crisprcas9-gene-editing-to-make-conditional-mutants-human-malaria

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite <em>P. falciparum</em>

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

University of Georgia

Export of parasite proteins into the host erythrocyte is essential for survival of  during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by  in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70,  Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most  species and is found only in  and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in  to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the  ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of  to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor  erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of .  Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of  spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, . An outstanding question in the field is how  undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the  species causing human disease, the chaperone PfHsp70x is unique to , and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784-1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.

The Exported Chaperone PfHsp70x Is Dispensable for the  Intraerythrocytic Life Cycle.

The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum.

Maintenance of tissue integrity during development and homeostasis requires the precise coordination of several cell-based processes, including cell death. In animals, the majority of such cell death occurs by apoptosis, a process mediated by caspase proteases. To elucidate the role of caspases in tissue integrity, we investigated the behavior of Drosophila epithelial cells that are severely compromised for caspase activity. We show that these cells acquire migratory and invasive capacities, either within 1-2 days following irradiation or spontaneously during development. Importantly, low levels of effector caspase activity, which are far below the threshold required to induce apoptosis, can potently inhibit this process, as well as a distinct, developmental paradigm of primordial germ cell migration. These findings may have implications for radiation therapy in cancer treatment. Furthermore, given the presence of caspases throughout metazoa, our results could imply that preventing unwanted cell migration constitutes an ancient non-apoptotic function of these proteases.

Anat Florentin

Department of Cellular Biology,
Center for Tropical and Emerging Global Diseases

University of Georgia

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Anat Florentin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cellular Biology,Center for Tropical and Emerging Global Diseases

University of Georgia

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Department of Cellular Biology,
Center for Tropical and Emerging Global Diseases