Increased Abundance of Proteins Involved in Resistance to Oxidative and Nitrosative Stress at the Last Stages of Growth and Development of Leishmania amazonensis Promastigotes Revealed by Proteome Analysis

Leishmania amazonensis is one of the major etiological agents of the neglected, stigmatizing disease termed american cutaneous leishmaniasis (ACL). ACL is a zoonosis and rodents are the main reservoirs. Most cases of ACL are reported in Brazil, Bolivia, Colombia and Peru. The biological cycle of the parasite is digenetic because sand fly vectors transmit the motile promastigote stage to the mammalian host dermis during blood meal intakes. The amastigote stage survives within phagocytes of the mammalian host. The purpose of this study is detection and identification of changes in protein abundance by 2DE/MALDI-TOF/TOF at the main growth phases of L. amazonensis promastigotes in axenic culture and the differentiation process that takes place simultaneously. The average number of proteins detected per gel is 202 and the non-redundant cumulative number is 339. Of those, 63 are differentially abundant throughout growth and simultaneous differentiation of L. amazonensis promastigotes. The main finding is that certain proteins involved in resistance to nitrosative and oxidative stress are more abundant at the last stages of growth and differentiation of cultured L. amazonensis promastigotes. These proteins are the arginase, a light variant of the tryparedoxin peroxidase, the iron superoxide dismutase, the regulatory subunit of the protein kinase A and a light HSP70 variant. These data taken together with the decrease of the stress-inducible protein 1 levels are additional evidence supporting the previously described pre-adaptative hypothesis, which consists of preparation in advance towards the amastigote stage.


Introduction
American cutaneous leishmaniasis (ACL) is caused by 15 species of the genus Leishmania (Kinetoplastida: Trypanosomatidae), which includes species from the Leishmania and the Viannia subgenera. The latter is also able to cause mucocutaneous (MCL) leishmaniasis. L. (L.) amazonensis is one of the major etiological agents of ACL and it is grouped into the "L. mexicana complex". The initial ACL lesions are crater-shaped and they become ulcerated in the center with the progression of the disease. The species L. mexicana, L. pifanoi and L. amazonensis are also able to cause an infrequent form of the disease termed anergic diffuse cutaneous leishmaniasis (ADCL) [1], which is characterized by diffuse nodular lesions and total or partial anergy. ACL and ADCL are not life threatening, as a difference with visceral leishmaniasis (VL) caused by other species, but they are responsible for considerable morbidity.
According to the WHO epidemiological records, about 1.5 millon cases of CL are registered worldwide anually. Most ACL cases are concentrated in Brazil, Bolivia, Colombia and Peru [2,3]. A recent increase in the cases of leishmaniasis has been observed globally, which may be explained by many interrelated factors as scarce control, poverty, poor hygiene, migrations, return to rural areas, penetration into the jungle, changes in ambient humidity, deforestation, etc. ACL is a zoonosis and the major reservoirs are rodents when the etiological agents belong to the L. mexicana complex [2].
The life cycle of the parasite involves an invertebrate host that injects the motile promastigote stage of the parasite to the mammalian host when feeding from venules of the dermis. The vector belongs to the genus Lutzomyia (Psychodidae: Phlebotominae) in the case of New World species of the parasite like L. amazonensis. Promastigotes are engulfed by host phagocytes and differentiate to amastigotes. When a sand fly feeds from blood of an infected mammal, amastigotes are released from phagocytes, transform into undifferentiated (procyclic) promastigotes and begin a developmental process from the procyclic to the more infective metacyclic stage.
A remarkable number of trypanosomatid genome sequences were assembled and annotated [4][5][6], which allowed for studying differential gene expression in different species, thus contributing to the knowledge about the biology of the parasite [7]. This is essential for future discovery of drug targets and vaccine candidates. In the case of L. amazonensis, two-dimension electrophoresis (2DE) maps of promastigotes were described [8,9]. In another study, the effect of culture passage and the resulting decrease of infectivity in the proteome profile of stationary phase promastigotes were evaluated by the same approach [10]. Also, a transcriptome analysis focused in the search of genes associated to antimony resistance was performed in promastigotes at the mid-logarithmic phase of growth in axenic culture [11]. However, differential gene expression throughout growth and development of the promastigote stage of L. amazonensis in culture and within the sand fly gut has not been still explored. The aim of this study is comparing different time points of growth of promastigotes in culture by 2DE and subsequent matrixassisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF).

