Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity Maria Fernanda Laranjeira da Silva 1 , Ricardo Andrade Zampieri 1 , Sandra M. Muxel 1 , Stephen M. Beverley 2 , Lucile M. Floeter-Winter 1 * 1 Departamento de Fisiologia, Instituto de Biocie ˆncias, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 2 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America Abstract In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg 2 L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg 2 mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol- targeted ARG lacking the glycosomal SKL targeting sequence (argDSKL) restored growth but failed to restore infectivity. Further study showed that the ARGDSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration. Citation: da Silva MFL, Zampieri RA, Muxel SM, Beverley SM, Floeter-Winter LM (2012) Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity. PLoS ONE 7(3): e34022. doi:10.1371/journal.pone.0034022 Editor: Fabio T. M. Costa, State University of Campinas, Brazil Received September 21, 2011; Accepted February 20, 2012; Published March 30, 2012 Copyright: ß 2012 da Silva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP), Conselho Nacional para o Desenvolvimento Cientı ´fico e Tecnolo ´ gico (CNPq) and National Institute of Health (AI 21903; AI 29646). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lucile@ib.usp.br Introduction Leishmaniasis is the second most important infection caused by a protozoan and affects 12 million people worldwide (WHO). Considered to be one of the most neglected tropical diseases, leishmaniasis is endemic in almost all tropical and subtropical areas and causes a wide range of devastating and potentially deadly diseases. There is no vaccine to prevent leishmaniasis, and the drug arsenal to treat this disease presents many problems, including toxicity and increasing parasite resistance to common chemotherapies. Therefore, the study and validation of new drug targets are important for the development of effective chemother- apies. The causative agents of leishmaniasis are protozoan parasites of the genus Leishmania, family Trypanosomatidae. The life cycle of Leishmania includes the extracellular promastigote form that resides in the midgut of the phlebotomine sand fly vector. The obligate intracellular amastigote form inhabits the phagolysosomes of mononuclear phagocytes (mainly macrophages) within the mam- malian host. Leishmania is able to survive and replicate in these different environments by adapting to a wide range of tempera- tures, pH levels and nutrient availability and also by escaping the anti-proliferative defense molecules produced by the host cell [1,2]. The amino acid L-arginine appears to play a key role in the survival of Leishmania in the mammalian host [3–5]. The modulation of the Th1/Th2 balance can induce either the death or proliferation of intracellular Leishmania in the macrophages [6– 8], and L-arginine plays an important role in this process as a common substrate of the inducible nitric oxide synthase (iNOS) and the ARG of the host [9]. These enzymes are competitively regulated by type 1 (Th1) and type 2 (Th2) cytokines, with increased iNOS and decreased ARG levels contributing to parasite control in Th1 responses, and decreased iNOS and increased ARG levels contributing to parasite survival in Th2 responses. Host cell metabolism of L-arginine is further complicated by the fact that Leishmania parasites also express a highly active uptake pathway for arginine and their own ARG. L. amazonensis [10] and L. mexicana [11] ARG proteins and enzyme activities have been biochemically characterized, as have their coding genes. Charac- terization of mutant parasites lacking ARG (arg 2 ) in L. mexicana and L. major demonstrated that the ARG pathway is essential for in vitro proliferation of these parasites, rendering arg 2 parasites auxotro- phic for polyamines [11,12]. The L. mexicana arg 2 parasites showed attenuated infectivity in BALB/c mice, which was attributed to an PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34022