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

Asparagine repeats in Plasmodium falciparum proteins: good for nothing?

To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell. 

PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes.

Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis. 

The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

Malaria is a common and life-threatening disease endemic in large parts of the world. The emergence of antimalarial drug resistance is threatening disease-control measures that depend heavily on treatment of clinical malaria. The intracellular malaria parasite is particularly vulnerable during its brief extracellular stage of the life cycle. Wilson et al. describe a screen targeting these extracellular parasite stages and make the surprising discovery that clinically used macrolide antibiotics are potent inhibitors of parasite invasion into erythrocytes.See research article: http://www.biomedcentral.com/1741-7007/13/52. 

Teaching old drugs new tricks to stop malaria invasion in its tracks.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC) of 0.11 ± 0.02 μM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC of 0.6 ± 0.01 μM), the agent of malaria, and Cryptosporidium parvum (EC of ∼65 μM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

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.

Article	Year
Asparagine repeats in Plasmodium falciparum proteins: good for nothing? PLoS pathogens\| PubMed ID: 23990777	2013
PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes. Nature\| PubMed ID: 25043010	2014
The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism. PloS one\| PubMed ID: 26010880	2015
Teaching old drugs new tricks to stop malaria invasion in its tracks. BMC biology\| PubMed ID: 26349580	2015
In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites. Antimicrobial agents and chemotherapy\| PubMed ID: 27895021	2017
The Exported Chaperone PfHsp70x Is Dispensable for the Intraerythrocytic Life Cycle. mSphere\| PubMed ID: 28959740	2017
PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum. Cell reports\| PubMed ID: 29141210	2017

Vasant Muralidharan

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