Materials and Methods Parasites
100 IU/ml penicillin (Life Technologies). The initial cell density was adjusted to 2 x 10 6 cells/ml in all cultures and growth was registered daily at the light microscope using a Neubauer chamber. Culture samples containing 10 8 promastigotes were obtained at the early-logarithmic, mid-logarithmic, late-logarithmic and stationary phases.

Total protein extracts
Promastigotes (10 8 per sample) were harvested at 2,000 g for 10 min and washed once in PBS. Then, the sediment was carefully resuspended in 150 μl lysis buffer (8.4 M urea, 2.4 M thiourea, 5% CHAPS, 50 mM DTT, 1% Triton X-100, 50 μg/ml DNase and Mini EDTA-free Protease Inhibitor Cocktail according to the manufacturer's instructions -Roche, Mannheim, Germany) and immediately mixed by mild rotation at 4°C for 30 min. Then, the protein extracts were centrifuged at 8,000g for 10 min. The supernatant was recovered, precipitated with methanol/ chloroform [13], dried at room temperature for 5 min and resuspended in 2X rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.003% bromophenol blue). Quantification was performed with the RC DC protein assay kit (BioRad) following the manufacturer's instructions and the results were compared with SDS-PAGE results as described [14].

Two-dimensional electrophoresis (2DE)
Fifty μg of total protein per sample were diluted to a final volume of 140 μl in 2X isoelectrofocusing (IEF) buffer (18.2 M DTT and 0.5% IPG buffer solution pH 3-10, BioRad). Then, samples were subject to IEF on 7 cm IPG pH 3-10 non-linear gradient strips (BioRad) in a Protean IEF Cell system (BioRad) following the manufacturer's instructions. Each run comprised seven steps (50 V for 12h, 250 V for 1h, 500 V for 1h, 1000 V for 1h, 2000 V for 1h, linear ramp to 8000 V for 1h and 8000 V up to 3500 VÁh), reaching more than 12,000 VÁh in every one. Then, the strips were run by 12% SDS-PAGE in a pre-cooled MiniProtean 3 Dodeca Cell system (BioRad) at 0.5 W/gel for 30 min and then at 1.5 W. The run was stopped 5 min after the diefront reached the bottom edge. The gels were stained with SYPRO Ruby (BioRad) and the gel images were acquired with EXQuest Spot Cutter (BioRad) according to the manufacturer's instructions.
The gel images were processed and analyzed with PDQuest 2D Advanced 8.0.1 software (BioRad) according to the manufacturer's instructions. First, the images were cropped and spots were detected automatically with the Spot Detection Parameter Wizard at a sensitivity value of 3.5. Then, all detected spots were manually checked by observation of 3D density graphs, single spot quantitation histograms and 2DE gel images. Normalization was performed by the Total Quantity in Valid Spots algorithm. Normality was checked by the Kolmogorov-Smirnov test and statistical inference of differential protein abundance was performed by the Student's t-test at the 0.05 significance level. Reproducibility was assured by preparing three independent biological replicate samples. However, all 2DE runs were performed simultaneously, which is essential for technical reproducibility across gels. For this purpose, all samples were stored at -80°C until use.
MS/MS. The data obtained were analyzed with ABI 4000 Series Explorer software 3.6 (Life Technologies). Peptide mass fingerprinting (PMF) and MS/MS spectra of fragment ions were smoothed and corrected to the zero baseline using routines embedded in ABI 4000 Series Explorer Software v3.6. Each PMF spectrum was internally calibrated with the mass signals of trypsin autolysis ions to reach a typical mass measurement accuracy < 25 ppm. Known trypsin and keratin mass signals, as well as potential sodium and potassium adducts (+21 Da and + 39 Da) were removed from the peak list. Protein identifications were performed with MASCOT 2.1 using Global Protein Server Explorer 4.9 (Life Technologies). The following search parameters were introduced: enzyme, trypsin; allowed missed cleavages, 1; carbamidomethyl cystein as fixed modification by treatment with iodoacetamide; variable modifications, oxidation of methionine; mass tolerance was set to ± 50 ppm for precursors and to ± 0.3 Da for MS/MS fragment ions. The confidence interval for protein identification was set to ! 95% (p < 0.05) and only peptides with an individual ion score above the identity threshold (52) were considered correctly identified. The reference template for MASCOT identifications was the genome sequence of L. mexicana (http://tritrypdb.org/common/downloads/release-9.0/LmexicanaMH OMGT2001U1103/fasta/data/) because the genome of L. amazonensis has not been sequenced yet and this species is included in the "L. mexicana complex". All data were also run against against individual L. amazonensis sequences within the NCBInr database. The identifications are available in the PRIDE repository of the ProteomeXchange Consortium [15] with the accesion number PXD002939.
