Secreted Proteins of Leishmania Alter Macrophage Figure 4. Scanning electron microscopy of L. mexicana parasites during TS. Following TS, parasites rapidly shrink in size and exhibit an increased number of exovesicles on the surface. (A) shows a parasite at 25uC. Increased numbers of exovesicles start to appear on the surface, after 1 h and 4 h of TS repectively (B and C). (D) Close-up of a parasite after 4 h of TS (E) Exovesicles released by L. mexicana after 4 h of TS collected by ultracentrifugation are within the size range of exosomes (40–100 nm). doi:10.1371/journal.pone.0018724.g004 Observing similar patterns of proteins in our silver staining results, we are not surprised that most of the proteins identified in our mass spectrometry analysis have been previously reported to be secreted by Leishmania. Indeed, this might indicate that the mechanism underlying augmentation of protein and exovesicle release is a general stress response to TS. Nevertheless, 11 proteins (15%) had not been reported before to be secreted from Leishmania within short times of 4 to 6 h. These proteins include nuclear proteins, cytosolic enzymes and also a number of hypothetical proteins. It is possible that low amount of secretion at 25uC has kept these proteins below the detection limit and the TS-induced augmentation has made them detectable. One third of the identified proteins were characterized by SecretomeP to be secreted nonconventionally (Suppl. Table S1). Although SecretomeP has been trained by databases of mammalian proteins [31], its coverage of nonconventionally secreted proteins of Leishmania is impressive. Indeed, this high level of coverage could suggest to what extent the patterns and mechanisms of nonconventional protein secretion have remained conserved among eukaryotes through evolution. Overall, we have shown that protein secretion from L. mexicana is increased within 4 h in response to TS. This augmentation seems to be at least partially mediated by an increase in budding of surface vesicles. TS-induced early released proteins and exovesicles of L. mexicana modulate various signalling molecules of the macrophage such as PTPs and transcription factors and they can inhibit production of NO. These modulations result in deactivation of the macrophage and its unresponsiveness to external stimuli such as LPS allowing the parasite in establish- ment of infection. Our results highlight the importance of early hours of infection and the effects of secreted proteins of L. mexicana on the alteration of the macrophage in the parasite’s benefit. PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e18724