Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.
The protozoan T. cruzi, the etiological agent of Chagas’ disease, affects a wide range of mammalian hosts, including humans. It is estimated that 8 million people are infected and 25 million are at risk, most of them living in areas of poor socioeconomic development [1]. The cell cycle of T. cruzi involves an invertebrate vector (triatomine bugs) and a mammalian host, and well-defined developmental stages (epimastigotes, metacyclic trypomastigotes, amastigotes and blood trypomastigotes). T. cruzi trypomastigotes, the classical infective stage, invade almost all mammalian cell and tissue types, and invasion is an obligatory step in their life-cycle in mammals. An essential step immediately prior to the mammalian cell invasion is the interaction of the parasite with the surrounding extracellular matrix. The extracellular matrix (ECM) is a highly dynamic non-cellular three-dimensional macromolecular network, which regulates different cellular functions, such as growth, differentiation or survival [cf. 2]. Its composition includes structural components (“matrisome”, [3]) and elements that can interact with or remodel the ECM [4]. Collagens, proteoglycans and glycoproteins (laminins, fibronectins, thrombospondins, tenascins, among others) constitute the main core of the ECM proteins. ECM-affiliated proteins (such as mucins, syndecans, plexins) and ECM-regulators (such as lysyl oxidases, sulfatases, extracellular kinases, proteases and secreted factors, such as TGFβ, cytokines) were classified as matrisome-associated proteins [4]. Cell-ECM interactions occur mainly by integrins present at the cell surface, which connect the extracellular signals and the intracellular response by the activation of specific signaling pathways [5,6] and dysregulation of the ECM is associated with the development of several pathological conditions [4,7]. Adhesion of Trypanosoma cruzi and other parasites to distinct elements of ECM has been described to involve different surface proteins from the infective stage of the parasite, of which the gp85/transialidase family plays an essential role (rev. [8]). T. cruzi binds to collagen [9], fibronectin [10–14], laminin [11,15,16,17], thrombospondin [11], [18], heparan sulfate [11,12,14,19], galectin-3 [20,21], as well as TGF-β [22]. Also, remodeling of ECM was observed during infection, with modifications in collagen and fibronectin content, as well as reorganization of laminin [13], at least partially due to T. cruzi proteolytic enzymes [23–25]. Despite the relevance of ECM for T. cruzi infection, the signaling pathways triggered by the trypomastigote-ECM interaction are less well known. Recently, we demonstrated a decrease in S-nitrosylation and nitration in the majority of the trypomastigote proteins when parasites are incubated with ECM [26]. In addition, dephosphorylation of proteins, such as α-tubulin, paraflagellar rod proteins (PFR or PAR), as well as ERK 1/2 was observed in trypomastigotes incubated with either laminin or fibronectin [27], although these do not reflect the entirety of possible interactions between parasite and ECM. To have a better understanding of the process, quantitative proteomic and phosphoproteomic approaches were employed to analyze changes in T. cruzi trypomastigote proteins when the parasites are incubated with ECM. Herein we show important changes in the T. cruzi trypomastigotes proteome, as well as in protein phosphorylation levels upon interaction with ECM. Proteins involved with metabolic processes, phosphatases, kinases and RNA/DNA binding elements were highly represented among the proteins modified by phosphorylation. In particular, a decrease of the glycolytic pathway was suggested by metabolite quantification and measurement of hexokinase, pyruvate kinase and lactate dehydrogenase activities, suggesting an extensive metabolic adaptation of trypomastigotes prior to host cell invasion. Taken together, our data show that not only structural adaptations but also important metabolic changes occur upon parasite interaction with ECM. Understanding these changes may further elucidate the adaptive mechanisms involved in parasite-host interaction.
