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.
Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made ...against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity.Graphic abstract:Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.
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.
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.
Abstract only
Malaria is a deadly disease caused by the apicomplexan parasite
Plasmodium
.
Plasmodium
asexual replication occurs in the red blood cell (RBC) and is what causes clinical symptoms of ...disease. In the RBC,
Plasmodium
moves through 3 developmental stages ending with schizogony and creating 16–32 daughter merozoites. At the end of schizogony, merozoites egress the host cell by breaking out of two membranes, the parasitophorous vacuole membrane and the RBC membrane. Schizogony and successful egress allows for exponential
Plasmodium
replication and RBC infection. Our lab recently identified a B3GLCT‐like protein in the
Plasmodium
ER (PfB3ER) that interacts with an essential ER chaperone and whose expression profile is similar to that of proteins required for egress. B3GLCT‐like proteins are known to act as glucosyltransferases and work in concert with protein O‐fucosyltransferases (POFUT2) to modify thrombospondin‐like repeats in mammalian cells. Surprisingly, the
P. falciparum
POFUT2 has been shown to be non‐essential while PfB3ER is predicted to be essential in the asexual stages. Based on PfB3ER homology to the mammalian B3GLCT and its predicted expression profile, we hypothesize that the O‐glycosylation function of PfB3ER is required for efficient egress of
P
.
falciparum
from RBCs. To test this, we have employed CRISPR/Cas9 gene editing to HA‐tag PfB3ER and create conditional mutants utilizing the TetR‐DOZI‐aptamer system. We show that PfB3ER localizes to the ER, unlike previous published results that suggest that PfB3ER is exported to the host RBC, and that PfB3ER is primarily expressed during schizogony. Further, we show that PfB3ER is essential for the asexual replication of
P. falciparum.
Future studies will focus on the specific stage of the intraerythrocytic lifecycle that are affected by PfB3ER knockdown and test the requirement of its glycosyltransferase activity. Together, these data have uncovered an essential function for a putative glycosyltransferase in the intraerythrocytic lifecycle of
P. falciparum.
Support or Funding Information
R01 AI130139/AI/NIAID NIH HHS/United States
During their intraerythrocytic development, malaria parasites export hundreds of proteins to remodel their host cell. Nutrient acquisition, cytoadherence and antigenic variation are among the key ...virulence functions effected by this erythrocyte takeover. Proteins destined for export are synthesized in the endoplasmic reticulum (ER) and cleaved at a conserved (PEXEL) motif, which allows translocation into the host cell via an ATP-driven translocon called the PTEX complex. We report that plasmepsin V, an ER aspartic protease with distant homology to the mammalian processing enzyme BACE, recognizes the PEXEL motif and cleaves it at the correct site. This enzyme is essential for parasite viability and ER residence is essential for its function. We propose that plasmepsin V is the PEXEL protease and is an attractive enzyme for antimalarial drug development.
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.
One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected ...patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.
The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid ...proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone–protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.
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