A Trypanosoma cruzi phosphatidylinositol 3-kinase (TcVps34) is involved in osmoregulation and receptor-mediated endocytosis.

TORRES, H.N.; SCHOIJET, A.C.; ALONSO, G.; DOCAMPO, R.; MIRANDA, K.; DE SOUZA, W.; FLAWIÁ, M.M.

Woods Hole, MA, USA

Congreso; 19th Annual Molecular Parasitology Meeting (MPM).; 2008

Marine Biological Laboratory Woods Hole

Trypanosoma cruzi, the etiological agent of Chagas’ disease, has the ability to respond to a variety of environmental changes during its life cycle. It has been found that cAMP levels increase when T. cruzi epimastigotes are subjected to hyposmotic stress. We described a membrane-associated cAMP-specific phosphodiesterase, TcrPDEC2, which is inhibited by the same inhibitors that affect the regulatory volume decrease (RVD) of the parasites after hyposmotic stress. Since inhibitors of TcrPDEC2 affect osmoregulation in T. cruzi, and TcrPDEC2 possesses a FYVE domain able to bind to PI 3-P, we hypothesized that PI 3-P production might have a role in osmoregulation. Here we report the functional characterization of TcVps34, the first class III PI 3-kinase from T. cruzi. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. While inhibition of TcVps34 with specific PI3K inhibitors resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were subjected to severe hyposmotic stress. In addition, in pTREX-TcVps34 overexpressing cells the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked whereas fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Finally, we have demonstrated that TcVps34 interacts with TcVps15, a serin-theronine protein kinase that is part of a membrane-associated complex together with Vps34 in yeast. Moreover, depletion of TbVps15 in procyclic T. brucei by RNA interference resulted in inhibition of cell growth and a distorted cellular morphology.