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Abstract

The malaria parasite Plasmodium falciparum has a unique, complex life cycle and exports a variety of proteins into the erythrocyte to alter the physiological properties of its host cell. These modifications are crucial for survival and render the parasite a master of immune evasion, which is one of the main reasons why there has not been developed an efficient vaccine to eradicate this devastating disease. Many exported proteins are thought to play a crucial role in protein export and host cell modifications. However, scientific breakthroughs in this field were hampered due to a lack of efficient genetic systems for the blood stages of the parasite. Over the last years, several new genetic tools were established for P. falciparum that allow the investigation of essential genes. In this study, four predicted essential proteins were analyzed using the glmS ribozyme system, an inducible knockdown method on mRNA level that is activated by glucosamine-6-phosphate. Coupled with selection-linked integration (SLI), it allows faster generation of transgenic cell lines than conventional subcloning methods. The glmS system worked efficiently in all cell lines studied with significant downregulation of the proteins of interest with 2.5 mM glucosamine. Concerning the development and morphology of the parasites, there was no growth defect observed via microscopy or SybrGreen-based growth assays. PF3D7_0220300 and PF3D7_0220600 are predicted to be membrane-bound proteins partly co-localizing with the Maurer’s clefts marker SBP1, and the erythrocyte membrane, respectively. PF3D7_0113300 and PF3D7_0301600 are predicted to be soluble proteins with the second partly co-localizing with SBP1. Using an in vitro static cytoadherence assay, I could show that binding to chondroitin sulfate A was significantly reduced in all cell lines except CS2PF3D7_0113300glmS with the strongest effect observed upon downregulation of PF3D7_0301600 and PF3D7_0220300. Quantification of the mean fluorescence intensities of PfEMP1 VAR2CSA via flow cytometry only showed significantly less signal after downregulation of PF3D7_0220300, further demonstrating the importance of the studied exported proteins in host cell modification, specifically cytoadherence and presentation of PfEMP1 on knobs. These results should be further investigated to get a more comprehensive understanding of the parasites’ biology. Moreover, inducible knockout systems using a dimerizable Cre recombinase could circumvent some disadvantages of the glmS system resulting from remaining protein levels or the cytotoxicity of glucosamine and should be considered in further studies.