Role of gene dosage in the acquisition of antimony resistance in L.donovani: experimental evidences

L. donovani parasites resistant to sodium stibogloconate (SSG) have developed higher survival skills in comparison to the wild-type SSG-sensitive parasites. Previous genomic and metabolomic studies undertaken within MPU revealed that SSG-resistance is clearly a phenomenon involving multiple molecular adaptations likely occurring in cascade. We hypothesize that gene dosage plays a major role in it. Identification of the ‘driver’ adaptations (and distinction from the ‘passenger’ ones) requires experimental studies. The aim of this PhD is to assess the functional importance of gene dosage and define its impact on parasite phenotype, in particular drug resistance. To address this research question, we will apply two approaches. On one hand, we will experimentally induce resistance in two different life stages of L. donovani: axenic promastigotes and intracellular amastigotes. The parasites obtained will be compared to naturally resistant strains at genome and phenotype levels. This will allow (i) identifying if the same molecular adaptations occur as in natural populations, (ii) describing their order of occurrence and (iii) detecting if any other adaptations occurred. We will pay a particular attention to gene dosage and its correlation with drug resistance and metabolite levels. On the other hand, we will experimentally over-express the most relevant genes identified in previous work for testing their functional impact. We will focus on the genes present in two circular episomes found to be over-expressed in Nepalese field isolates that are clinically resistant to antimonials. One episome contains essential regulatory genes, MAPK1 and a secreted acid phosphatase (SAcP). This particular episome is always combined with a second episome, containing the H-Locus that is related to drug resistance in the close species L. infantum. These two episomes are always jointly present in Nepalese strains and their copy number is correlated. We are planning to modify these two episomes and to manipulate strains which do not contain these episomes. With these transfected parasite lines we will establish the complete phenotype (drug resistance, metacyclogenesis, resistance to apoptosis, host response and metabolome profile) and a genomic analysis. In order to see if MPK1 overexpression play a role on the phenotype we will complete this analysis by a comparative phosphor-proteomic analysis. We will also pay particular attention to SAcP and its role in the host disturbance.

Keywords:B780-tropical-medicine