An insight into the proteome of Crithidia fasciculata applied to the search of distintive features of Leishmania species involved in cutaneous and visceral leishmaniasis

The Leishmania infantum tyrosine aminotransferase (TAT) has been identified as an important catabolic enzyme in the degradation pathway of the aromatic amino acids with a broad substrate range. Prior studies demonstrated that the end-product of the tyrosine degradative pathway (phydroxyphenyl lactic acid) is excreted in large amounts by the promastigote, indicating that this pathway is highly activated. During the life cycle of the parasite, the protein is over-expressed in the early-logarithmic stage of the procyclic promastigote with a reduced basal expression at the stationary phase. This may be consistent since the expression of the protein is highly dependent on the availability of the nutrients. However, the protein is over-expressed at the protein level in the more infective PNAstationary-phase promastigotes. Interestingly, the subcellular location of the protein is different between the procyclic promastigotes and the more infective PNA- promastigotes. L. infantum TAT is also expressed in the amastigote-like axenic population (pH 4.5, 37°C, 5%CO2). Pulled-down assays in native conditions using a specific affinity-purified polyclonal antibody were developed in order to analyze the proteins that presumably interact with TAT in the procyclic stage. The results indicate that the protein is interacting with a cytoplasmic decarboxylating malic enzyme responsible of the conversion of the cytosolic malate produced during the glycolysis to pyruvate, which is then converted to alanine by amino acid transaminases. This interaction suggests that TAT protein is acting as an alanine aminotransferase using pyruvate as amino acceptor and yielding NAD(P)H which can protect against oxidative stress. This is in agreement with the reactivity of the leishmanial TAT, since its preferred amino acceptor for the transamination is the pyruvate. Moreover, the comparison of the expression in L. chagasi between two isolated strains, one resistant to nitric oxide (NO) and other sensitive, reveals the overexpression of the protein in the first one. The increased expression of the protein in the more infective and resistant subpopulations of L. infantum promastigotes suggests that the TAT is playing an important role in the resistance against reactive oxygen species through the regeneration of reducing agents. Provided its expression in the amastigote like form, the leishmanial TAT may be considered as a possible target in the development of future inhibitors.Peer Reviewe