Proteosome 26s in Plasmodium falciparum, implication towards physiology and drug resistance

Malaria remains one of the leading causes of mortality worldwide, especially in tropical regions. This disease is caused by a protozoan parasite of the Plasmodium genus, with P. falciparum being the most virulent species capable of infecting humans due to its ability of cytoadherence, obstructing perfusion and, consequently, evading immune system clearance. Multiple therapies have been used over the years, with artemisinin-based combination therapies (ACTs) being the most widely used today. Activated artemisinin presumably generates cellular damage by reacting with the susceptible groups of biomolecules. These are posteriorly marked with ubiquitin to be recognized and degraded by the 26S proteasome. However, artemisinin resistance has emerged, and it is necessary to understand its mechanisms not to allow its spread while it is very located. A partner group laboratory found a single nucleotide variant (SNV), causing a non-synonymous mutation, the E738K, in the rpn2 gene, on ACT resistant P. chabaudi parasites. Therefore, this project aimed to evaluate the impact of the E378K variant, located in the rpn2 gene, a subunit of the 19S regulatory particle of the 26S proteasome, on the proteasome function in the parasite life cycle and in the artemisinin response. Two plasmids were constructed bearing the E738 and 738K variants, using parts of the P. falciparum rpn2 gene and the part of the P. chabaudi rpn2 gene where the variant is located. These chimeric genes were created due to a 78.6% homology between the proteins of both species and the impossibility to culture P. chabaudi parasite in vitro. Drug susceptibility assays showed that the 26S738K parasites presented a higher IC50 and higher parasite survival in the ring-stage survival assay (RSA), compared to the 26SE738 parasites, in the artemisinin response. Additionally, they presented a stabilization in the proteasome activity when challenged to dihydroartemisinin (DHA) and consequent reduction of polyubiquitinated proteins accumulation. On the contrary, E738 parasites showed a decrease in the proteasome activity and increased polyubiquitinated proteins upon reaction with DHA. These results suggest that the 738K variant confer DHA resistance to the parasites and that the proteasome is involved in this DHA resistance. Moreover, it demonstrates that the ubiquitin-proteasome pathway plays an important role in the DHA mechanism of action.

Basic Science
Drug Resistance

Apr 2023 — Mar 2027

Portugal