Reverse genetics reveals new genes involved in host cell modification by the malaria parasite P. falciparum

Abstract text

Following invasion of human erythroyctes, the malaria parasite P. falciaprum massively changes the biochemical and biophysical properties of the host cell. This process directly and indirectly underpins the pathology associated with malaria infection, and is driven by a large number of proteins encoded by the parasite, and which are transported to the host cell. To understand to role specific exported proteins play in host cell modification, we initiated a medium-scale reverse genetic analysis of a number of genes of interest (GOI) we believed may be involved in this process. Over the course of 6 years we generated 76 genetically modified parasite lines using a number of techniques. In contrast to previous studies, we find that a large majority of the GOI (14/18) are resistant to both direct inactivation, or introduction of the GFP coding sequence. Use of a smaller tag, 3xHA, allowed us to generate 5 further parasite lines which also included a glmS ribozyme to allow modulation of protein levels. Additionally, we obtained 2 knockout lines. A detailed analysis of three parasite lines revealed involvement of the GOI/POI in a number of host cell modification processes and uncovered novel mutant phenotypes never before reported. Further analysis of one specific parasite line lacking an exported J-domain protein highlighted a potential role for residual human HSP70 proteins in host cell modification processes, the first time this has been experimentally demonstrated. Taken together, our data highlight the importance of exported proteins for parasite growth, survival and pathology in the human host and host cell, and underpin the complex nature of the host parasite interaction at a molecular level.