Translational regulation in Plasmodium falciparum

Sammanfattning: Plasmodium falciparum is the causative agent of the most malignant form of human malaria, which remains as one of the most devastating infectious diseases. In face of a continuous international effort to eliminate the disease, the parasite not only has evaded a total obliteration, but has now evolved resistance to many of the available drugs. Next generation rational drug design is in urgent need and the key of such will lie on the successful identification of the parasite’s ‘Achilles heel’. While many existing and outstanding drugs have shown the promises of targeting the parasite translation machinery, the translation dynamics as well as the translational regulatory mechanisms are poorly understood. The studies described in this thesis aim to further our understanding on the translational regulation in P. falciparum, at both the global and gene-specific levels. Pregnancy associated malaria (PAM) is commonly seen with excessive sequestration of infected red blood cells in the placenta, the phenomenon is widely considered as the result of the specific ligand-receptor binding between the parasite derived PfEMP1- VAR2CSA proteins and the CSA proteoglycans. Translation of VAR2CSA protein is repressed by an upstream open reading frame, and a predicted trans factor is required for de-repression of var2csa translation. By using a spontaneously derived mutant that fails to efficiently translation the V AR2CSA proteins, we identified PTEF (Plasmodium translation enhancing factor) as the putative trans acting factor that allows efficient VAR2CSA translation. PTEF binds to the ribosomes and can enhance translation in a E. coli system. Importantly, higher PTEF expression was invariably observed to be associated with PAM in previous studies. Furthermore, PTEF function requires the processing by a calpain protease, blockage of the processing abolishes PTEF function in a reporter assay. Our data strongly suggest PTEF is an important regulator of PAM and raises potential therapeutic opportunity. It has been well described that codon usage bias could have a profound effect on translation efficiency. Codon usage is extremely biased in P. falciparum and cumulated to frequent insertions of asparagine homorepeats in up to one fourth of the proteome. However, the biological effect of this codon usage bias has not been studied. By using rationally recodonized GFP sequences, we showed that the increased use of GU wobble codon could reduce translation efficiency. We also demonstrated that the GU wobble- rich codon context underlying the asparagine homorepeats could impart significant influence on the translational output and transcript stability of the host gene. Despite this, GU wobble codons are overrepresented in the genome. Bioinformatics analyses suggested the high content of GU wobble codon might serve as a global regulatory mechanism. We thus offered new insight on the genome evolution of the parasite. RIFIN is the largest variable surface antigen family in P. falciparum. Its research profile has been much uplifted recently, as report showed that it might have a crucial link with severe malaria. While there is a sufficient interest to investigate the regulatory mechanisms associated with the RIFIN family, functional study of RIFIN is often marred by the lack of robustly verified reagents. By using RNA-sequencing and ultra- dense peptide microarray, we were able to authenticate specific RIFIN antibodies that exhibit some degree of intra-family cross-reactivity but minimal non-specific reactivity with other antigens. The derivation of these reagents will be important for future studies.

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