Genetic Analysis of Plasmodium Falciparum (2022)

Genome sequencing of Plasmodium falciparum is almost complete but the functional analysis of the Plasmodium falciparum genome is restricted because of our limited ability to genetically manipulate this malaria parasite. Gene regulation is one of the most intriguing and enigmatic aspects of malaria parasite biology. The ability of the parasite to control differential gene expression is poorly understood and so far only a few Plasmodium promoters have been identified that share no significant homology with either themselves or with other eukaryotic promoters. <p> <em>maebl</em> is a paralogue of the ebl gene family in P. falciparum and is expressed at a different time point in the blood stages of the parasite as compared to the other well characterized members of the family, baebl and eba-175. Transcript analysis by northern blot hybridizations and Random amplification of cDNA ends (RACE) idenitifed a much longer 5' untranslated region for maebl when compared to baebl and eba-175, suggesting the possibility of post-transcriptional regulation for maebl. A single transcription start site was mapped for eba-175 and multiple transcription start sites were identified for baebl and maebl. In order to test the promoter activity of the 5' regions of baebl, eba- 175 and maebl, a modified Chloramphenicol acetyl transferase (CAT) reporter assay was developed for P. facliparum that is more accurate and user-friendly compared to the conventional CAT assays. The 5' regions of all three genes expressed CAT from episomes in P. falciparum blood stages confirming the presence of promoter elements in 5' regions. So far, only a few promoters are used for transgene expression in P. falciparum and our results show that the 5' regions of these three ebl genes could be used for the same. </p><p> MAEBL is expressed in the mid-trophozoite stages of blood-stage development and is located on the surface of merozoites just before the invasion of new host erythrocytes. To understand the role of MAEBL in erythrocyte invasion, maebl was disrupted in P. falciparum NF54 parasites by single homologous recombination. There was no apparent phenotypic effect of maebl disruption in these parasites. Erythrocyte invasion assays performed on different enzymatically-treated erythrocytes showed that maebl is not essential for blood-stage development and the disruptant clones had a similar invasion phenotype to that of wild-type parasites. It is possible that the function of MAEBL is complemented by another parasite protein hence masking the effect of maebl disruption. In the rodent malaria parasite, P. berghei, MAEBL has been shown to be essential for mosquito salivary gland invasion by malarial sporozoites. In future, we would like to confirm a similar role for MAEBL in P. falciparum using the maebl disruptant clones and also investigate the possible role for MAEBL in the sporozoite invasion of human liver cells. </p><p>Application of new technologies has produced a wealth of information in recent years about the genomes, proteomes, and other aspects of the basic composition of the malaria parasites. Many aspects of the parasites' biology can be inferred through these approaches and yet our ability to utilize this new information to reveal the complex biology of Plasmodium has been painstakingly slow due to the lack of robust and user-friendly molecular genetic tools. One of the most important genetic tools that are missing in Plasmodium is an efficient transposon-mediated mutagenesis system. piggyBac transposable element has been widely used in eukaryotic metazoa to perform various genetic analyses. We tested the ability of piggyBac transposition system to transform P. falciparum blood stages. We were able to transform P. falciparum blood stages with a helper-dependent piggyBac transposition system at considerably high transformation efficiency. Multiple integrations were obtained through out the genome of the parasite at TTAA target sites. Our results also confirmed the suitability of this system for large-scale genetic analysis of P. falciparum which will provide new insight into the complex genetic structure of the malaria parasite and greatly accelerate efforts to develop novel intervention strategies.</p>