Trypomastigotes (ECM-treated for 120 min or control) were fixed in 2% paraformaldehyde for 15 minutes at room temperature, pelleted by centrifugation (4,000 x g for 5 minutes), washed twice in PBS, resuspended in PBS, added to a coverslip and dried at room temperature. After permeabilization of the parasites with PBS containing 1% BSA and 0. 1% Triton X-100 for one hour at 37°C, anti-phosphoserine, anti-phosphothreonine, anti-phosphotyrosine (Invitrogen–dilution 1: 200 for each antibody), anti-PAR monoclonal antibody (1: 200) or anti–TcHexokinase (kindly provided by Dr. Ana Cáceres, Universidad de Los Andes, Venezuela) were added and incubated for 1 h at room temperature. After three washes with PBS containing 0. 1% Triton X-100, the correspondent secondary antibodies were added (anti-rabbit or anti-mouse-Alexa 555 conjugated (1: 5000); followed by one hour incubation at 37°C. After three washes in PBS-0. 1%-Triton X-100, the coverslips were faced under a solution containing 50% glycerol, 50% milliQ H2O 2 mM sodium azide, and 20 μg/mL of 4' , 6-diamidino-2-phenylindole, dilactate (DAPI-Invitrogen). The images were taken on an ExiBlue™ camera (Qimaging®) coupled to a Nikon Eclipse E 600 optical microscope and deconvoluted using the software Huygens Essential (Scientific Volume Imaging). Frozen pellets of trypomastigotes (1 x 109 MTy or Ty) were resuspended in 1 mL of Lysis Buffer (30 mM Tris-HCl, pH 7. 6,1 mM EDTA, 0. 1% Triton e 0. 25 M sucrose, containing phosphatase and protease inhibitors, as described above, disrupted by ultrasonic for 4 x 10 s (frequency of 40%, Thomas GEX 600 apparatus). After centrifugation (10 000 x g, 15 min), the supernatant was separated and employed to measure the enzymatic activities (Hexokinase, Pyruvate kinase and Lactate dehydrogenase). In all cases, the amount of NADH / NADPH was measured spectrophotometrically at 340 nm and its concentration calculated (extinction coefficient = 6. 220 M-1 cm-1) [32]. Three independent biological samples were employed. Due to the possible presence of ECM proteins in the MTy samples, the enzymatic activities were expressed by 1x108 parasites. The number of parasites in each experimental point was also estimated by a calibration curve using the amount of paraflagellar rod protein in the Western blotting (anti-PAR monoclonal antibody 1: 2000).
Previous studies demonstrated that there is a significant decrease in protein phosphorylation upon parasite interaction with fibronectin or laminin, both components of the ECM. Thus we decided to investigate possible changes in protein phosphorylation that could occur upon trypomastigote interaction with the entire ECM. The experiments outlined in the scheme (Fig 1A) were performed in order to analyze both the proteome and the phosphoproteome of the parasites incubated with ECM. The proteome and phosphoproteome analyses identified 3,093 proteins and 7,880 phosphopeptides, respectively, with FDR values less than 1% and p scores less than e-7 for peptide identification (Fig 1B). Thirty six proteins (57%) and 212 phosphopeptides (67%) correspond to proteins with unknown function (hypothetical proteins), as described by others in different trypanosomes [27,35–42], which is in accordance with the number of proteins (49. 2%) with unknown functions predicted by the genome sequence of T. cruzi [43] (Fig 1B). Sixty-three non-unique proteins from the proteome data exhibited significant variations, with only 5 showing reduction and 58 showing an increase in their protein level (Fig 1B, S1 Fig, S1 Table). Seventeen proteins showed changes where MTy/Ty ≥1. 5, of which nine were hypothetical, including the one with the greatest change (MTy/Ty ≥12. 9). Among the proteins with increased expression, it is worth emphasizing members of the gp85/trans-sialidase (TS) family, involved in host cell infection by T. cruzi, (MTy/Ty = 2. 2 for one member of group II and Mty/Ty = 4. 5 for one member of group IV); small GTP-binding protein rab6 (Mty/Ty = 3. 8); ribosomal RNA processing protein 6 (MTy/Ty = 1. 98; the splicing factor 3a; and the 2Fe-2S iron-sulfur cluster binding domain containing protein (Mty/Ty = 1. 77). In the phosphoproteomic analysis, only phosphopeptides having MTy/Ty ratio below 0. 8 or above 1. 2, with p values less than 0. 05, have been considered to evaluate the effect of ECM on trypomastigote protein phosphorylation. Among the 303 phosphopeptides selected by these criteria, 69 showed an increase and 234 a decrease in their phosphorylation levels. Of these, 91 (33%) are proteins with known function, of which 19 showed an increase and 72 a decrease in their phosphorylation levels (S2 Table; Fig 2A and 2B). Taken together the data indicate that adhesion of trypomastigotes to ECM overall leads to protein dephosphorylation, in agreement with previous observations with T. cruzi trypomastigotes incubated with ECM components, fibronectin and laminin [27]. Phosphorylation site analysis identified 371 differentially phosphorylated amino acid residues: 275 (74. 