LProline, D Glucose and the intracellular cycle of Trypanosoma cruzi

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L-Proline, D- Glucose and the intracellular cycle
of Trypanosoma cruzi

• Laboratório de Bioquímica de Parasitas - Depto.
  de Bioquímica. Instituto de Química - USP

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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).

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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.

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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

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Trypomastigote bursting x L-proline
Trypomastigotes x 106 mL-1
Days Post infection
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Different time post-infection x intracellular
stages
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Scheme of experiment
200 mM L-Proline
200 mM L-Proline
0 mM L-Proline
0 mM L-Proline
200 mM L-Proline
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Trypomastigote bursting x L-Proline in different
intracellular stages
Epimastigote
Amastigote



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Trypomastigotes x 106 mL-1
-
-

-
0,2 mM L-Proline
- 0 mM L-Proline
Days Post infection
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Intracellular stages x L-Proline
Amastigotes
Intracellular epimastigotes
Trypomastigotes
Intracellular forms (106 mL-1)
L-Proline (mM)
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Transporte de L-Prolina
L-proline uptake x substrate concentration
System A
Vo (nmols / 20 x 106 cells min)
System B
L-Proline (mM)
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Proline uptake x T. cruzi mammalian host stages
Trypomastigotes
Amastigotes
Intracellular epimastigotes
Vm (nmols / 20 x 106 cells min)
L-Proline (mM)
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Intracellular concentration of free proline in
different stages
Pro (mM)



Intracellular Amastigote

6,61
0,01


Intracellular Epimastigote

0,73
0,01


Trypomastigote

2,74
0,01


Extracellular Epimastigote

6,76
0,04

CHO-K1


0,27
0,03


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Transporte de L-Prolina
Proline uptake in the different environment where
T. cruzi lives
Insect vector intestinal content
LIT 10 FCS
Vo (nmols / 20 x 106 cells min)
Human serum
LIT
CHO Cytoplasm
L-Proline (mM)
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D-Glucose uptake x life cycle stages of T. cruzi
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L-Proline, D-Glucose and the intracellular cycle
a model
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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.

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Identification, cloning and functional
characterization of amino acid transporters of
Trypanosoma cruzi.
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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.
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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.
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Algorithm used for the detection of genes coding
for putative amino acid transporters
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PATs identified in T. cruzi
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Alignment of PATs corresponding to group 1 as an
example
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Analysis of copy number per group and tandem
repeats
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Analysis in sillico of the presence of
trans-membrane spanners
Determinantion of the number of putative
trans-membrane helices
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Phenogram corresponding to the PATs herein
described as well as to other amino acid
transporters from other protozoans
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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.
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Conclusions (in brief)

• Aproximately 1.120.000 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.

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Acknowledgements
Dr. Maria Júlia Manso Alves Dr. Walter Colli
Dra. Silvia Uliana Dr. Claudio Pereira Lic. León
Bouvier
Dr. Renata Rosito Tonelli Marcela
Martinelli Camila Galvão Lopes
Support
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(No Transcript)
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Proline as carbon and energy source
Scheme of the intermediary metabolism in
trypanosomatids
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Modelos de transportadores
Uniport
Simport
Substrato
Ion
ATP
Substrato
Ion
ADP
Antiport
Uniport
Substrato
ATP
Substrato
Ion
ATP
ADP
Ion
ADP
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Proline uptake x two intracellular prolin
concentrations
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Vmax for proline uptake x cations (Na, K or
cholin).
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L-proline uptake x Temperature
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Inhibition of proline uptake by competitors
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Proline uptake x pH
System A
System B
Vm (nmols / 20 x 106 cells min)
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Proline uptake x Ionophors and other
mitochondrial inhibitors
System A
System B
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Northern Blot x life cycle stages of T. cruzi
Seqüência da região codificante de transportador
de glicose de T. cruzi 1
atgccatcca agaagcagac tgatgtgagt gttggggaca
ggcagcccga cgagactctc 61 acattttgct
cgttggagaa cctgaaggtt gcacaagtgc aggtggttgg
tggaacactg 121 aacggattct caattggctt
tgttgccgtg tatgcttatt tctacctgat gtccacggac
181 tgctcgatgt acaagaagga ggtggcgtgc aacagggtat
tgaacgcgga gtgttcttgg 241 aacaaaacac
gtggagaatg cggctggaac ggctttacct gctttttggg
gcacggtaag 301 gataagacgc catgtttgga
tgatagcagg tgcaagtggg tgtacagcga cgaagagtgc
361 aagaatccga ctgggtacag ctcgtcctat aacggcatct
ttgctggtgc gatgattgtt 421 ggcgcaatga
ttgggtcgat ctatgccggg cagtttgccg cgaggtttgg
tcacaaggtg 481 tcgttcctga tcgtcggcat
cgttggcgtt gtgtcatccg tgatgtacca tgtgtcctcg
541 gcaacgaatg agttttgggt gctgtgcgtt ggtcgtctac
tgataggtgt tgtgctcggt 601 cttgtgaacg
ttgcatgccc catgtatgtc gaccagaacg cccacccgaa
gttccttcac 661 gtggacggtg ttctgtttca
ggtgttcacc acgtttggca ttatgtttgc tgcagcgatg
721 gggttggcta ttgggcaaag cgtcaacttt gacaaggaca
tcaaaatgga tgcccgcatg 781 cagggctact
gtgccttttc tacgctgttg tcggtgctca tggttgcgct
tggtatcttc 841 ttgggcgaga gcaagacgaa
gtttacgagc ggcaagcacg aggacgatgg cactgcgctg
901 gacccgaacg agtacagcta cttgcagatg cttggacctt
tggcgatggg actagtgact 961 tccgggacgc
tgcaactgac tggcatcaat gccgtgatga attacgcgcc
aaagattatg 1021 ggcaacttgg ggatggtgcc
tcttgtgggc aacttcgtgg tgatggcgtg gaactttgtg
1081 acaactctcg tctcgattcc acttgcccgg gtcctcacaa
tgcgccagct gtttcttggt 1141 gcctcgcttg
tggcgtcggt ctcgtgtctg ctcctgtgcg gggtccctgt
gtaccccggc 1201 gttgccgata agaacgtgaa
gaatggcgtt gcgatcactg gaattgccgt attcatcgcc
1261 gcgtttgaga ttggccttgg accgtgcttc tttgtgcttg
cccaggagct gttcccacgc 1321 tctttccgtc
cgaggggttc gtccttcgtg ctcttgacga atttcatctt
taatgttatc 1381 atcaacgtct gctacccaat
cgcgacggag ggcatctctg gcggcccgtc tggcaaccag
1441 gacaagggtc aggcagtcgc gttcatcttt tttggctgca
ttggtcttgt ctgcttcgtt 1501 ctgcaggtgt
tcttcctgta cccgtgggag gagagcactc ctcagaacca
cggagacacc 1561 aacgaagagt ccgcacttcc
agaacggcag tcgccgattg aggttgccac ccctggcaac
1621 cgtcaagccg cgtga
Northern Blot a epimastigota b
tripomastigota c amastigota
a
b
c
2.7 kpb -
2.1 kpb -
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Algoritmo proposto para a identificação e
caracterização de PATs
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