Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function Kasra Hassani1,2, Elisabeth Antoniak1, Armando Jardim3, Martin Olivier1,2* 1 Department of Microbiology and Immunology, McGill University, Montre´al, Que´bec, Canada, 2 The Research Institute of the McGill University Health Center, Montre´al, Que´bec, Canada, 3 Institute of Parasitology, McGill University, Montre´al, Que´bec, Canada Abstract Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25–26uC) to the mammalian host (37uC). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-kB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection. Citation: Hassani K, Antoniak E, Jardim A, Olivier M (2011) Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function. PLoS ONE 6(5): e18724. doi:10.1371/journal.pone.0018724 Editor: Gordon Langsley, Institut national de la sante´ et de la recherche me´dicale - Institut Cochin, France Received October 3, 2010; Accepted March 9, 2011; Published May 3, 2011 Copyright: 2011 Hassani 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 Canadian Institutes of Health Research grant no. MOP-12671 to M.O. (www.cihr-irsc.gc.ca). Research in M.O. laboratory is supported by operating grants from the Canadian Institute of Health Research (CIHR). M.O. and A.J. are members of the FQRNT Centre for Host-Parasite Interactions. K.H. is the recipient of a studentship from the Research Institute of the McGill University Health Centre. 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: martin.olivier@mcgill.ca Introduction Parasites of genus Leishmania are the causative agents of leishmaniasis, a spectrum of diseases ranging from self-healing wounds known as cutaneous leishmaniasis (caused by Leishmania major and Leishmania mexicana) to a potentially lethal form known as visceral leishmaniasis (caused by Leishmania infantum and Leishmania donovani). The life cycle of Leishmania comprises two stages. The motile and flagellated promastigotes, live in the midgut of sandfly and are transferred to the mammalian host during a blood meal. In the mammalian host, promastigotes are internalized by macrophages where they transform into non-motile amastigotes and reside in the phagolysosome [1]. Leishmania is known to modulate the host’s innate immune response to allow the parasite to multiply in the macrophage phagolysosome (reviewed in [2]). The parasite utilizes various strategies and virulence factors to alter the host cell signalling, favouring its survival. We have previously shown that JAK/STAT, IRAK-1 and MAP kinases signalling pathways are rapidly altered by Leishmania, leading to partial inactivation of various transcrip- tion factors such as STAT-1a, AP-1 and NF-kB [2,3,4]. In addition, we have recently reported that Leishmania cleaves and activates host protein tyrosine phosphatases (PTPs) in a process that involves the surface metalloprotease gp63 [5]. Activation of PTPs is pivotal to the pathogenesis of Leishmania. Activated PTPs can dephosphorylate key kinases such as JAK2 and IRAK-1 that could otherwise induce production of leishmanicidal molecules. The presence of promastigotes is not necessary for this phenomenon as conditioned parasite culture medium also induced cleavage of various PTPs (e.g. PTP-1B, SHP-1 and TC-PTP ), suggesting that Leishmania secreted proteins may be important for virulence [5]. Protein secretion in eukaryotes occurs both conventionally through the Golgi apparatus and nonconventionally via pathways such as direct membrane translocation, exovesicle blebbing, secretory lysosomes and exosomes. These pathways partly explain the presence of proteins that lack a signal peptide in the exoproteome of eukaryotes [6,7]. Interestingly, such nonconven- tional secretion pathways have been recently observed in Leishmania and Trypanosoma parasites [8,9,10,11,12]. The transfer from the insect vector to the mammalian host involves a temperature shift (TS) from ambient temperature to 37uC, and contact with the host cell molecules. After internaliza- tion into the macrophage, the environmental pH of the parasites PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e18724