1%) serine, 83 (22. 4%) threonine and only 13 (3. 5%) tyrosine residues, in accordance with the abundance of serine/threonine kinases found in T. cruzi [44], the fact that T. cruzi does not express receptor coupled tyrosine kinases but dual specificity kinases [44], as well as with the phosphoproteome data described for different stages of the parasite [42,45, rev. 46]. In silico analysis, as described below, suggested the involvement of different kinases (CAMK, TKL, CMGC, CK1, AGC and others). To understand better the role of the proteins controlled by phosphorylation during the parasite response to the ECM, GO-terms enrichment analysis was performed. Additional information was obtained on the molecular function and/or sub-cellular localization of 29 proteins and 126 phosphopeptides previously labeled as hypothetical (unknown function) based on version 40 of the TriTrypDB. The data are shown in S1 Table and S2 Table. Most of proteins identified are related to structural function, pathogenicity, metabolism and protein phosphorylation. Both cytoplasm and axoneme seem to be the main localization of the identified phosphopeptides. Among the proteins identified, gp85/trans-sialidase (TS) family members, involved in infection of host-cells by T. cruzi, were enhanced in trypomastigotes incubated with ECM (MTy/Ty = 2. 2 for one member of group II and MTy/Ty = 4. 5 for one member of group IV). Small GTP-binding protein rab6 (MTy/Ty = 3. 8), ribosomal RNA processing protein 6 (MTy/Ty = 1. 98), splicing factor 3a and 2Fe-2S iron-sulfur cluster binding domain containing protein (MTy/Ty = 1. 77) were also identified in the group of the proteins with an increased expression. The following proteins are highly represented in the pool of proteins modified by phosphorylation (Fig 2B and S2 Table): proteins involved in metabolic processes (19 phosphopeptides); in phosphorylation/dephosphorylation (such as kinases and phosphatases, 20 phosphopeptides); structural proteins (such as flagellum and microtubule related proteins, 19 phosphopeptides); transport-associated proteins; and RNA/DNA binding elements. This suggests an extensive metabolic adaptation occurring in trypomastigotes prior to host-cell infection. Of note, enzymes that participate in glucose metabolism, in addition to adenylosuccinate lyase (ADSL) and alanine aminotransferase (ALT) (Table 1; Fig 2C), lipid metabolism (3-oxo-5-alpha-steroid 4-dehydrogenase; putative (pseudogene) and ethanolamine phosphotransferase) (Fig 2B, S2 Table) are modified by phosphorylation. Since a significant number of enzymes from the glucose metabolism were modified by phosphorylation, their role was further investigated. Seven enzymes involved in carbohydrate metabolism were identified in the phosphoproteomic analysis: hexokinase (HK); 6-phosphofructo-2-kinase/fructose-2; 6 bisphosphatase (PFK2); 6-phospho-1-fructokinase (PFK1) (pseudogene); phosphoglucomutase (PGM); pyruvate phosphate dikinase (PPDK); phosphoglycerate kinase (PGK) and NADH-dependent fumarate reductase (FRD), in addition to adenylosuccinate lyase and alanine aminotransferase (Fig 2C, Fig 3, Table 1, S2 Table). Except for NADH-dependent fumarate reductase (MTy/Ty = 1. 5) and adenylosuccinate lyase (MTy/Ty = 2. 2), all the others showed decrease in their phosphorylation levels when trypomastigotes were incubated with ECM (Table 1 and Fig 3). Most of these enzymes are localized in the glycosomes, a peroxisome-like organelle essential for trypanosomatids survival and characterized for containing most of the glycolytic/gluconeogenic pathways in kinetoplastids, in addition to enzymes of other metabolic pathways, such as the pentose phosphate pathway, beta-oxidation of fatty acids, and biosynthesis of pyrimidines (rev. [47–49]). Likewise, phosphorylation of enzymes involved in carbohydrate metabolism was described in the proteome and phosphoproteome of the glycosomes in T. brucei and Leishmania donovani [40,41,50]. Since the data suggested possible changes in the metabolism of T. cruzi, the metabolite content was analyzed by GC/MS to understand better the response of trypomastigotes upon adhesion to ECM. GC-MS analysis allowed the identification of 21 metabolites with a significant variation (p < 0. 05) in the MTy/Ty ratio (S5 Table), from which significant changes were found for carbohydrate, lipid and amino acid metabolites. Some of these metabolites are substrates or products of the enzymes modified by phosphorylation that are found, although not exclusively, inside the glycosomes (Fig 3A and 3B, Table 1). The following metabolites from the glycolytic pathway were modified in parasites upon incubation with ECM: increase in glucose/galactose (molecules indistinguishable in the GC-MS methodology) with ECM (MTy/Ty = 1. 