Evaluation of resistance to nitric oxide (NO) by the [3'-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay Logarithmic and stationary phase promastigotes were washed with PBS and at 26°C for 4 h at a cell density of 10 6 in a final volume of 100 μl in the presence of different concentrations (1:2 serial dilutions from 16 to 0.25 mM) of NaNO 2 (NO donor) in PBS pH 5.0. Next, suspensions were centrifuged at 700g for 10 min and the sediments were resuspended in 100 μl PBS pH 7.0. Then, 10 μl of 5 mg/ml MTT were added and the cells were incubated at 25°C for 4 h. The reaction was stopped and formazan solubilized by adding 100 μl of 0.4N HCl in 50% isopropanol. Finally, A 490nm was measured to assess formazan accumulation, which is related to viability [16]. The differences were statistically assessed with the paired Student's t-test from three biological replicates of the experiment.

2DE-MS/MS analysis of L. amazonensis promastigotes
Total protein was extracted from L. amazonensis axenic cultures of promastigotes at early-logarithmic (day 2), mid-logarithmic (day 3), late-logarithmic (day 5) and stationary (day 7) phase ( Fig 1). Average protein concentrations (mg/ml) were: 4.4 ± 0.2 (day 2 promastigotes); 4.5 ± 0.4 (day 3); 4.4 ± 0.1 (day 5); 4.5 ± 0.2 (day 7). (Fig 2). The spots were automatically detected with the Spot Detection Parameter Wizard. Sensitivity was adjusted to 3.5 in order to filter out the non-identifiable faintest spots according to experimental setup with the mass spectrometer. An average of 202 spots were detected per gel and the cumulative non-redundant number is 339 (S1 Table). The cut-off ratio values were 1.7 for up-regulation and 0.6 for downregulation (1.7 and -1.7 fold changes), respectively (S1 Fig, S1 Table). Statistical significance was inferred with the Student's t test (p < 0.05) because the outcome of the Kolmogorov-Smirnov test was p = 0.318, 0.280 and 0.273 for the comparisons day3:day2, day5:day2 and day7: day2, respectively. Ratios are relative to early-logarithmic phase promastigotes (day 2). Sixty three differentially expressed proteins out of 339 could be identified by MALDI-TOF/TOF ( Fig  2, Table 1, S1 Table) against the TriTrypDB L. mexicana genome annotations. All selected proteins were identified and the MASCOT score values were significant, except for one spot that could not be identified with the L. mexicana reference genome sequence but the NCBInr database instead (Table 2, spot Lam1601, soluble promastigote surface antigen fragment PSA-31S, part of the PSA-38S). In fact, this L. amazonensis gene had been previously characterized [17]. Random identification of constitutively expressed proteins was also performed ( Table 2), which occasionally allowed for retrieving additional information about relative expression in different pathways (see below). In certain cases, different spots contain the same protein, which may be due to protein processing, post-translational modifications or protein aggregation. In the next sections, this has been cited as "protein variant". In principle, the nature of the variations is unknown but the 2DE-resolved variants could be separately analyzed and identified. The comparison of experimental and/or predicted molecular weight and isoelectric point provides insight (Table 3). For example, three variants of the protein LmxM.15.1160 were found. Two of them present a similar MW but differ in the estimated pI value in 0.5 pH units, whereas the MW of the third one is about 40% lower and the pI 8.5, which strongly suggests that a considerable portion of the protein sequence has been removed during intracellular protein processing. The most striking changes in experimental MW and pI (Table 3) are discussed below according to changes in relative abundance of variants of the same protein.
The differential gene expression results at the protein abundance level in the promastigote differentiation process studied herein are compared below with expression profiles reported in other species. Experimental variation between studies has been considered. In other cases, the information is complemented with reported data about the differentiation process of promastigotes to amastigotes.

