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.
Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H(+) gradient, but not on a Na(+) or K(+) gradient at the cytoplasmic membrane level.
KeywordsMetabolism; tyrosine aminotransferase and aspartate amino transferase.
ABSTRACTTrypanosoma cruzi, the etiological agent of Chagas disease, lacks genes that encode canonical branched-chain aminotransferases. However, early studies showed that when epimastigotes were grown in the presence of 14 C 1 -DL-leucine, the label was incorporated into various intermediates. More recently, our studies provided evidence that T. cruzi epimastigotes display a single ATPdependent and saturable transport system that enables epimastigotes to uptake branched-chain amino acids (BCAAs) from the culture media. To extend our knowledge of the first step of BCAA catabolism, the ability of this parasite's noncanonical broad specificity aminotransferases, such as tyrosine aminotransferase (TAT) and aspartate aminotransferase (ASAT), to transaminate these amino acids was investigated. Indeed, our results show that TAT and ASAT utilize BCAAs as substrates; however, both enzymes differ in their catalytic competence in utilizing these amino donors. For instance, ASAT transaminates isoleucine nearly 10-fold more efficiently than does TAT. This unique characteristic of TAT and ASAT allows to explain how BCAAs can be oxidized in the absence of a BCAA transaminase in T. cruzi.
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