HIV Aspartyl Peptidase Inhibitors Interfere with Cellular Proliferation, Ultrastructure and Macrophage Infection of Leishmania amazonensis Lı´via O. Santos1, Fernanda A. Marinho 2 , Ellen F. Altoe´ 1, Bianca S. Vito´ rio1, Carlos R. Alves1, Constanc¸ a Britto1, Maria Cristina M. Motta3, Marta H. Branquinha2, Andre´ L. S. Santos2, Claudia M. d’Avila-Levy1* 1 Laborato´ rio de Biologia Molecular e Doenc¸as Endeˆmicas, Instituto Oswaldo Cruz (IOC), Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Rio de Janeiro, Brazil, 2 Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Go´ es, Centro de Cieˆncias da Sau´ de (CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Funda˜o, Rio de Janeiro, Brazil, 3 Instituto de Biof´ısica Carlos Chagas Filho, CCS, (UFRJ), Centro de Cieˆncias da Sau´ de (CCS), Bloco K, Ilha do Funda˜o, Rio de Janeiro, Brazil Abstract Background: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. Methodology/Principal Findings: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs) on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb) and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. Conclusions/Significance: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania, HIV and macrophages. In addition, there are many unresolved questions related to the management of Leishmania-HIV-coinfected patients. For instance, the efficacy of therapy aimed at controlling each pathogen in coinfected individuals remains largely undefined. The results presented herein add new in vitro insight into the wide spectrum efficacy of HIV PIs and suggest that additional studies about the synergistic effects of classical antileishmanial compounds and HIV PIs in macrophages coinfected with Leishmania and HIV-1 should be performed. Citation: Santos LO, Marinho FA, Altoe´ EF, Vito´ rio BS, Alves CR, et al. (2009) HIV Aspartyl Peptidase Inhibitors Interfere with Cellular Proliferation, Ultrastructure and Macrophage Infection of Leishmania amazonensis. PLoS ONE 4(3): e4918. doi:10.1371/journal.pone.0004918 Editor: Adam J. Ratner, Columbia University, United States of America Received December 17, 2008; Accepted January 31, 2009; Published March 26, 2009 Copyright: 2009 Santos 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 study was supported by the Brazilian agencies: MCT/CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), FAPERJ (Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro), FUJB (Fundacao Universitaria Jose Bonifacio), CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior) and FIOCRUZ (Fundacao Oswaldo Cruz). A.L.S.S, C.M.D., C.R.A., C.B., M.C.M.M., M.H.B. are CNPq research fellows. Competing Interests: The authors have declared that no competing interests exist. * E-mail: davila.levy@ioc.fiocruz.br Introduction Leishmaniasis is among the most neglected of the tropical diseases: more than 12 million people are currently infected worldwide, there are 2 million new cases every year (a number that is growing), and 350 million people are considered to be at risk. Two basic clinical forms are recognized – cutaneous leishmaniasis (CL), a disfiguring and stigmatizing disease, and visceral leishmaniasis (VL) or kala-azar, which is fatal without treatment [1]. The development of the HIV/AIDS pandemic during the last 20 years has modified the spectrum of leishmaniasis in both the clinical and epidemiological fields [2]. Cutaneous leishmaniasis, for instance, has attracted more attention due to reactivation, or even visceralization, in the immunocompromised hosts [2]. Currently available therapeutic options range from topical treatment to systemic therapy for more complex cases. These agents are expensive, not active orally, require long-term parenteral administration and produce serious side effects (e.g. cardiac and renal toxicity); moreover, resistance to these compounds has become a severe problem [3]. PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4918