Differential protein abundance in L. amazonensis promastigotes
Regulation of gene expression. Proteins involved in translation and post-transcriptional regulation of gene expression, play a key role in the biology of trypanosomatids because these organisms lack most transcriptional regulation mechanisms (reviewed in [18]). The abundance of some of them changes in the growth and differentiation process of L. amazonensis promastigotes. First, three variants of the translation elongation factor 2 (EF2) are up-regulated in midlogarithmic with respect to early-logarithmic phase promastigotes, as well as two different DEAD/DEAH RNA helicases and the alanine-tRNA synthetase (Table 1). One EF2 variant is considerably lighter and slightly more basic (60.3 KDa, pI 6.9) than the others (Table 3), which suggests that it is partially proteolyzed during processing. The two other variants may have been post-translationally modified, according to the differences in the theoretical MW values (Tables 1 and 3). A heavier variant of the EF2 is constitutively expressed (Tables 2 and 3). This  constitutive variant may contain additional post-translational modifications, probably glycosylation regarding to the increase in MW ( Table 3). The increase in abundance of the EF2 variants (Tables 1 and 2) suggests changes in translational regulation at an intermediate point of growth and differentiation. The EF2 was described to be an immunostimulatory protein in L. donovani [19], but the importance of this protein for differentiating promastigotes may rely more directly in regulation of translation rather than preparation in advance for colonization of the host cell within the mammalian host (see pre-adaptative hypothesis below). An alternative possibility could be the decrease of the steady-state levels in case the immune response elicited by the protein leads to progression of the disease. Another protein involved in gene expression regulation is the endoribonuclease L-PSP (pb5), which was found to be down-regulated in amastigotes of species causative of cutaneous (L. mexicana, L. major) and visceral (L. donovani, L. infantum) leishmaniasis [20][21][22][23][24]. However, information about differential abundance of this protein throughout the promastigote growth and differentiation process is not available so far. In the case of L. amazonensis, this endoribonuclease is over-expressed in stationary phase promastigotes, therefore at the end of the differentiation process (Table 1).
Protein folding. The heat shock proteins HSP70 and HSP83-1 were described to be abundant and constitutive throughout the main stages of the life cycle of Leishmania spp. [25,26]. Differential expression in the promastigote stage of L. infantum was not detected at the transcript and protein levels [14,20]. In the case of L. amazonensis, distinct variants of both chaperones are constitutively or differentially expressed in promastigotes throughout growth and differentiation (Tables 1 and 2). Two HSP70 (LmxM.28.2770) variants of the same MW but different estimated pI (5.4 and 6.2) are less abundant at late-logarithmic phase (Tables 1 and  3). The HSP70-related protein 1 (HSP70-rel1), encoded by a different gene (LmxM29.2490), is slightly heavier (Table 3) and its relative abundance is analogous, whereas a lighter variant of the LmxM.28.2770 HSP70 (44.5 KDa) of estimated pI around the theoretical value (5.2) is highly up-regulated (24-fold) in stationary phase promastigotes (Tables 1 and 3). Finally, a heavier variant (97.6 KDa) is constitutive (Tables 2 and 3). Therefore, alternation of HSP70 variants originated by protein processing or post-translational modifications is observed at the last stages of the growth and differentiation process of L. amazonensis promastigotes. Simultaneously, the chaperonin HSP60 is down-regulated (late-logarithmic phase), whereas the differentially regulated variants of the HSP83 are down-regulated at mid-logarithmic phase, an earlier point of growth and differentiation. According to our results, the overall HSP70 levels increase in the ongoing of differentiation of promastigotes considering all variants together. As HSP70 proteins of L. infantum and L. donovani were described to be immunostimulatory [19,27], they may be involved in colonization of the mammalian host phagocytesby differentiated promastigotes, though it may lead to susceptibility or resistance of the host depending on the type of immune response elicited. According to previous data, the stress-inducible protein 1 homolog (STI1) is constitutively expressed in the developmental process of stationary phase promastigotes to amastigotes in the case of L. infantum [28] but no information is available about growth and development of promastigotes. In the case of L. amazonensis, the STI1 is down-regulated at late logarithmic phase (Table 1), simultaneously to some HSPs mentioned above.
