Secreted Proteins of Leishmania Alter Macrophage Figure 3. Effect of TS on secretion of selected proteins. (A) Western blot analyses of the L. mexicana exoproteome show that secretion of gp63, LACK, peroxiredoxin, CPB, HSP70 and HSP83 is augmented in response to TS. However, release of the glycosomal proteins hexokinase and HGPRT remains unchanged. MW denotes molecular weight. (B) Phosphatase activity of the exoproteome increases following TS was measured by phosphate analog pNPP assay (p-value = 0.0005). doi:10.1371/journal.pone.0018724.g003 attributed to an elevated level of exovesicle budding, detected in SEM of L. mexicana promastigotes. TS mimics one of the key signals informing the parasite of its new environment. Thus, one can suppose that similar increases in protein and exovesicle release are anticipated to occur in vivo. We speculate that this phenomenon could be a strategy utilized by L. mexicana to paralyze the resident and recruited macrophages even before parasite contact and phagocytosis would occur. Both SHP-1 and PTP-1B, which are important regulators of inflammation [25,26], are rapidly cleaved and activated upon infection or contact with the exoproteome. Furthermore, our results are indicative of modula- tion of other PTPs following exoproteome treatment. Activated PTPs can in turn switch-off many key kinases such as JAK2, IRAK-1 and MAP kinases hindering macrophage activation [3,27,28]. In addition, similar to infected macrophages, exopro- teome-treated macrophages were also unresponsive to external stimuli such as LPS [29]. These stimuli could have otherwise activated AP-1 and NF-kB for production of inflammatory cytokines such as TNF. This is in line with our observation on inhibition of NO production, a crucial antimicrobial product of the macrophage. Therefore an important outcome of exopro- teome release could be taming down the inflammation following infection and paralyzing the macrophage, actions that facilitate the establishment of Leishmania infection. The mechanism by which Leishmania secreted proteins access the host cytoplasm and nucleus has been a matter of debate. Several mechanisms have been proposed and our group has proposed that surface-shed GPI-anchored gp63 can enter the host cytosol through lipid raft microdomains, where it cleaves a number of host phosphatases [5]. More recently we proposed that gp63 is granted access to the nucleus probably via a nuclear localization signal (NLS)-like motif where it can cleave the AP-1 components Jun D, c- Fos, Fra-1 and Fra-2 [24]. Silverman et al have also extensively studied exosome production by L. donovani and proposed that exosomes fuse with the plasma membrane and their content is released into the cytoplasm of the macrophage [11]. Their findings showed induction of IL-8, but not TNF, by L. donovani exosomes; however a mechanism of action by which these effects were induced in macrophage was not advanced. Since L. mexicana exosomes bud from the plasma membrane and their surface contains GPI- anchored gp63, interaction of the exosomes with the macrophage plasma membrane and its fusion could be another route for entrance of gp63 into the host macrophage. In addition to gp63, we observed that other Leishmania virulence factors are also released from L. mexicana. We are currently performing more in depth studying of the role of gp63 and CPB in modulation of the macrophage by exosomes using various knocked-out strains of Leishmania. A recent study by Silverman et al [30] has shown evidence for the interaction of L. donovani exosomes with macrophages and dendritic cells both in vitro and in vivo by examining cytokine production profiles. However, it is important to emphasize that in these studies, exosomes were collected from L. donovani parasites culture for 24 h. However, having observed the dramatic and rapid increase in protein release from L. mexicana, we intentionally looked at very earlier times and studied the impact of the rapidly released proteins on macrophage signalling. From our point of view and with regard to relatively fast internalization and differentiation of Leishmania parasites, studying exosome/exopro- teome release in longer time points should be done on amastigotes rather than promastigotes. PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e18724