Vesicular trafficking in Trypanosoma cruzi: characterization of Vps15 protein kinase as a regulatory subunit of TcVps34 PI 3-kinase.

SCHOIJET AC; FLAWIÁ MM; ALONSO GD

Mar del Plata, Buenos Aires.

Congreso; IX Congreso Argentino de Protozoologia y Enfermedades Parasitarias.; 2011

Sociedad Argentina de Protozoología

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its different stages in the insect vector and in the vertebrate host. As T. cruzi passes through its complex life cycle, it comes across with different environmental stressors. One of the most relevant is the extreme fluctuations in osmolarity that occur within the gut of the vector, and also as the infective form passes out of the vector excreta (600-700 mOsm) and suddenly encounters the interstitial fluid of the mammalian host (300 mOsm). Previous studies on the response to hyposmotic stress have shown that both insect and vertebrate stages possess a strong regulatory volume decrease (RVD) mechanism that completely reverses cell swelling (Rohloff and Docampo, 2008). Moreover, it has been reported that cAMP levels increase when epimastigotes are subjected to hyposmotic stress, stimulating the traffic of an aquaporin from the acidocalcisomes to the contractile vacuole complex (CVC). Concomitantly, an increase in osmotically active metabolites occurs in the acidocalcisomes, which are transferred to the CVC creating an osmotic gradient that stimulates water entry into this organelle. Finally, the excess of water is released out of the cell through the flagellar pocket (Rohloff et al., 2004; Rohloff and Docampo, 2008). Previously, we have functionally characterized a membrane-associated cAMP-specific phosphodiesterase (Alonso et al., 2006), named TcrPDEC2, which showed to be localized in the CVC and involved in osmoregulation. TcrPDEC2 possesses a FYVE domain, which is characteristic of proteins recruited to phosphatidylinositol 3-phosphate- (PI 3-P) containing membranes, and we demonstrated that this domain it is crucial for its localization and activity (Schoijet et al., 2011). Due to the phosphoinositide PI 3-P is one type of phospholipid involved in receptor signaling and several membrane trafficking events, and in particular, Class III phosphoinositide 3-kinase (PI3K), known as Vps34 [Vps (vacuolar protein sorting)], are essential regulators of vesicular transport, autophagy and nutrient sensing, we continued studying this signal pathway. In this sense, we have functionally characterized the first class III PI3K reported in Trypanosoma cruzi, named TcVps34, which has a role in vital processes for the survival of this parasite such as osmoregulation, acidification, and receptor-mediated endocytosis (Schoijet et al., 2008). It is worth to remark that TcVps34 overexpressing cells showed enlarged and functional contractile vacuoles and defects in the flagellar pocket and the citostome. In yeast and other organisms, Vps34 exerts its lipid kinase activity by forming a protein complex with a Ser/Thr protein kinase known as Vps15. However to date, the role of Vps15 in the regulation of Vps34 has not been extensively studied. Recently, we have initiated the characterization of TcVps15, a Vps15 homologue in T. cruzi. TcVpsl5 gene encodes a 147 kDa protein which exhibits a significant degree of similarity to the catalytic domain of the Ser-Thr family of protein kinases. The recombinant TcVps15 was able to phosphorylate both comertial histone mix (H2As) and myelin basic protein (MBP), showing a cation preference for Mn+2 over Mg+2. Moreover, transgenic parasites overexpressing TcVps15 showed an increase in their kinase activity. Subcellular fractionation of epimastigotes overexpressing TcVps34 or TcVps15, followed by western blot analysis, showed that both enzymes are localized mainly at the membrane fraction. Interestingly, the localization of these proteins was not affected by the treatment of epimastigotes with wortmannin, a PI3K inhibitor. Further experiments will be performed to address the mechanism by which TcVps15 regulates TcVps34. Rohloff, P., and Docampo, R. (2008) A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi. Exp Parasitol 118: 17–24. Rohloff, P., Montalvetti, A., and Docampo, R. (2004) Acidocalcisomes and the contractile vacuole complex are involved in osmoregulation in Trypanosoma cruzi. J Biol Chem 279: 52270–52281. Alonso, G.D., Schoijet, A.C., Torres, H.N., and Flawia, M.M. (2006) TcPDE4, a novel membrane-associated cAMPspecific phosphodiesterase from Trypanosoma cruzi. Mol Biochem Parasitol 145: 40–49. Schoijet, A.C., Miranda, K., Medeiros, L.C., de Souza, W., Flawiá, M.M., Torres, H.N., Pignataro, O.P., Docampo, R., Alonso, G.D. (2011) Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi. Mol Microbiol 79(1):50-62. Schoijet, A.C., Miranda, K., Girard-Dias, W., de Souza, W., Flawiá, M.M., Torres, H.N., Docampo, R., Alonso, G.D. (2008) A Trypanosoma cruzi phosphatidylinositol 3-kinase (TcVps34) is involved in osmoregulation and receptor-mediated endocytosis. J Biol Chem 283(46):31541-50.