Two variants of the η subunit of the T-complex protein 1 (TCP1η) are down-regulated in late logarithmic phase promastigotes (Table 1), whereas the TCP1β subunit is constitutively expressed (Table 2). Therefore, the down-regulation of the TCP1η, the STI1 and the HSPs mentioned above is simultaneous and takes place at one of the final stages of growth and differentiation of L. amazonensis promastigotes in culture. The exact function of the TCP complex is not known in Leishmania spp., although it has been suggested that the TCP1γ subunit may participate in maintaining the structural dynamics of the cytoskeleton [29]. Changes in cell morphology throughout promastigote differentiation and flagellar movement demand continuous re-organization of the cytoskeleton. Differential expression of the TCP1η has not been reported in L. infantum promastigotes differentiating in culture so far, although the encoding gene is up-regulated at the transcript level in metacyclic promastigotes isolated from the sand fly anterior midgut [30]. A cyclophilin variant is down-regulated in mid-logarithmic phase L. amazonensis promastigotes, whereas another variant is constitutively expressed. Cyclophilins are able to prevent aggregation of adenosine kinase domain-containing proteins [31,32] like nucleoside diphosphate kinases (NDK). Two NDKb variants encoded by the same gene but very different in MW (presumably due to processing) are up-regulated at the beginning of growth and differentiation of promastigotes (early-logarithmic phase) (Tables 1 and 3). Up-regulation of the NDKb at the transcript level in procyclic L. infantum promastigotes [33] agrees with the profile found in L. amazonensis. The NDKs are involved in nucleotide biosynthetic processes, which may occur preferentially at the initial stages of growth and differentiation of promastigotes as suggested by the results. The 14-3-3 protein 1 relative abundance profile is the same as for the NDKb in L. amazonensis promastigotes. The exact functions of 14-3-3 proteins are unknown, although they have been related to many cellular processes. An intrinsic NDK activity has been associated to these proteins in general [34] and antiapoptotic properties of the L. donovani orthologs have been reported [35]. In fact, the 14-3-3 protein is able to prolong the lifespan of the infected host phagocyte. However, given the multiple functions of this protein, the implications of its increased levels at the beginning of promastigote growth and differentiation are not known so far. The decrease of Cph may cause an increase in aggregation of NDKb and 14-3-3, which may be a clue to explain their decrease at an earlier stage.
Proteolysis. Two components of the ubiquitin-proteasome system are differentially abundant throughout growth and differentiation of L. amazonensis promastigotes: the ubiquitinconjugating enzyme E2 (UbqC-E2) is down-regulated in late-logarithmic phase, whereas the proteasome α-3 subunit is up-regulated in stationary phase promastigotes ( Table 1). None of them have been detected in differential gene expression analysis during development of promastigotes of any Leishmania species in culture so far. However, it has been described that the the E2 steady-state transcript levels are higher in cultured promastigotes than in promastigotes obtained from the sand fly gut [30,36]. The 14-3-3 protein is associated to other components of the proteasome [37]. As mentioned above, the abundance of this protein decreases at the last stages of growth and development of L. amazonensis promastigotes (late-logarithmic phase promastigotes), which is simultaneous to the decrease of the UbqC-E2.
Other proteases of unknown function are up-regulated in mid-logarithmic phase of growth and development of L. amazonensis promastigotes: two mitochondrial protein peptidase variants, a metal-dependent aminoexopeptidase of the M17 family and two variants of a mitochondrial processing peptidase of the M16 family that present similar MW and pI analogous to the theoretical value (Table 3). Last, the calpain LmxM.20.1280 is down-regulated in late-logarithmic phase promastigotes, whereas the paralog (LinJ.20.1230) was found to be up-regulated in L. infantum metacyclic promastigotes [33]. The physiological meaning of all these changes in proteasome components and proteases of unknown function is not known so far, although those changes at a mid time point of promastigote differentiaton suggest that they are involved in degradation of self proteins during this process.
Signaling. The protein kinase A (PKA) of L. amazonensis was described to be involved in autophagy [38,39], which is a critical process in promastigote differentiation. This is in agreement with 24-fold increase of the regulatory subunit (rPKA) in stationary phase promastigotes of this species (Table 1). The rPKA is also up-regulated at the transcript level at the same growth phase of L. infantum promastigotes [20]. Therefore, these data point to an increase of abundance of the rPKA upon growth and differentiation of the promastigote stage, which may be critical for survival.
