Protein Turnover and Differentiation in Leishmania

International Journal for Parasitology 37 (2007) 1063–1075 www.elsevier.com/locate/ijpara Invited Review Protein turnover and differentiation in Leishmania Se´bastien Besteiro a,1,2, Roderick A.M. Williams a,2, Graham H. Coombs b, Jeremy C. Mottram a,* a Wellcome Centre for Molecular Parasitology and Division of Infection & Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK b Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, UK Received 18 February 2007; accepted 16 March 2007 Abstract Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life-cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of L. major has facil- itated the identification of the parasite’s vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks. Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Leishmania; Differentiation; Protease; Peptidase; Genome 1. Introduction are present in the insect’s midgut; non-dividing, but mammalian-infective, metacyclic promastigotes in the thoracic Leishmania are protozoan parasites with a complex life- midgut and proboscis of the sandfly. The metacyclic prom- cycle, involving several developmental forms (Fig. 1a). astigotes, when inoculated into a mammalian host through These forms represent an adaptation to the changing envi- a sandfly bite, differentiate (after being phagocytosed by a ronmental conditions encountered by the parasites within macrophage) into the intracellular aflagellate amastigote their two hosts: the mammalian host, to which they are form (Fig. 1b, right). This form of the parasite resides pathogenic, and the sandfly insect vector. In the sandfly, within a vacuole with lysosomal features that is termed Leishmania replicate as extracellular and actively motile the parasitophorous vacuole. flagellated cells known as promastigotes (Fig. 1b, left), During transition through these different extra- and which reside primarily in the insect’s alimentary tract. intracellular environments, Leishmania are exposed to Two main forms can be distinguished (although several many changes in their living conditions: for example, other intermediate forms have been reported (Bates and there are variations in the availability and type of nutri- Rogers, 2004; Gossage et al., 2003)): multiplicative, but ents, pH, temperature, as well as the availability of oxy- not mammalian-infective, procyclic promastigotes that gen. The strategy adopted by the parasites to survive these changes is to develop into highly specialised and adapted forms. These developmental forms are distin- * Corresponding author. Tel.: +44 141 330 3745; fax: +44 141 330 8269. guished by their nutritional requirements, their growth E-mail address: [email protected] (J.C. Mottram). rate and ability to divide, the regulated expression of their 1 Present address: CNRS UMR 5235, Universite´ de Montpellier 2, Place Euge`ne Bataillon, Baˆtiment 24, CC 107, 34095 Montpellier cedex 05, surface molecules, and also their morphology. Metacyclic France. promastigotes are different from the procyclic forms in 2 These authors contributed equally to this work. that they are pre-adapted for survival in the mammalian 0020-7519/$30.00 Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.03.008 1064 S. Besteiro et al. / International Journal for Parasitology 37 (2007) 1063–1075 Fig. 1. Changes in cell shape during the Leishmania life-cycle. (a) Scanning electron microscope images of the main Leishmania major life-cycle stages, the procyclic and metacyclic promastigotes were grown in culture, the amastigote was isolated from an infected macrophage isolated from a mouse. (b) Schematic representation of the main intracellular organelles from Leishmania promastigote (left) or amastigote (right) forms. The flagellar pocket marks the anterior end of the cell. host: for instance, they express stage-specific surface mol- compartment: as they are intracellular, non-motile forms, ecules and become complement-resistant. Amastigotes they have a reduced size and have a much-reduced flagel- multiply within the parasitophorous vacuole in macro- lum that does not emerge from the flagellar pocket phages and are highly adapted morphologically to this (Fig. 1b, right). They are also acidophiles, adapted to S. Besteiro et al. / International Journal for Parasitology 37 (2007) 1063–1075 1065 the low pH of this compartment, and have an adapted myces cerevisiae and Plasmodium falciparum, but consider- energy metabolism. ably less complex than that of mammals (Table 1). The The two differentiation events mainly studied with findings for Leishmania were subsequently confirmed when Leishmania are the procyclic to metacyclic differentiation the genomes of Leishmania infantum and Leishmania bra- of promastigotes (also called metacyclogenesis) and the ziliensis were fully sequenced (http://merops.sanger.ac.uk/), metacyclic promastigote to amastigote transformation showing that all three species have a very similar array of inside the host macrophage. Some factors triggering these peptidases. Such database searches are extremely informa- events in vitro have been characterised. For instance, low tive in predicting, based on homologies, gene contents, but pH, lack of oxygen and nutritional depletion of tetrahydro- it should not be discounted that Leishmania contains as yet biopterin can trigger metacyclogenesis. Conditions mim- unidentified peptidases that have roles in the cell that have icking a phagolysosome-like environment, such as low not yet been described in other organisms. pH, a temperature of 37 °C and elevated CO2, can induce the promastigote to amastigote differentiation (Barak 2.1. Aspartic peptidases and metallopeptidases et al., 2005). Although, these environmental factors triggering Leishmania differentiation in vitro were recognised sev- Leishmania major has just two aspartic peptidases. One eral years ago, relatively little is known about the has sequence similarity to presenilin 1 (PS1) which is a molecular processes that mediate the cellular remodelling. multi-pass membrane peptidase that cleaves type I mem- It is likely that a series of changes in gene expression are brane proteins, such as the amyloid precursor protein of instrumental in the morphological changes associated with Alzheimer’s disease and the Notch receptor that is involved differentiation to the individual developmental forms. in signalling during differentiation and development (Xia However, in Leishmania protein-coding genes are tran- and Wolfe, 2003). There is also the suggestion that PS1 scribed as polycistronic RNAs and they are apparently has a role in autophagy (see Section 4). The second is an not regulated at a transcriptional level (Campbell et al., enzyme that has sequence identity to an intramembrane 2003), which makes the identification of stage-specific signal peptide peptidase (SPP) that cleaves signal peptides genes problematic. Recent transcriptomic and proteomic within their transmembrane region. Whilst SPP appears approaches to identify stage-regulated genes and proteins to be ubiquitous in eukaryotes, PS1-like peptidases have are promising, but the studies have been carried out on dif- been described in mammals, worms and Dictyostelium, ferent Leishmania species and are therefore difficult to com- but not detected in yeast or other protists such as Plasmo- pare (Holzer et al., 2006; McNicoll et al., 2006; Saxena dium. In contrast, pepsin-like aspartic peptidases such as et al., 2007; Walker et al., 2006). Some of the most clear- the plasmepsins, which are so abundant in Plasmodium cut stage-specific markers include peptidases, some of and other apicomplexan parasites (Coombs et al., 2001), which have been known to be associated with the mamma- are apparently entirely absent from Leishmania. lian virulence of Leishmania for a long time (Mottram There are 16 families of metallopeptidases represented in et al., 2004), and whose functions range from nutrient L. major, including a dipeptidyl-peptidase III homologue acquisition to cellular reshaping and recycling (Mottram (family M49) that appears to be absent from trypano- et al., 2004; Williams et al., 2006). Thus these peptidases, somes. Metallopeptidases include many aminopeptidases, and probably others too, are instrumental to the differenti- carboxypeptidases and dipeptidases, very few of which ation of the parasite. Their involvement in these processes have been studied in Leishmania. One exception is leish- is the focus of this review. manolysin (also known as gp63 or MSP). Leishmania has multiple members of this gene family, which is the major 2. The degradative capacity of Leishmania major glycosyl phosphatidyl inositol (GPI)-anchored surface protein and is thought to have a role in the parasite’s virulence Peptidases are a structurally and functionally diverse set and pathogenesis (Yao et al., 2003). of enzymes that hydrolyse proteins and they can be grouped into distinct

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