Leishmania mexicanaMutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

Abstract

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (⌬GPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other nonprotein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ⌬GPI8 mutant. Significantly, the ⌬GPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth. INTRODUCTIONLeishmania are protozoan parasites that cause a spectrum of diseases in humans. These parasites alternate between a flagellated promastigote stage that proliferates within the midgut of the sandfly vector and a nonmotile amastigote stage that invades mammalian macrophages, where they occupy the phagolysosome compartment. The cell surface of the promastigote stage is coated by a protective glycocalyx that comprises a number of glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a complex GPI-anchored lipophosphoglycan (LPG), and a family of free GPIs (termed glycoinositolphospholipids [GIPLs]) Beverley and Turco, 1998; Figure 1). Components in this glycoclayx are thought to be essential for parasite survival and infectivity in the diverse host Beverley and Turco, 1998). The GPIanchored glycoproteins include an abundant metalloproteinase, termed gp63 or leishmanolysin, and the promastigote surface antigen (PSA2)/gp46 family of glycoproteins Murray et al., 1989;Frommel et al., 1990;Lohman et al., 1990). Gp63 has been shown to be proteolytically active against a wide variety of different peptide substrates and has been reported to act as a ligand for macrophage receptors, either directly or after opsonization with complement, to protect the parasites from complementmediated lysis and also to contribute to the pathology of lesion development (see Alexander and Russell, 1992;Joshi et al., 1998). However, the fact that these proteins are encoded by multicopy polymorphic genes (Button et al., 1989;Lohman et al., 1990;Symons et al., 1994) has hindered elucidation of their function by genetic analysis (Joshi et al., 1998). Moreover, surface expression of gp63 and PSA2 is dramatically down-regulated in the amastigote stage of some Leishmania species and variable within particular parasite populations of others (Bahr et al., 1993;Handman et al., 1995) such that the precise function of these parasite pro...

GPI transamidase of Trypanosoma brucei has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits

“…GPI8 homologues of S. cerevisiae, S. pombe, A. thaliana, and P. falciparum are type I transmembrane proteins. In contrast, D. melanogaster, C. elegans, and L. major GPI8 homologues do not have a transmembrane domain (27). Therefore, the presence or absence of a transmembrane domain does not correspond to two groups of GPI transamidases.…”

SETH1andSETH2, Two Components of the Glycosylphosphatidylinositol Anchor Biosynthetic Pathway, Are Required for Pollen Germination and Tube Growth in Arabidopsis [W]

“…GAPs were found not to be required for lymphocyte development in mice, but were essential for normal lymphocyte function and maintenance of a normal peripheral lymphoid compartment (Bessler et al, 2002). Similarly, GAPs are not essential to Leishmania for survival within mammalian host cells (Hilley et al, 2000), but parasite growth depends of the availability of free GPIs (Ilgoutz et al, 1999). Furthermore, transamidation-deficient mutants in yeast accumulate complete GPI lipids as well as GPI precursor proteins and show cell growth arrest (Meyer et al, 2000).…”

Manipulación genética y el estudio del parásito protozoario Leishmania.

“…Igualmente, la eliminación de los genes que codifican para las proteínas SHERP/HASP (proteína hidrofílica en membrana del retículo endoplásmico y mitocondria/proteína de superficie hidrofílica acilada) abundantes en estadio metacíclico infectivo (64), y la ausencia de proteínas que intervienen en la síntesis de diferentes glicoconjugados implicados en la virulencia tuvieron un efecto pequeño en la pérdida de virulencia (76,77,80,81,90).…”

“…Las aproximaciones genéticas también se han realizado por medio de complementación génica, en la que se empieza a partir de un fenotipo mutante o variante para identificar el gen involucrado (49,72,(76)(77)(78)(79)(80)83,88). Los genes responsables de los defectos son identificados por una transfección en masa de los mutantes con una librería cósmida de ADN de la especie de Leishmania silvestre y se seleccionan por recobrar la función a través de la complementación.…”

“…Los mutantes carentes de ambos genes no sobrevivieron en el ambiente hidrolítico dentro del intestino medio del insecto; además, la unión del parásito al intestino medio del insecto y el mantenimiento de la infección luego de la excreción de la sangre del infectado se vieron comprometidas (83,88). Los estudios subsecuentes han extendido esta aproximación para la identificación de más de 10 genes involucrados en la síntesis de LPG y glicoconjugados relacionados (72,(76)(77)(78)(79)(80)(81)90).…”