The receptor of the activated protein kinase C (RACK) of Leishmania spp. (LACK) is upregulated in early-logarithmic phase promastigotes in L. amazonensis (Table 1). Constitutive expression throughout growth of L. infantum promastigotes has been reported [40]. The motile stage of Crithidia fasciculata, a monogenetic trypanosomatid that does not infect mammals in its life cycle, also increases the levels of the RACK orthologue CACK in early logarithmic phase [41] like L. amazonensis, according to 2DE-MALDI-TOF/TOF results obtained by the same procedure. The LACK antigen is able to protect partially against canine leishmaniasis when the encoding gene is administered in a mammalian expression plasmid vector either containing antibiotic resistance genes or alternative selection markers [42]. This protein is located in the particulate fraction of the cytoplasm near the plasma membrane and it is up-regulated in L. infantum amastigotes [40]. RACK proteins belong to the WD40 repeat family. In particular, LACK is able to bind sequences present in certain proteins involved in DNA replication and RNA synthesis and the β chain of the MHC-II. RACKs are able to translocate PKCs to the required intracellular locations [24] in order to participate in certain signal transduction pathways that have been characterized in mammalian cells [25]. The current knowledge on signal transduction pathways is still reduced in trypanosomatids. However, progress has been made with particular proteins. For example, at least one of the rPKA functions could be determined (autophagy, involved in metacyclogenesis) and it is known that LACK is a good vaccine candidate [42]. The signaling proteins 14-3-3 and putative protein phosphatase in Table 1 are more abundant in early logarithmic phase. The 14-3-3 protein is also involved in protein folding. As mentioned above, antiapoptotic function able to prolong the lifespan of the infected macrophage has been also described [35].
Catabolism and biosynthesis of surface molecules. Glycolysis is less active in amastigotes than in promastigotes [43]. The opposite has been observed for β-oxidation of fatty acids. Most glycolytic enzymes are compartmentalized in the glycosome and some of them have been considered as potential drug targets [44][45][46]. Thiol groups of glycolytic enzymes are modified under nitrosative stress in Escherichia coli and Mycobacterium tuberculosis, leading to an appropriate conformational state of the protein that somehow allows for resistance [47,48]. These findings suggest an interesting hypothetical association between steady-state levels of glycolytic enzymes in the appropriate conformational state and resistance to NO. M. tuberculosis and certain invasive E. coli strains are facultative intracellular pathogens, whereas the amastigote of Leishmania spp. is an obligate intracellular stage. Promastigotes are also able to survive in the presence of NO. On these grounds, we propose a hypothetical association between differential abundance of glycolytic enzymes and resistance to nitric oxide (see discussion about nitric oxide resistance below). However, this has not been experimentally tested. The triose phosphate isomerase (TPI), the enolase and the bisphosphoglycerate-independent phosphoglycerate mutase (PGM BPI ) are differentially regulated in L. amazonensis promastigotes (Table 1), whereas the glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) is constitutively expressed ( Table 2). Constitutive expression of the gGAPDH was also described in the differentiation process of L. infantum promastigotes to amastigotes [28]. The abundance of the TPI, a potential vaccine candidate [49], decreases in mid-logarithmic phase promastigotes, whereas the PGM BPI is up-regulated at late-logarithmic phase. The PGM BPI was reported to be down-regulated in L. infantum and L. donovani amastigotes [20,23,24,50] at the transcript and protein levels, respectively. Additionally, this protein is considered a promising drug target candidate [51]. Differential expression of the TPI and the PGM BPI has not been detected across promastigote growth and differentiation of these visceral leishmaniasis-causative species in other studies. The enolase is down-regulated at the final stages of growth and differentiation of L. amazonensis (late-logarithmic phase) (Fig 3), which is analogous at the transcript level (over-expressed in metacyclics) in the case of L. infantum [33]. This protein is down-regulated in the amastigote stage of L. mexicana, L. donovani and L. infantum [21,23,24,33]. The expression profile in L. infantum stationary phase metacyclic promastigotes is comparable at the transcript level [33].