2); decrease in pyruvic acid (MTy/Ty = 0. 2) and lactic acid (MTy/Ty = 0. 34). Interestingly, metabolites derived from the glycolytic pathway branch and common to TCA cycle: succinic acid (MTy/Ty = 1. 37), malic acid (MTy/Ty = 1. 21) and fumaric acid (MTy/Ty = 1. 15), were increased in parasites incubated with ECM. Succinate is considered the main source of reducing equivalents to the respiratory chain through the action of a NADH-dependent fumarate reductase and it is also (in addition to alanine) one of the main products excreted by trypanosomatids [48,51], although lactate excretion by T. cruzi [52] may increase, depending on metabolic adaptations. These changes in metabolite level do not appear to correlate with changes in metabolic enzyme levels (S1 Table). Rather, they may reflect modulation of metabolic enzyme activity. Although less representative in the metabolite quantification, an increase in free amino acids in MTy (tyrosine, glycine or isoleucine, MTy/Ty ratio ≈1. 4) and fatty acids (mainly palmitic acid, MTy/Ty ratio ≈1. 4) may also indicate wider changes in the metabolism of MTy (S5 Table). To analyze the potential role of metabolic enzyme phosphorylation in modulating the glycolytic pathway in MTy, enzymatic activities of hexokinase/glucokinase (HK/GK) and pyruvate kinase (PK) were determined, as well as for lactate dehydrogenase (LDH). Although PK and LDH were not detected in our phosphoproteomic analysis (S2 Table), these enzymes were included because significant depletion of pyruvate and lactate were observed in MTy (S5 Table). Further, as pointed out before, the changes in the metabolite levels seem to be independent of the relevant metabolic enzyme expression levels accordingly to the proteomic data (S1 Table). The enzymes HK and GK catalyze the formation of glucose-6-phosphate, the first reaction of the glycolytic pathway and both lack the regulatory allosteric inhibition by glucose-6-phosphate, common to other organisms. Both are localized inside the glycosomes, of which HK presents the highest activity [53,54]. A distinct HK, which phosphorylates glucose and fructose, as well as GK activity were also described in the cytosol [53]. The affinity of HK for glucose is higher than that of GK (Km values of 0. 06 mM and 0. 7 mM, respectively) in addition to the higher amounts of HK over GK in the parasite [55]. However, in the present work we could not separate the activities of both enzymes and, thus, they are collectively represented by HK/GK activities. The three phosphorylated residues of T. cruzi hexokinase (S161, T153, T159) (Table 2, S1 Table, S2 Fig) were located in the same peptide identified by LC-MS/MS and presented similar changes in the phosphorylation level (MTy/Ty ratio = 0. 76; 0. 76 and 0. 57, respectively). The identified phosphorylated peptide is localized in the catalytic domain or in the substrate-binding site of the enzyme, according to the alignment of T. cruzi-HK (TcCLB. 508951. 20), T. brucei (Tb927. 10. 2010) and the three isoforms of human-HK (P52790; P19367; P52789), performed by ClustalW platform (S2B Fig). HK/GK activities from MTy and Ty homogenates were measured spectrophotometrically in the presence of a coupling system containing glucose-6-phosphate dehydrogenase. HK/GK activity is clearly reduced (by approximately 46%) in MTy relative to Ty (Fig 4A, a). Previous treatment of Ty and MTy homogenates with alkaline phosphatase significantly reduced the activity (approximately 45% for Ty and 59% for MTy extracts, Fig 4A, c, d). Also, the activity was drastically reduced (approximately 70%) when the homogenate was previously treated at 56°C for 1 h. Since HK is inhibited by small phosphate molecules, such as PPi present in distinct organelles including glycosomes [53,56,57], the experiment was repeated with the parasite extract previously immunoprecipitated with anti-HK antibodies. Similar results have been obtained, confirming the relevance of phosphorylation for HK/GK activity (S4 Fig). Inhibition of the enzymatic activity by dephosphorylation is consistent with the accumulation of glucose detected in MTy. However, one cannot rule out other possibilities, such as changes in glucose transport in MTy, alterations of HK oligomerization, which is usually tetrameric [54] or somehow by contributing to the hysteretic and