Presentation on theme: "L-Proline, D- Glucose and the intracellular cycle of Trypanosoma cruzi Laboratório de Bioquímica de Parasitas - Depto. de Bioquímica. Instituto de Química."— Presentation transcript:
L-Proline, D- Glucose and the intracellular cycle of Trypanosoma cruzi Laboratório de Bioquímica de Parasitas - Depto. de Bioquímica. Instituto de Química - USP
Why to study L-Proline in T. cruzi ? Because it is a main carbon and energy source, together with glucose, aspartic acid and glutamic acid. Because it is involved in the differentiation process from the epimastigote to the trypomastigote forms (metacyclogenesis).
Our goals 1. To study the relevance of D-Glucose and L-Proline in the intracellular cycle of Trypanosoma cruzi. 2. To characterise the transport of L-Proline and D- Glucose among the different stages of the life cycle of T. cruzi.
Experimental infection model Description: Cells: CHO-K1 (auxotrophic for L-proline) Parasites: Trypomastigotes, CL strain clone 14 This model allows: The possibility of modulating the intracellular concentration of proline The possibility of obtaining synchronic cultures
Trypomastigote bursting x [L-proline] Days Post infection Trypomastigotes x 10 6 mL -1
Different time post-infection x intracellular stages
Scheme of experiment 0 M L-Proline 200 M L-Proline 200 M L-Proline 200 M L-Proline 0 M L-Proline
Amastigote Trypomastigote bursting x L-Proline in different intracellular stages + 0,2 mM L-Proline - 0 mM L-Proline Epimastigote Trypomastigotes x 10 6 mL -1 Days Post infection
Intracellular stages x [L-Proline] [L-Proline] ( M) Intracellular forms (10 6 mL -1 ) Amastigotes Intracellular epimastigotes Trypomastigotes
Transporte de L-Prolina L-proline uptake x substrate concentration [L-Proline] (mM) Vo (nmols / 20 x 10 6 cells min) System A System B
Proline uptake x T. cruzi mammalian host stages Vm (nmols / 20 x 10 6 cells min) [L-Proline] (mM) Amastigotes Intracellular epimastigotes Trypomastigotes
Transporte de L-Prolina Proline uptake in the different environment where T. cruzi lives Human serum LIT Insect vector intestinal content CHO Cytoplasm LIT 10% FCS Vo (nmols / 20 x 10 6 cells min) [L-Proline] (mM)
D-Glucose uptake x life cycle stages of T. cruzi
L-Proline, D-Glucose and the intracellular cycle: a model
Conclusions We established that in our model proline is a differentiation factor in the intracellular cell cycle of T. cruzi. We propose that proline in the extracellular medium (host-cell cytoplasm) is required as an energy source for the intracellular differentiation from the intracellular epimastigote to the trypomastigote stages. We propose a metabolic switch along the mammalian-host cycle between glucose and proline comsumption, controled by the glucose and proline transporters.
Identification, cloning and functional characterization of amino acid transporters of Trypanosoma cruzi.
Background The metabolism of amino acids is relevant along the parasite life cycle (metacyclogenesis, differentiation inside the mammalian host-cells). The metabolite transporters of T. cruzi are proteins basically unexplored from a molecular point of view. In fact, the single transporter gene that has been cloned up to now, and which function was biochemically demonstrated is the hexose transporter. No amino acid transporters were cloned and functionally characterized in trypanosomes up to date.
Goals 1.To identify genes coding for amino acid transporters (with particular interest for those coding for Proline, Gluatamate and Aspartate) in trypanosomatids. 2. To characterize the products of these genes from a biochemical, molecular and functional point of view.
Algorithm used for the detection of genes coding for putative amino acid transporters
PATs identified in T. cruzi
Alignment of PATs corresponding to group 1 as an example
Analysis of copy number per group and tandem repeats
Determinantion of the number of putative trans-membrane helices Analysis in sillico of the presence of trans-membrane spanners
Phenogram corresponding to the PATs herein described as well as to other amino acid transporters from other protozoans
Confirmation that the PATs correspond to real and expressed sequences PCR from genomic DNA and RT-PCR from total RNA from infective and non-infective stages.
Conclusions (in brief): Aproximately sequences corresponding to ESTs and genomic sequences were analysed. Fifteen thousand sequences corresponded to partial ORFs coding for putative amino acid transporters (PATs). We could identify 60 ORFs corresponding to PATs. All the identified genes match with the AAAP family in the classification of the TC The genes coding for PATs are arranged in tandem repeats in the T. cruzi genome.
Acknowledgements: Dr. Maria Júlia Manso Alves Dr. Walter Colli Dr. Renata Rosito Tonelli Marcela Martinelli Camila Galvão Lopes Support: Dra. Silvia Uliana Dr. Claudio Pereira Lic. León Bouvier
Scheme of the intermediary metabolism in trypanosomatids Proline as carbon and energy source
Modelos de transportadores Substrat o Ion ATP ADP Ion Substrato Ion ATP ADP Ion Substrato ATP ADP Uniport Simport Antiport
Proline uptake x two intracellular prolin concentrations
Vmax for proline uptake x cations (Na+, K+ or cholin).