Three components of the pyruvate dehydrogenase complex (PDC), namely the E1β subunit, dihydrolipoamide dehydrogenase (DHLDH) and dihydrolipoamide acetyltransferase (DHLAT), are significantly up-regulated in early-logarithmic phase promastigotes (Fig 3,   Fig 3. Differential abundance of proteins involved in L. amazonensis metabolism. Constitutively expressed and differentially regulated proteins that participate in glycolysis, the Krebs cycle, amino acid metabolism and β-oxidation of fatty acids are depicted. Colour legend for up-regulation: green, day 2 (lag/early log); blue, day 3 (log); orange, day 5 (late log); red, day 7 (stat). The constitutively expressed proteins experimentally detected throughout the promastigote growth curve are highlighted in violet. Abbreviations not detailed in the text: ALD, fructose-1,6-bisphosphate aldolase; ECH, enoyl-CoA hydratase/isomerase; FMR, fumarase; MET6, methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; MDH, malate dehydrogenase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; SDH, succinate dehydrogenase.  Table 1). The aconitase is the only differentially abundant enzyme of the Krebs cycle found. This protein is over-expressed at the beginning of growth and differentiation of promastigotes (in early-logarithmic phase), like the PDC components mentioned above. On the opposite, the citrate synthase (CitS), isocitrate dehydrogenase (ICDH), succinyl-CoA synthetase α subunit (SCS) and GDP-forming succinyl-CoA ligase β chain (SCL) are constitutively expressed (Fig 3,  Table 2). The Rieske iron sulfur precursor (RISP) is a protein involved in the electron transport chain. RISP abundance decreases in late-logarithmic phase promastigotes, when growth and differentiation are at an advanced stage. Krebs cycle and iron-sulfur cluster-dependent enzymes have been associated to susceptibility to NO by growth inhibition depending on the redox state of sulfhydryl groups in pathogenic bacteria [52,53].
In summary, the differentially abundant glycolytic enzymes TPI and enolase, the DHLDH and DHLAT components of the PDC and the electron transport chain protein RISP are downregulated in the final stages of growth and differentiation of promastigotes, except for the increase of PGM BPI . These changes suggest higher demand of energy during the first stages of promastigote development and might be somehow associated to nitric oxide resistance, as proposed above. On the opposite, the acyl-CoA dehydrogenase, involved in β-oxidation of fatty acids, is up-regulated in stationary phase promastigotes (Fig 3, Table 1), suggesting alternation in the energy source at this stage.
The GDP-mannose pyrophosphorylase (GDP-MP) is up-regulated in late-logarithmic phase promastigotes, which is in agreement with a previous observation in L. infantum promastigotes by the same approach [14]. The GDP-MP is essential for virulence of promastigotes of L. mexicana [54], a species closely related to L. amazonensis. This enzyme is involved in biosynthesis of the lipophosphoglycan (LPG), the glycosylinositol phospholipids (GIPLs) and other glycoconjugates characteristic of the surface of these parasites. The up-regulation of the GDP-MP in late-logarithmic phase promastigotes suggests that higher amounts of these surface molecules are synthesized when the end of growth and differentiation of promastigotes approaches.
Up-regulation of proteins involved in resistance and survival at the last stages of growth and development of L. amazonensis promastigotes Trypanothione system. A processed protein variant (12.9 KDa, pI8.5) of the tryparedoxin peroxidase (TryP) is constitutively expressed throughout the growth curve of L. amazonensis promastigotes, whereas two heavier variants of estimated experimental MW and pI close to the theoretical values (22.0 KDa, pI6.0) are differentially abundant (Fig 4, Tables 1 and 3). One of the TryP heavy variants is up-regulated at day 3, whereas the other one is down-regulated at the same time point. The TryP light variant is highly up-regulated (>30-fold) in stationary phase promastigotes (day 7). In the case of L. infantum, the TryP decreases in amastigotes compared to promastigotes [28] but there are no available data about relative abundance during growth and differentiation process of amastigotes to date. The complex expression profiles of different TryP variants in L. amazonensis promastigotes (Fig 4) has not been found in Old World Leishmania species such as L. major or L. infantum so far and the enzyme was reported to be constitutively expressed in both species [20,55], whereas the non-pathogenic trypanosomatid C. fasciculata differentially regulates the TryP across growth and differentiation of the motile choanomastigote stage [41]. In addition to cellular detoxification of reactive oxygen species, the TryP has been involved in other processes such signaling proliferation and differentiation [56]. The complex expression pattern of different variants found in L. amazonensis promastigotes (Fig 4) suggests that the TryP is especially important throughout the growth and differentiation process studied herein.
The iron superoxide dismutase (Fe-SOD) is over-expressed in L. amazonensis and in C. fasciculata. This protein initiates elimination of the superoxide anion by conversion to hydrogen peroxide. The Fe-SOD is up-regulated in L. amazonensis late logarithmic phase promastigotes (day 5). The TryP is able to reduce this substrate thanks to the assistance of the tryparedoxin (TXN1), the trypanothione (T[S] 2 /T[SH] 2 ) and the NADPH-dependent enzyme trypanothione reductase (TryR). The TryR has not been identified between the selected spots of this analysis and the TXN1 is among the constitutively expressed ones (Table 2).
