A phosphodiesterase (TcrPDEC2) and a phosphatidylnositol 3-kinase (TcVps34) are involved in osmoregulation in Trypanosoma cruzi

SCHOIJET, ALEJANDRA; KILDARE, M.; ALONSO, GUILLERMO; TORRES, HÉCTOR N; FLAWIÁ, MIRTHA M.; DOCAMPO, ROBERTO

Woods Hole, Massachusetts, USA.

Congreso; Molecular Parasitology Meeting; 2007

Molecular Parasitology

During its development in different hosts, T. cruzi faces extreme fluctuations in osmolarity. Both insect and vertebrate stages of the parasite have been shown to possess a robust regulatory volume decrease (RVD) mechanism that reverse cell swelling under hypo-osmotic stress. Amino acid and K+ release account for 57% of the RVD of epimastigotes after hypo-osmotic stress. Volume recovery also involves aquaporin (TcAQP)-mediated water movement. In this work, we report the contribution of TcrPDEC2, a cAMP-specific phosphodiesterase and TcVps34, a class III phosphatidylinositol 3-kinase to osmoregulation and membrane trafficking in epimastigotes of T. cruzi. Over-expression of TcrPDEC2 or TcVps34 in epimastigotes caused differences in resistance of the parasites to hypo-osmotic stress when cells were incubated in the presence of type 4 PDE or PI3K inhibitors, respectively. Type 4 PDE inhibitors rolipram and etazolate induced an increase in volume recovery in wild type cells whereas PI3Ks inhibitors wortmannin and LY294,002 impaired this mechanism. No effects were observed in TcrPDEC2 and TcVps34 over-expressing cells. Immunofluorescence showed co-localization of TcrPDEC2 and calmodulin in the anterior portion of the parasite, near the contractile vacuole/flagellar pocket region. Structural analysis of TcVps34 over-expressing cells showed functional and large contractile vacuoles when compared to wild type cells. Kinetics of proton uptake in TcVps34 cells showed higher H+-ATPase and lower H+-PPase activities than in wild-type cells. Finally, endocytosis of FITC-transferrin was reduced in TcVps34 over-expressing cells. Taken together, these results suggest a potential role for TcrPDEC2 and TcVps34 in osmoregulation, vacuolar acidification, and membrane trafficking in T cruzi.