Characterisation of the role of epigenetic variation in the adaptation of malaria parasites to changes in the conditions of the human host

Abstract

Malaria is a vector-borne disease transmitted by female Anopheles mosquitoes and caused by Plasmodium parasites. The deadliest species, Plasmodium falciparum, is responsible for over 200 million cases and almost half a million deaths per year. These parasites possess a complex life cycle that takes place between the human and mosquito hosts and involves many different stages that develop within different tissues and cells. As a result, malaria parasites are exposed to a wide variety of environments which has led them to develop a huge adaptive capacity. An example of this is the fact that Plasmodium parasites have developed resistance against nearly all known antimalarial drugs, constituting a major challenge for malaria control and eradication programs.

In general, slow, long-term adaptation involves irreversible changes at the genetic level. However, within the human host circulation malaria parasites are exposed to fluctuating conditions that can vary both within the same or between different hosts. Adaptation to these fluctuations requires a speed and reversibility that can only be provided by changes at the transcriptional and posttranscriptional levels. Consequently, in addition to directed transcriptional responses, malaria parasites present a characteristic adaptive mechanism that revolves around the spontaneous generation of transcriptional diversity by the means of clonally variant gene expression. Clonally variant genes (CVGs) are located in facultative heterochromatin and are regulated at the epigenetic level so that they can stochastically switch between transcriptionally active or silent states. In this manner, within a genetically homogenous parasite population, different individual parasites can present different active or silent CVGs resulting in transcriptional and phenotypic diversity. Upon an environmental change, natural selection can act upon this pre-existing populational diversity and select those subpopulations of parasites with the most fit CVG expression patterns under the new set of conditions. This constitutes the basis of a bet-hedging adaptive strategy as the transcriptional diversity that precedes the selective pressure is what enables the population to survive.

CVGs participate in many host-parasite interactions and have been found to play an important role in two bet-hedging adaptive strategies: immune evasion through antigenic variation mediated by var genes and resistance to toxic compounds due to changes in the permeability of the membrane of infected red blood cells mediated by clag3 genes. In this thesis, we have further characterised the role of CVGs in the adaptation of P. falciparum to the fluctuating conditions of the human host circulation and their involvement in bet-hedging adaptive strategies. Firstly, we have continued with the study of the adaptive role of clag3 genes by identifying new antimalarial compounds that are susceptible to the resistance

mechanism mediated by changes in the expression patterns of these genes and also by generating a triple transgenic parasite line that can be used to analyse the expression dynamics of clag3 genes under different conditions. Secondly, we have determined how malaria parasites use their CVGs at the onset of a blood infection in a new human host and how the epigenetic memory behind the expression patterns of many of these genes is lost and re-established during the passage of parasites through transmission stages. Finally, we have found a new adaptation mechanism that enables malaria parasites to adapt to fluctuations in the availability of lipids in the culture medium simulating those they may encounter in the human circulation. This mechanism involves the loss of functional PfNDH2, a protein that forms part of the mitochondrial electron transport chain, suggesting at a possible link between the mitochondria and lipid metabolism under these conditions. In summary, we have further demonstrated the adaptive potential of CVGs as well as the huge adaptive plasticity of P. falciparum.

Los parásitos de la malaria presentan una gran plasticidad adaptativa que puede estar mediada a múltiples niveles. Para hacer frente a las presiones ambientales altamente fluctuantes en el hospedador humano, estos organismos poseen un mecanismo que permite adaptación rápida, reversible, a nivel transcripcional que está mediada por los llamados genes con expresión clonal variante. Estos genes regulados a nivel epigenético se caracterizan por presentar cambios estocásticos entre el estado transcripcional activo y silenciado. Esto constituye una fuente de diversidad transcripcional a nivel poblacional ya que distintos parásitos pueden presentar distintos genes con expresión clonal variante activos y silenciados. Cuando se produce un cambio en el ambiente, la selección natural puede actuar sobre esta diversidad preexistente y seleccionar aquellos individuos con el patrón de expresión de estos genes mas ventajoso bajo las nuevas condiciones. Esto representa un caso de lo que se conoce como estrategias adaptativas de tipo “bet-hedging” (estrategia de diversificación de la apuesta).

En esta tesis, hemos continuado con la caracterización del rol de los genes con expresión clonal variante en la adaptación de Plasmodium falciparum (la especie responsable de los casos mas severos de malaria) a las condiciones fluctuantes de la circulación del hospedador humano y su participación en las estrategias de adaptación de tipo “bet-hedging”. De esta manera, hemos seguido con el estudio del papel adaptativo de los genes clag3, identificando nuevos fármacos antimaláricos a los cuales el parasito puede desarrollar resistencia mediante cambios en la expresión de estos genes. También, hemos caracterizado los patrones de expresión de los genes con expresión clonal variante al comienzo de una infección en sangre en un nuevo hospedador humano. Esto nos ha permitido establecer que la memoria epigenética para la expresión de muchas familias génicas con expresión clonal variante se borra durante los estadios de transmisión. Finalmente, hemos descubierto un nuevo mecanismo de adaptación que permite a los parásitos de la malaria adaptarse a la falta de lípidos en el medio de cultivo simulando las fluctuaciones de este nutriente en la circulación humana. En definitiva, hemos afianzado la increíble plasticidad adaptativa de estos organismos.