Arginase. The arginase is a manganese-dependent enzyme that participates in the urea cycle by hydrolyzing L-arginine into urea and ornithine. These products are able to induce the alternative macrophage activation pathway [57]. The ARG of the host phagocytic cell competes with the inducible nitric oxide synthase (iNOS) for the substrate L-arginine [58]. For these reasons, parasite clearance is more effective when low levels of host ARG are expressed. The ARG is also annotated in the genomes of the Leishmania species, thus contributing to decrease the levels of NO synthesis by the iNOS of the host cell through substrate competence. This enzyme is essential for infectivity, proliferation and virulence of the parasite [59,60]. 2DE-MALDI-TOF/TOF analysis has revealed high ARG levels (29-fold) in L. amazonensis stationary phase promastigotes. L. infantum promastigotes overexpress the ARG in amastigotes with respect to stationary phase promastigotes [28]. A possible explanation for these findings combined is preparation in advance for differentiation to the amastigote stage and survival within the phagolysosome of the host cell. This statement is called pre-adaptation hypothesis and has been previously supported [20,[61][62][63]. The MTT test has confirmed that L. amazonensis stationary phase promastigotes are more resistant to NO than logarithmic phase promastigotes (Fig 5). Therefore, NO resistance increases in the ongoing of growth and development of promastigotes in culture.
Ornithine is an essential precursor in polyamine biosynthesis [54,55]. This pathway is essential for the biosynthesis of trypanothione because this molecule consists of two glutathione residues coupled through spermidine. Consequently, arginase up-regulation might be indirectly related to the TryP differential expression profile found (Fig 4).
Synopsis of differentially abundant proteins involved in survival and resistance. Decreased abundance of a variant of the STI1 chaperone simultaneous to the increase of the levels of the ARG, HSP70, TryP, Fe-SOD (stress-resistance proteins) and rPKA (indicative of differentiation) has been found when growth and differentiation of L. amazonensis promastigotes is complete. These changes, especially ARG up-regulation, may be linked to the increased The FE-SOD participates in ROS processing providing hydrogen peroxide, which is then reduced by the TryP with the assistance of the TXN1, trypanithione and TryR. TryP differential expression pattern is complex through the promastigote growth curve of L. amazonensis. A light TryP variant is up-regulated in stationary phase promastigotes. A heavier TryP variant is down-regulated at day 3 and another one up-regulated simultaneously. Finally, a fourth variant is constitutively expressed during promastigote growth. Colour legend for up-regulation: green, day 2 (lag/early log); blue, day 3 (log); orange, day 5 (late log); red, day 7 (stat). The constitutively expressed proteins experimentally detected throughout the promastigote growth curve are highlighted in violet.
doi:10.1371/journal.pone.0164344.g004 ability of differentiated promastigotes to resist NO with respect to the non-differentiated, which is in agreement with the pre-adaptation hypothesis towards survival within the host phagocyte and may explain unsuccessful clearance by the immune system. In addition, higher levels of the GDP-ManP have been observed at an advanced growth phase, which probably leads to increased amounts of glycoconjugates in differentiated promastigotes.

Conclusions
2DE-MS/MS has revealed that promastigotes of the New World species L. amazonensis, responsible for ACL, up-regulate proteins involved in survival and resistance throughout growth and development in culture: the TryP and the Fe-SOD, involved in defense against oxidative damage; the ARG, involved in nitrosative stress resistance within the phagocytic host cell, which is up-regulated in stationary phase promastigotes, thus supporting the pre-adaptative hypothesis; the immunostimulatory protein HSP 70, involved in protein folding; and the rPKA, involved in signaling and autophagy, thus related with metacyclogenesis. The LACK antigen is down-regulated throughout growth and simultaneous development of promastigotes in this species causative of ACL, whereas it was reported to be constitutively expressed in the VL agent L. infantum. The down-regulation of the STI1 and the up-regulation of proteins involved in resistance and survival (ARG, HSP70, Fe-SOD, TryP and rPKA) in fully grown and differentiated promastigotes suggest the pre-adaptative hypothesis, also proposed for other Leishmania species.