

Abstract

Introduction

Section snippets

Cell culture and transfection

Cell lysis, partial fractionation and GPI-PLC assay

Partial structures of amastigote GPIs

Deficiency of two amastigote GPIs is associated with cytostasis

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends. Microbiol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Cell Biol. Int. Rep.

J. Biol. Chem.

Mol. Biochem. Parasitol.

Methods Enzymol.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Biochem. Parasitol.