A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites.

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.

Nfya-1 functions as a substrate of ERK-MAP kinase during Caenorhabditis elegans vulval development.

A common bridge between a linear cytoplasmic signal and broad nuclear regulation is the family of MAP kinases which can translocate to the nucleus upon activation by the cytoplasmic signal. One pathway which functions to activate the ERK family of MAP kinases is the Ras signaling pathway which functions at multiple times and locations during the development of Caenorhabditis elegans including the development of the excretory cell, germ cells, male tail, and vulva. It has been most extensively characterized during the development of the vulva which is formed from the vulval precursor cells (VPCs), a set of six equivalent, epithelial cells designated P3.p - P8.p. Although LIN-1 appears to be a primary target of ERK MAP kinase during vulval development, it is likely that other developmentally important molecules are also regulated by ERK-mediated phosphorylation. The identification of physiological substrates of MAP kinases has been aided by the identification of docking site domains in substrate proteins that contribute to high-affinity interactions with kinases. Our laboratory has identified the C. elegans protein, T08D10.1/NFYA-1, as a potential ERK MAP kinase substrate in this manner, and we have initiated a characterization of its role during Ras-mediated development. T08D10.1 possesses significant homology to the CCAAT-box DNA-binding domain of the vertebrate nuclear transcription factor-Y, alpha (NF-YA) family of proteins. NF-Y proteins act as part of a complex to regulate the transcription of a large number of genes, in particular, genes that function in the G1/S cell cycle transition. T08D10.1/NFYA-1 is predicted to code for a protein containing multiple potential phosphorylation sites for ERK MAP kinase and a D-domain docking site. We demonstrate through biochemical analysis of purified NFYA-1 protein that it can act in vitro as a high affinity substrate for activated ERK MAP kinase. Growth factor activation of the Ras pathway in a tissue culture system has negligible effect on the protein's transactivation potential, however, the DNA-binding activity of the protein is reduced after treatment with activated ERK-MAP kinase. We demonstrate through mutant analysis that nfya-1 acts to inhibit vulval development and functions downstream or in parallel to let-60/ras. Both the NF-Y complex and the Ras signaling pathway play a fundamental role in cell proliferation and oncogenesis and the connection between the two is an important insight into the mechanisms of cell fate specification and cellular response.

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

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

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

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum 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 P. falciparum 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, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum 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 glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum 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 P. falciparum 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 P. falciparum. IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium 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, P. falciparum. An outstanding question in the field is how P. falciparum 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 Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, 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.