cooperative behavior of HK at low enzyme concentration described in T. cruzi epimastigotes [58]. The phosphorylation of HK could be by auto-phosphorylation [59] or by protein kinase (s). Of note, the same pattern of HK in MTy and Ty inside the glycosomes was shown by immunofluorescence using specific anti-HK antibodies (S2A Fig). These data also indicate that no significant changes in the number of glycosomes occur in MTy, in accordance with the literature, where approximately the same number was found in the different forms of T. cruzi, in contrast to the variability described during the life cycle of other species [60]. Pyruvate kinase (PK) was not detected in the phosphoproteome/proteome described herein, as pointed out above, but the low amount of pyruvate found in MTy led us to measure a corresponding enzymatic activity. In the cytosol, PK catalyzes the formation of pyruvate and ATP from phosphoenolpyruvate and ADP and, in the case of T. cruzi epimastigotes, PK is inhibited by millimolar concentrations of ATP and Pi and activated by micromolar concentrations of fructose 2,6-bisphosphate (rev. [48,61]) by the tetrameric stabilization of PK in response to the effector binding [62]. As shown (Fig 4B, a), PK activity is strongly inhibited in trypomastigotes incubated with ECM (approximately 65%). Treatment of the enzyme with alkaline phosphatase increased its activity, mainly in MTy homogenates (25% Ty and 125% MTy), as shown in Fig 4B, c, d. The decrease in pyruvate content observed could be attributed to the inhibition of pyruvate kinase in the cytosol and/or pyruvate phosphate dikinase in the glycosome, which would lead to an increase of dicarboxylic acids from the glycolytic branch (succinate, fumarate and malate) in the glycosome, (cf. Fig 3 and S5 Table). However, higher consumption of pyruvate, for example by its conversion to acetyl-CoA inside the mitochondria or to lactate in the cytosol, cannot be ruled out. Lactate dehydrogenase-like (LDH) activity was measured in the parasite homogenate due to the decrease observed in lactate content in MTy (S5 Table). In many organisms, a tetrameric form of the enzyme catalyzes the oxidation of lactate to pyruvate in the presence of NAD+ as hydrogen acceptor. LDH activity in T. cruzi epimastigotes was attributed to the isoenzyme I of ∂-hydroxyacid dehydrogenase localized inside the glycosomes and in the cytoplasm [63] and to an unknown protein in T. brucei [51], since a typical lactate dehydrogenase is absent from the genomes of trypanosomatids. The measurement of LDH activity showed approximately 33% reduction in MTy in comparison to trypomastigotes (Fig 4C, a), in agreement with the decrease of lactate detected in MTy. LDH was also inhibited by alkaline phosphatase treatment (approximately 65% for Ty and 41% for MTy extracts), reinforcing the role of phosphorylation in modulating LDH activity (Fig 4C, c, d). Alanine, rather than lactate, is usually the main product of the reduction of pyruvate in T. cruzi, a reaction catalyzed by alanine aminotransferase inside the glycosomes (rev. [48,49]). Lactate excretion by the parasite [52] may increase depending on metabolic adaptations, as described for the procyclic forms of T. brucei [51] and may explain the aforementioned results in trypomastigotes. Although no significant differences in alanine content between MTy and Ty were detected by GC/MS analysis, alanine aminotransferase is less phosphorylated in trypomastigotes incubated with ECM and may be responsible for the switch to the LDH reaction for NADH-reoxidation. Since T. cruzi possesses the enzyme repertoire for gluconeogenesis, this pathway may also be activated in MTy, resulting in higher consumption of pyruvate, lactate and glycerol, although no reserve polysaccharide was detected and gluconeogenesis has not been fully established in T. cruzi. Our data suggest that incubation of trypomastigotes with ECM triggers metabolic adaptations in the parasites, and that phosphorylation, or more specifically protein dephosphorylation, may be involved in these processes. In spite of the relevance of the dephosphorylation and the high representative number of phosphatases in the genome and proteome of T. cruzi [64,42], only two protein phosphatases with diminished phosphorylation levels in MTy were found: an endonuclease/exonuclease/phosphatase responsible for dephosphorylation of DNA sequences (TcCLB. 504073. 10) and PP1, a serine/threonine phosphatase (TcCLB. 507757. 50). Serine/threonine phosphatases are abundant in T. cruzi and constitute more than 50% of the 86 protein phosphatases in the genome. Protein phosphatase PP1 is dephosphorylated in MTy (MTy/Ty = 0. 