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Active transport of L-proline in the protozoan parasite Trypanosoma brucei brucei.

ABSTRACT: The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.

SUBMITTER: L'Hostis C

PROVIDER: S-EPMC1132516 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Project description:Drug resistance in pathogenic protozoa is very often caused by changes to the 'transportome' of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil(®)), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the "selectivity region" of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability.

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Active transport of L-proline in the protozoan parasite Trypanosoma brucei brucei.

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