Functional genomics through transposon-mediated mutagenesis to unravel virulence gene regulation in Plasmodium falciparum – PlasmoPiggyBac

Mutant library construction
Transposon-mediated mutagenesis has been used in various genetic analyses performed extensively in Drosophila and numerous mammalian cell lines that have led to a better understanding of complex gene expression in these organisms. piggyBac insertions into the P. falciparum genome are being obtained by co-transfection of parasite erythrocytic stages with a transposon plasmid and a transposase-expressing helper plasmid. The transposon plasmid carries a drug-resistance cassette (hdhfr) flanked by the ITR1 and ITR2 sequences of the piggyBac element. The ITRs are oriented such that upon transposition, they will carry the drug-resistant cassette into the Plasmodium genome without any of the plasmid backbone. Following transfection with piggyBac plasmids, drug resistant parasite populations are being established and parasites clones are being obtained by limiting dilution .
Genetic screen for factors involved in var gene activation, repression and switching
Concerning the screens, we have conceived an assay that allows us to easily identify mutants affected in var gene regulation. We are performing the forward functional genomic approach in a transgenic parasite line in which a var promoter drives expression of a drug-selectable marker. The versatility of the piggyBac system allows us to transform any strain, including these genetic modified parasites.
Identification and study of candidate factors.
The most promising candidates will be chosen for a deeper analysis. Genomic distribution, cellular location, association with noncoding RNA and other proteins will be studied for the encoded proteins. The methods and skills needed are well established in our group.

A high efficient transfection technology is essential for the success of the project. Although this technology is well established in our lab, we tried to improve it using the protocol learnt during my visit to Dr. Adam´s laboratory (which developed the piggyBac transposon mutagenesis) and other alternative protocols. We have generated around 100 mutant clones and currently we are performing the screening to identify those affected in var gene regulation.
The main goal of this project is to identify factors involved in var gene regulation. To simplify the screening, we have performed the mutant library in a transgenic parasite line in which a var promoter drives expression of a drug-selectable marker. In order to make even easier the screening, we are generating a parasite line in which two different var promoters drive expression of two drug-selectable markers. Since intact var gene regulation implies that only one var gene is expressed at a time, mutant clones able to growth in the presence of both drugs have a misregulated monoallelic var gene expression. Therefore, this improvement provides an efficient and straightforward method (just growing in the presence of both drugs) to identify the desired mutants.

1) We have got a high-efficiency transfection but we believe that we can still improve it so we are planning to incorporate some new few changes in the technique.
2) We met Dr. Marcelo Jacobs-Lorena, a malaria investigator at Johns Hopkings Bloomberg School of Public Health in Baltimore. We benefited from his experience with the piggyBac transposon mutagenesis since he has already successful applied this approach to indentify genes involved in gametocytes formation in malaria parasites. piggyBac insertions into the P. falciparum genome are obtained by co-transfection with a transposon plasmid and a transposase expression helper plasmid . Dr. Jacobs-Lorena has kindly given us improved versions of the plasmids. We are going to use these plasmids for next transfections since allow us to generate not only gene-disruption mutagenesis but also gain of function mutants and other useful features for the screening and the identification of insertion sites.
3) Once we have identified the genes in which a loss-of-function or gain-of-function mutation gives a misregulation of antigenic variation, we are going to characterize them and to investigate their exact role in the regulation of the var gene family. The most appropriate set of experiments will be different in each case, but there are several strategies that we can advance. Chromatin structure and nuclear organization of the var gene family are paramount players in var gene regulation. Hence, we are going to analyze both aspects in the mutant clones to investigate for disparities with wild type parasites. Chromatin immunoprecipitation studies will give us the enrichments in particular histone modifications associated with the var genes and fluorescence in situ hybridizations, the nuclear organization of var genes

After 6 months, we have not published anything.

Pathogens have evolved countermeasures to avoid immune clearance and prolong the period of infection in their vertebrate hosts. The type and degree of immune escape strategies depends on the in vivo ‘lifestyle’ the pathogen has adopted. Protozoan parasites use different strategies to coordinate their expression of phenotypic variation, which is used in many cases to fool the immune system, or to successfully invade new host cells. Recent insights using modern molecular biology techniques show that this is achieved via a coordinated manner of action of different genetic and/or epigenetic factors.

The protozoan pathogen Plasmodium falciparum, which infects up to 300 million people causing more than two million lives each year, undergoes antigenic variation to establish persistent blood stage infection. Several clonally variant gene families undergo antigenic variation in P. falciparum and are expressed during blood stage infection at the surface of infected erythrocytes. One family encoded by 60 var genes expresses the major virulence adhesion surface molecule causing severe malaria (capillary blockages in the brain and other organs mediated by infected erythrocytes). Switching expression between the 60-member var gene families avoids parasite immune clearance and prolongs the period of infection and transmission to the mosquito.

The first event that initiates mutually exclusive expression of any clonally variant gene family remains elusive and represents the Holy Grail in the field of antigenic variation of pathogens as well as in other research areas. Transcription of a single var gene occurs at a unique perinuclear expression site. To this end, this proposal aims to identify factors associated with mono allelic expression (proteins, DNA enhancer element or ncRNA) by using an innovative strategy. To achieve this ambitious goal, we will create a transposon-mediated mutant library in a transgenic parasite line that allows us to efficiently identify mutants affected in virulence gene regulation. Identified candidate molecules will be validated using state-of-the-art methods for malaria parasites, many developed in our laboratory, to investigate nuclear mechanisms that control epigenetic regulation of antigenic variation. We expect using an empirical approach to obtain new exciting insights into the underlying principles of ‘gene counting’ of virulence gene families and other important molecular aspects of antigenic variation. The successful application of the above approach will open avenues in the field of gene regulation in malaria parasites. The project is likely to reveal an Achilles Heel of the parasite’s immune evasion strategy, which could be targeted for future drug intervention strategies.