65), which would be expected to increase its enzymatic activity and consequently contribute to the dephosphorylation of proteins [65] (Table 2; S2 Table). Other protein phosphatases, such PP2A, PP2C or dual specific phosphatase were described in the proteome / phosphoproteome of T. cruzi by different groups or in the glycosomes of T. brucei [41] or L. mexicana [50], which might also contribute to the decrease of the phosphorylation level of the proteins. To understand further the signaling pathways activated in the parasite upon contact with host ECM, the kinases capable of phosphorylate the peptides detected by LC-MS/MS were predicted by the GPS 2. 1 software. Only the higher scores for each phospho-residue (S, T and Y) in the peptide were selected, totaling 378 putative kinases for the 303 phosphopeptides identified by LC-MS/MS (S3 and S4 Tables). Most of the identified sites correspond to modifications by serine/threonine kinases and the phosphorylation of tyrosine was attributed to the dual-specificity kinases, in agreement with the absence of conventional tyrosine kinases in the genome of trypanosomatids [44]. Of the 303 phosphopeptides analyzed, 78. 5% and 21. 1%, respectively, presented one or two phosphorylated sites and only one phosphopeptide showed three phosphorylated-sites (Fig 5A and 5B). The phosphorylated sites identified herein are predicted to be modified mainly by elements of the CMGC kinases superfamily (68), STE kinases (62), TKL kinase (61), AGC kinase (31), CK1 family (31) (Fig 5D, Tables 1 and 2, S3 and S4 Tables). Kinases that do not belong to any characterized family are grouped as “Others”. Only the catalytic subunit of protein kinase A, a member of the AGC kinase superfamily showed in MTy a slightly increase in its phosphorylation level (MTy/Ty = 1. 27). The phosphorylation of T197 of mouse PKA, located in the activation loop, which corresponds to T35 described herein for T. cruzi, increases PKA activity, indicating that upon interaction with ECM, PKA is activated [66,67]. Proteins responsible for a plethora of biological phenomena in T. cruzi have been described as PKA substrates, such as kinases (type III PI3 kinase-Vps34, PI3 kinase, mitogen-activated extracellular signal-regulated kinase), cAMP-specific phosphodiesterase (PDEC2), putative ATPase, DNA excision repair protein, aquaporin, hexokinase and members of gp85/TS [68,69]. The role of PKA during metacyclogenesis (differentiation of epimastigotes to metacyclic trypomastigotes [70]) or during the amastigogenesis (differentiation of trypomastigotes into amastigotes [42]) is well established, with the stimulation of adenylyl cyclase and increment in cAMP concentration during the process. Albeit the relevance of PKA in the physiology of the parasite, their specific role in trypomastigotes incubated with ECM was not determined. In contrast to PKA, reduction in phosphorylation was detected in the majority of the kinases, for example in glycogen synthase kinase 3 (GSK3) and ERK1/2, presumably leading to a decrease of their activities in MTy: Y187 from GSK3 (MTy/Ty = 0. 78) corresponds to human Y216 also located in the activation loop and whose phosphorylation is necessary for activity [71]; T190 and Y192 from mitogen-activated protein kinase (MTy/Ty = 0. 75), corresponds to human T185/Y187, whose phosphorylation is necessary for ERK1/2 activation [72]. Dephosphorylation of ERK1/ 2 was also observed in the incubation of trypomastigotes with laminin or fibronectin [27]. Decrease in phosphorylation of other kinases was also found: protein kinase ck2 regulatory subunit (MTy/Ty = 0. 76), inositol-related signaling kinases; inositol polyphosphate kinase-like protein (MTy/Ty = 0. 67) and phosphatidylinositol-4-phosphate 5-kinase type II beta (MTy/Ty = 0. 37) (Table 2). Whether this is a reflection of their activation status remains to be determined. The phosphopeptides assigned to particular kinase families by the GPS 2. 