A new mechanism of antimalarial drug resistance regulated at the epigenetic level

Abstract

Malaria is responsible of almost half a million deaths every year. Currently, campaigns for the control and elimination of malaria are implemented in malaria endemic areas. However, drug resistance is one of the major impediments to achieve malaria elimination. In this thesis we have investigated how P. falciparum parasites develop resistance to some toxic compounds by functional variation linked to epigenetic regulation of clag3 genes. These genes present clonally variant expression and determine the formation of the main channel for the transport of solutes at the membrane of the infected RBC: Plasmodium Surface Anion Channel (PSAC). Hence, we hypothesized that P. falciparum parasites can modify the permeability of the membrane to specific solutes by epigenetic regulation of clag3 genes expression; this way, parasites could develop resistance to antimalarial drugs. To test this hypothesis, we have investigated the role of switches in clag3 expression in the acquisition of resistance to the antibiotic BS, the dynamics of clag3 genes expression in human infections and we have tested drugs susceptible to failure by this drug resistance mechanism.

First, we show that BS pressure at low concentrations selected for parasites expressing clag3.1, whereas parasites exposed to higher concentrations of BS had repressed the expression of both clag3 genes. We did not find any mutation in the genome of these parasites that could explain the change in their phenotype. Thus, we concluded that parasites can develop resistance to toxic compounds through epigenetic regulation of clag3 genes. Then, we found that parasites collected from patients with uncomplicated malaria predominantly express one of the two paralogues, consistent with the property of mutually exclusive expression, previously described in lab-adapted parasite lines. Adaptation to culture conditions or selection with toxic compound results in isolate-dependent changes in clag3 expression, implying functional differences between the proteins encoded. We also observed that samples collected at day 9 post-infection in human experimental infections (when parasites had been in the peripheral blood for approximately one erythrocytic cycle) showed a mix of parasites expressing either clag3.1 or clag3.2, suggesting that the epigenetic memory of clag3 genes is reset during transmission stages. Finally, we tested whether other drugs, that are suspected to require facilitated transport to reach the cell, could be susceptible of failure by this drug resistance mechanism. We found that the antimalarial compounds T3 and T16 (bis-thiazolium salts) require the product of clag3 genes to enter the infected erythrocyte and that P. falciparum populations can develop resistance to these compounds by selection of parasites with dramatically reduced expression of both genes. The rest of the drugs that we tested might use alternative routes in which clag3 genes are not involved.

We have described for the first time an antimalarial drug resistance mechanism regulated at the epigenetic level in P. falciparum parasites. This phenomenon may be of relevance for parasite adaptation to the presence of toxic compounds in human blood, selecting rapidly those parasites that present the less permeable phenotype and developing drug resistance in a single infection.

Actualmente, la resistencia a los medicamentos antimaláricos es uno de los principales impedimentos para lograr la eliminación de la malaria. En esta tesis hemos investigado cómo los parásitos de P. falciparum desarrollan resistencia a algunos compuestos tóxicos por variación funcional relacionada con la regulación epigenética de los genes clag3 (clag3.1 y clag3.2), los cuales presentan expresión clonal variante y mutuamente exclusiva (en condiciones normales sólo uno de los dos genes está en estado activo). clag3 determinan la formación del canal principal para el transporte de solutos a través de la membrana del eritrocito infectado: PSAC.

En este trabajo, primero observamos que la aplicación de bajas concentraciones del antibiótico blasticidina en cultivos de P. falciparum resultó en la selección de parásitos que expresan clag3.1, mostrando una IC50 a este compuesto más elevada que aquellas líneas que expresan clag3.2. Por otro lado, parásitos expuestos a concentraciones más altas de blasticidina reprimieron la expresión de ambos clag3 y mostraron altos niveles de resistencia al fármaco. No encontramos ninguna mutación en el genoma de estos parásitos que explicase el cambio de fenotipo, sugiriendo que se trata de un mecanismo regulado a nivel epigenético. El estudio de clag3 en parásitos recolectados de pacientes con malaria no complicada mostró que P. falciparum en infecciones naturales expresa predominantemente uno de los dos parálogos: clag3.2, indicando que este patrón de expresión confiere una ventaja fenotípica en sangre humana. Por otro lado, el análisis de muestras recogidas de infecciones humanas experimentales determinó que la memoria epigenética de los genes clag3 se restablece durante las etapas de transmisión, seleccionándose en pocos ciclos aquellos parásitos que presentan el patrón de expresión más favorable en sangre humana: clag3.2. Finalmente, probamos si otros fármacos que requieren transporte facilitado para llegar a la célula podrían ser susceptibles de fracaso terapéutico a través de este mecanismo de resistencia. Hayamos que los compuestos antipalúdicos T3 y T16 (sales de bis-tiazolio) requieren el producto de los genes clag3 para ingresar en el eritrocito infectado y que poblaciones de P. falciparum puedan desarrollar resistencia a estos compuestos mediante la selección de parásitos con expresión reducida de ambos genes.