Figure 2. GO analyses of the TS-induced exoproteome of L. mexicana. Pie chart representation of distribution of GO terms in the proteins identified to be secreted after 4 h of TS. (Molecular function, level 2). doi:10.1371/journal.pone.0018724.g002 measure phosphatase activity in the exoproteome induced by TS. As shown in Figure 3B, TS induces a significant augmentation in phosphatase activity further reinforcing rapid augmentation of protein release upon TS. TS induces an amplification of exovesicle release from the surface of L. mexicana In order to study the possible routes of protein secretion from L. mexicana we performed scanning electron microscopy (SEM) of TS and non-TS parasites. SEM analysis of L. mexicana promastigotes cultured at 25uC showed that the parasite surface was covered with budding exovesicles. Interestingly, as parasites decreased in size, the quantity of the budding exovesicles increased notably following TS (Figure 4A–C). Exovesicles isolated by ultracentri- fugation of 4 h of TS exoproteome are 40–100 nm in diameter, similar to the size range of exosomes (Figure 4E) [22]. TS-induced exoproteome of L. mexicana cleaves and activates macrophage PTPs Having previously seen the impact of the conditioned medium of Leishmania parasites on modulation of host signalling [5], we examined the effect of proteins released by L. mexicana on macrophage PTPs. Incubation of B10R macrophages with exoproteome preparations induced cleavage of SHP-1 and PTP1-B, however to a lesser extent than that observed with L. mexicana infection (Figure 5A). PTP in-gel assay shows that the pattern of active PTPs in the macrophages is heavily modulated after infection or exoproteome treatment. White spots in Figure 5B Secreted Proteins of Leishmania Alter Macrophage represent the results of enzymatic activity of SHP-1 and PTP-1B cleavage fragments as well as modulation of other PTPs of the macrophage (Figure 5B). Finally the pNPP assay results directly illustrate that PTP activity in the macrophage significantly increases following incubation with the exoproteome. We have shown previously that inhibition of PTPs completely blocks pNPP hydrolysis in macrophages. Thus the phosphatase activity measured by this assay belongs mainly to PTPs [23]. Overall, the exoproteome of L. mexicana induces an increase in the PTP activity within the macrophage and specifically induces cleavage and activation of host PTPs, specifically SHP-1 and PTP-1B (Figure 5C). TS-induced L. mexicana exoproteome modulates translocation of transcription factors to the nucleus Previous reports that examined the early events in the host following Leishmania infection pointed to alteration in the translocation of transcription factors such as AP-1 and NF-kB in response to agonists [4,24]. To investigate whether the exopro- teome had a similar effect on these transcription factors, macrophages were treated with the exoproteome for 16 h followed by 1 h stimulation with LPS. Electromobility shift assays (EMSAs) were performed with macrophage nuclear proteins and oligonu- cleotides corresponding to the AP-1 and NF-kB binding site. L. mexicana infection results in induction of a noncanonical form of NF-kB as previously reported [4] as well as complete disappear- ance of AP-1 (Figure 6). Furthermore, Figure 6A shows that incubation with the exoproteome as well leads to strong inhibition of AP-1 translocation in response to LPS. As shown in Figure 6B, exoproteome was found to cause reduction of translocation of the canonical NF-kB; interestingly, the noncanonical form of NF-kB previously found to be involved in induction of chemokines but not inflammatory cytokines by Leishmania [4], was observed to be induced. TS-induced L. mexicana exoproteome partially inhibits LPS-induced NO production Survival of Leishmania parasites within the macrophage relies on their ability to inhibit NO production. In Figure 7 we show that both infection with L. mexicana or incubation with the exoproteome failed to induce NO production by the macrophage. Of interest, was the finding that LPS-induced NO production was, however, further inhibited following infection or incubation with the exoproteome. As AP-1 and NF-kB are the transcription factors inducing transcription of inducible nitric oxide synthase (iNOS), this result is in line with our observation that the exoproteome of L. mexicana inhibits translocation of the latter transcription factors in response to LPS. Overall, our results strongly suggest that proteins secreted by L. mexicana during the initial hours of infection are capable of modulating macrophage signalling and function promoting intracellular parasite survival. Discussion The exoproteomes of trypanosomatids have attracted significant attention and multiple studies have resulted in long lists of proteins that are secreted from multiple species [8,9,10,11,12]. However the context by which these proteins are secreted, in nature as well as their potential effect on the host has been neglected. The present study aimed to shed light on the mechanisms by which proteins secreted by Leishmania modulate signalling and function of the macrophage. Here we show that protein release by L. mexicana is rapidly augmented in 2 to 4 h after TS. Increase in protein release was PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e18724