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Event 

Title:
On the mechanism of chloroquine resistance in Plasmodium falciparum
When:
19.11.2010 - 19.11.2010 16.00 h - 16.30 h
Where:
Aula 7 (Ed. Fermi) -
Category:
Seminars

Description

On the mechanism of chloroquine resistance in Plasmodium falciparum 

Mauro Chinappi, Allegra Via, Paolo Marcatili, Anna Tramontano

The spread of chloroquine-resistant P.falciparum strain is a devastating health problem in most part of the world. In the last decades, due to its effectiveness and reasonable cost, chloroquine represented the best and widely used antimalarial drug; however, within a decade of its introduction, P. falciparum parasite resistance to chloroquine was initially observed in Southeast Asian and South America and subsequently dramatically spread to most of the malaria-endemic countries. In its erythrocyte stage, P. falciparum invades the red blood cells where it forms a lysosomal isolated acidic compartment known as the digestive vacuole (DV). The parasite grows by ingesting haemoglobin from erythrocyte cytosol and by depositing it in the DV, where haemoglobin is degraded and the toxic haem is incorporated into inert haemozoin cristal. Chloroquine is a diprotic weak base and, at physiological pH (~7.4), can be found in its un-protonated (CQ), mono-protonated (CQ+) and di-protonated (CQ++) forms. The uncharged chloroquine CQ is the only membrane permeable form of the molecule and it freely diffuses into the erythrocyte and the parasite cytoplasms, up to the DV where it is believed to interfere with heam detoxification. It has been observed that chloroquine sensitive (CQS) parasites accumulate much more chloroquine in the DV than chloroquine resistant (CQR) strains. The reduced chloroquine accumulation in CQR is associated to mutations in PfCRT (P. falciparum Chloroquine Resistance Transporter), a protein embedded in the vacuolar membrane.
Several experiment have been performed in order to understand the mechanism of PfCRT action. It has been experimentally shown that mutated PfCRT transports chloroquine outside the vacuole. Experimental results, however, can be explained by two alternative models for PfCRT [1]: (a) the channel model (i.e. a passive channel that enables charged chloroquine to leak out of the food vacuole down its electrochemical gradient) or (b) the carrier model (i.e. an active efflux carrier extruding chloroquine from the food vacuole). In this study we carried out a multilayered approach grounded both on a mathematical model for the chloroquine transport and accumulation into the infected erythrocyte and on a sequence and structure analysis of PfCRT, which allowed us to propose a three-dimensional model of the protein. As for the analitical model, combined hypotheses for chloroquine-heme binding inside the vacuole and PfCRT function (channel or carrier) were examined. The comparison of mathematical model predictions with experimental results allowed us to reduce the number of hypotheses. In particular, we were able to exclude that PfCRT transports unprotonated chloroquine (CQ) and we showed that experimental results could be interpreted both in term of channel and carrier model. Moreover our analysis strongly suggests that the form of chloroquine interacting with heme (or heme related) species inside the parasite vacuole is the unprotonated one. On the other hand, the computational analysis of the PfCRT sequence and its three-dimensional model allowed us to propose that the mutated protein acts as an active carrier of chloroquine. In particular, our study confirms that PfCRT is a member of the drug/metabolite transporter superfamily. The combination of these two rather diverse techniques has allowed us to narrow down significantly the number of plausible hypotheses and led us to conclude that the unprotonated form of chloroquine binds the heme or related species inside the vacuole and that the PfCRT mutated protein confers resistance by carrying either the mono or di-protonated chloroquine out of the vacuole.

Venue

Venue:
Aula 7 (Ed. Fermi)

Description

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