Identification and characterization of the Trypanosoma cruzi Pantothenate Transporter (TcPPT1) and its role in survival under stress conditions

RUIZ DANIELA; FRACCAROLI, LAURA VIRGINIA; BALCAZAR DARIO; LAROCCA LUCIANA; TORRES PABLO; CARILLO CAROLINA

Capital Federal

Congreso; 18th ICID abstract supplement 2018; 2018

Chagas disease is an endemic parasitosis originallyfrom Latin America, caused by the protozoan Trypanosomacruzi. The current therapies are limited in efficacy and show multiple sideeffects. Thus, there is a need to identify new targets to develop novel, moreeffective and specific trypanocidal strategies.Vitamins are essential micronutrients for all livingcells; in particular, B vitamins are relevant in the biology of T. cruzi. Provided that pantothenate,the precursor of Coenzyme A, is involved in numerous essential cellularreactions and that our previous in silicostudies suggested that T. cruziis auxotrophous for this vitamin, the aim of this work was to studypantothenate role in T. cruziepimastigotes and to identify and characterize potential pantothenate-transporters.In order to approach this aim, we evaluated T. cruzi epimastigotes (Y-GFP strain)proliferation and viability performing growth curves and MTT assay in a culturemedia with low concentration of pantothenate. Provided that our bioinformaticstudies yielded the identification of a putative pantothenate transporter (TcPPT1), we constructed a mutant Y-GFPstrain that overexpress TcPPT1 fusedto a mCherry tag. Then we evaluated differential proliferative response underdifferent stressing conditions: nutrient deprivation (20nM pantothenate),oxidative stress (0-150uM H2O2) and trypanocidal drugs(0-100uM Benznidazole and Nifurtimox).T. cruzi epimastigotes proliferation ratewas significantly decreased in culture media with low concentration ofpantothenate. This effect was reverted, in a concentration dependent manner,when the vitamin was added to the media. The pantothenate proliferative effectevidenced on T. cruzi epimastigotes wasnot observed in other axenic cultures of related trypanosomatids (T. brucei, L. mexicana and C. fasciculata).TcPPT1 overexpression was assessed by PCR, RT-PCR andfluorescence microscopy in mutant Y-GFP clones. Proliferation rate of TcPPT1-mCherry epimastigotes wassignificantly higher than mCherry epimastigotes (control) in low pantothenateconditions (p<0.05) (35.0±2.3 million/ml vs 21.7±2.9 at 4th day,respectively). Additionally, TcPPT1-mCherryclones cultured 24 and 48 h in media with H2O2 or trypanocidaldrugs showed increased proliferation compared with the control counterpart.The findings presented herein suggest thatpantothenate and its transporter (TcPPT1)are relevant for T. cruziepimastigotes survival and proliferation, especially under stressingconditions.