decreases to 5.5. TS and the subsequent reduction in pH induce the transformation of the vector promastigote form to the mammalian host amastigote form. Morphological changes, cell- cycle arrest, and alteration of the gene expression profile are among the main developmental changes that occur during this transformation [13]. In contrast to the long term effects of TS on the pathogenesis of Leishmania, the early events following TS have not been addressed. Here we provide evidence that TS induces a dramatic increase in protein release by L. mexicana within a few hours. We have seen by electron microscopy that this increase is concurrent with an augmentation in budding of surface exovesicles. Finally, in line with our previous studies, we observed that the exoproteome of L. mexicana upon TS is able to modulate macrophage signalling and functions such as activation of PTPs, modulation of transcription factors and inhibition of nitric oxide (NO) production. Results Protein release from L. mexicana is augmented by TS To examine the profile of protein secretion triggered by TS, L. mexicana promastigotes were incubated for 2 or 4 h at 25uC or 37uC. The exoproteome of the parasites after these conditions are depicted in Figure 1. The exoproteome of L. mexicana contains a wide range of proteins and the levels of secretion are augmented by the TS as soon as 2 h. Densitometry analysis on total released proteins and selected bands shows a 1.7–2 fold increase in protein release upon TS within both 2 and 4 h. Direct measurement of protein content of the exoproteome showed that within 4 h approximately 1.2 mg/ml and 1.9 mg/ml of protein is secreted by 108/ml parasites at 25uC and 37uC respectively. As a control, we also measured protein content of total parasites after 4 h of TS and observed that this increase in secretion is not due to a TS-induced general increase in protein production (data not shown). We assessed the integrity of the parasites during the 4 h incubation by flow cytometry using propidium iodide (PI) staining. Our results show that the percentage of PI-positive parasites was negligible (,3%) and in line with what reported in similar studies. This indicates that presence of proteins in the culture supernatant was a result of secretion and not disruption of the plasma membrane due to parasite damage. As a control, treatment with H2O2, a reagent known to cause cell damage, lead to detection of ,90% PI-positive parasites (Supplementary Figure S1). Analysis of the TS-induced proteome released by L. mexicana We performed Liquid Chromatography-Mass Spectrometry (LC/MS/MS) to identify the proteins that were rapidly released by L. mexicana upon 4 h of TS. In addition, MS analysis was performed on the most prominent bands visualized on silver- stained SDS-PAGE of the exoproteome to further corroborate the proteins identified by LC/MS/MS analysis of the total exopro- teome. Together 72 proteins were identified, 11 (,15%) of which were not reported in other studies [8,9]. We used Gene Ontology (GO) analysis under the molecular function filter to look at the exoproteome more closely. A high percentage of the proteins exhibit catalytic (GO:0003824) and binding (GO:0005488) activities (Figure 2), the latter term representing interactions among molecules. Using the SignalP server, signal peptides were detected on two proteins (,3%) suggesting their secretion to be through the conventional pathway. On the other hand, using the SecretomeP server, a program for prediction of nonconventionally secreted proteins from mammalian cells, we predicted 24 proteins Secreted Proteins of Leishmania Alter Macrophage Figure 1. Protein secretion augments following TS. Stationary parasites were washed 3 times in PBS and resuspended in serum free medium to final density of ,26108/ml. Parasites were incubated at 25uC or 37uC for 2 and 4 h. Following incubation, parasites were removed by centrifugation and the supernatant immediately precipi- tated with TCA/acetone. Precipitated proteins were run on SDS-PAGE and visualized by silver staining. Clear augmentation of protein release in response TS can be seen. doi:10.1371/journal.pone.0018724.g001 (,33.5%) as candidates for nonconventional secretion (Supple- mentary Table S1, Supplementary file S1). To analyze the effect of TS on protein secretion from different compartments of the cell, we examined the levels of a subset of the detected proteins by Western blot analysis. This panel of proteins have different subcellular localizations: the metalloprotease gp63 is a plasma membrane GPI-anchored and conventionally secreted [14], cysteine protease b (CPB) is a lysosomal and flagellar pocket enzyme [15], Leishmania homolog for activated c-Kinase (LACK) is a cytosolic protein [16] , peroxiredoxin is a cytosolic and mitochon- drial enzyme [17], heat shock protein 70 (HSP-70) is a cytoplasmic and mitochondrial protein [18], heat shock protein 83 (HSP-83) is a cytosolic protein [19], hexokinase [20] and hypoxanthine-guanine phosphoribosyltransferase are glycosomal enzymes [21]. Figure 3A shows an increase in the release of all proteins, except for the glycosomal proteins. These results support our previous observation on general augmentation of protein release in response to TS. They also show that the pathway by which glycosomal proteins are released is unaffected by TS. More importantly, they confirm that the augmentation of protein release upon TS is not due to parasite damage, since the release of glycosomal proteins does not increase. To validate the presence of a protein phosphatase in the MS analyses, we performed a p-nitrophenyl phosphate (pNPP) assay to PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e18724