1 software (Fig 3D) were used for the construction of sequence logos, which correspond to sequence alignment with the central point as the likely phospho-amino acid residue (Fig 3C). For the AGC kinase families," other" and CK1, no consensus was observed in the amino acid sequences surrounding the S/T residue. For the" Atypical" , TK and STE families some amino acids were identified with a high level of conservation. The analysis of the sequence logos (Fig 3C) indicates some conservation relative to the sites already characterized for humans, such as the group of peptides phosphorylated by CMGC, where a proline next to the phosphorylation site was also identified; lysine and arginine near the phosphorylation site for AGC; residues of aspartate and glutamate for CK1 family substrates and the conservation of phenylalanine and proline residues near the phosphorylated site for the atypical kinases. Incubation of T. cruzi trypomastigotes with the extracellular matrix results in important and more extensive changes than the ones previously described by the incubation of trypomastigotes with fibronectin or laminin [27]. Reduction in the phosphorylation level of proteins seems to be a general event in trypomastigotes incubated with ECM (Tables 1 and 2, S2 Table). Kinases, except for PKA, PP1 phosphatase and enzymes from the glycolytic pathway and probably the glycolytic branch exemplify these decreases (S2 and S3 Tables). Strikingly, correlating with the observed decrease in phosphorylation level of the enzymes, a significant inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase-like were detected. (S2 and S5 Tables, Fig 4). These results, in association to the slightly increase of glucose and drastic reduction of pyruvate and lactate strongly suggest that ECM triggers important reduction in the glycolytic pathway in T. cruzi trypomastigotes. Although hexokinase is among the many substrates described for PKA [69], a possible correlation between these enzymes has not been explored herein. Interestingly, trans-sialidases, surface glycoproteins belonging to the T. cruzi gp85/trans-sialidase family, are also one of the substrates for PKA [69] and PKA activity and trans-sialidase expression has been associated with differentiation and invasion of host cells by T. cruzi [73]. A similar coincidence of increase in PKA activity and expression of two members of the gp85/trans-sialidase family were observed upon incubation of trypomastigotes with ECM (MTy/Ty = 2. 2 and 4. 5, S1 Table), perhaps preparing the parasites for an efficient invasion of the host cell. The possibility that members of the large gp85/ trans-sialidase family interact with different components of ECM to trigger all the modifications described here remains to be determined. Taken together, the data presented herein suggest reprogramming of the metabolism of trypomastigotes triggered by their interaction with the extracellular matrix, an obligatory step before cell invasion and differentiation into amastigotes, the multiplicative stage of T. cruzi in the vertebrate host. The reduction in glycolytic enzyme activity in trypomastigotes by phosphorylation/dephosphorylation events seems to be part of this reprogramming, with the involvement of yet to be identified protein kinases and phosphatases.
Adhesion of Trypanosoma cruzi to distinct elements of ECM involving different surface proteins from the infective stage of the parasite has been described. Despite the relevance of ECM for T. cruzi infection, the signaling pathways triggered in trypomastigotes upon interactions with ECM are less well understood. In previous work we demonstrated the dephosphorylation of proteins, such as α-tubulin, paraflagellar rod proteins and ERK 1/2 in trypomastigotes incubated with either laminin or fibronectin. Further, we described changes in the S-nitrosylation and nitration pattern of proteins from trypomastigote incubated with ECM. To expand our knowledge on ECM triggered parasite signaling we applied quantitative proteomic and phosphoproteomic studies to trypomastigotes incubated with ECM (MTy) compared to controls (Ty). Our results indicate relevant changes in total protein and phosphoprotein profiles in MTy. The kinases implicated in the modifications were suggested by bioinformatic analyses, as well as the number of modifications and the frequency of amino acids per peptide that have been modified. Proteins involved in metabolic processes, including enzymes from the glycolytic pathway, phosphatases and kinases were the most representative groups among the proteins modified by phosphorylation. Quantification of metabolites in MTy and Ty also indicated that glucose metabolism is impaired in trypomastigotes incubated with ECM. The significant inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy associated with phosphorylation levels, strongly suggests that trypomastigotes reprogram their metabolism in response to interaction with the extracellular matrix, an obligatory step prior to host cell invasion.
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Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix