Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential ...proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field's understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.
The difficulty of faithfully recapitulating malarial protein complexes in heterologous expression systems has long impeded structural study for much of the Plasmodium falciparum proteome. However, ...recent advances in single-particle cryo electron microscopy (cryoEM) now enable structure determination at atomic resolution with significantly reduced requirements for both sample quantity and purity. Combined with recent developments in gene editing, these advances open the door to structure determination and structural proteomics of macromolecular complexes enriched directly from P. falciparum parasites. Furthermore, the combination of cryoEM with the rapidly emerging use of in situ cryo electron tomography (cryoET) to directly visualize ultrastructures and protein complexes in the native cellular context will yield exciting new insights into the molecular machinery underpinning malaria parasite biology and pathogenesis.
With half the world’s population currently at risk, malaria remains a significant global health burden.The difficulty of expressing many malarial protein complexes in heterologous systems has precluded structural and biochemical studies, impeding efforts to elucidate the functions and molecular mechanisms of many important but poorly understood biological pathways, including potential therapeutic targets.Recent and ongoing advances in structure determination of macromolecular complexes using cryo electron microscopy (cryoEM) provide new avenues for structural study of the P. falciparum proteome, much of which previously resisted structure determination.CryoEM of endogenously derived macromolecular complexes, enabled by the significantly reduced sample requirements of cryoEM studies, will lead to the discovery of so far unknown native substrates, binding partners, and modifications.
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.
The ability of eukaryotic parasites from the phylum Apicomplexa to cause devastating diseases is predicated upon their ability to maintain faithful and precise protein trafficking mechanisms. Their ...parasitic life cycle depends on the trafficking of effector proteins to the infected host cell, transport of proteins to several critical organelles required for survival, as well as transport of parasite and host proteins to the digestive organelles to generate the building blocks for parasite growth. Several recent studies have shed light on the molecular mechanisms parasites utilise to transform the infected host cells, transport proteins to essential metabolic organelles and for biogenesis of organelles required for continuation of their life cycle. Here, we review key pathways of protein transport originating and branching from the endoplasmic reticulum, focusing on the essential roles of chaperones in these processes. Further, we highlight key gaps in our knowledge that prevents us from building a holistic view of protein trafficking in these deadly human pathogens.
Objective: Comorbidity between posttraumatic stress disorder (PTSD) and substance use disorders (SUD) is common, and both are associated with cognitive dysfunction. However, few studies examine the ...impact of cognitive deficits on treatment outcomes. Here, we leverage data from a randomized clinical trial of integrated versus phased psychotherapy for SUD and PTSD to examine the relation of cognitive functioning to treatment response. Method: One-hundred and thirteen veterans with co-occurring PTSD and SUD completed Penn Computerized Neurocognitive Battery tests assessing attention, executive control, memory, and spatial processing. Linear mixed-effects models examined interactions between cognitive functioning and time in predicting primary PTSD and SUD outcomes across both treatments. Results: Significant verbal immediate memory by time interactions were found for both PTSD symptoms (p = .01, f
2 = 0.020) and percent heavy drinking or drug use days (p = .004, f
2 = 0.020). There was a significant working memory by time interaction for percent heavy drinking or drug use days (p = .007, f
2 = 0.016). Participants with better verbal memory had greater reductions across time in PTSD symptoms and drinking/drug use, while those with better working memory had lesser reductions in their drinking/drug use across time. Conclusions: Individuals with lower verbal memory functioning had less robust PTSD and SUD symptom reductions in PTSD/SUD psychotherapy, with differences that were generally small in magnitude. Those with better working memory functioning had worse SUD outcomes. Together with prior literature, findings suggest that neurocognitive functioning may impact the effectiveness of PTSD and SUD treatment.
What is the public health significance of this article?
This study suggests that psychotherapy for veterans with PTSD and substance use disorders may be less effective in individuals with lower verbal memory functioning.
The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related ...organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the
ndoplasmic
eticulum-resident
alcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of
Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.
The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.
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.
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•PfClpC and PfClpP are Clp orthologs in the apicoplast of Plasmodium falciparum•Conditional inhibition of the PfClpC results in growth arrest and apicoplast loss•Addition of apicoplast-derived metabolite IPP rescues the growth phenotype•PfClpC chaperone activity is required to stabilize the PfClpP protease
Plasmodium falciparum contains a unique organelle, the apicoplast. Using genetic and phenotypic assays, Florentin et al. characterize the apicoplast Clp chaperone and protease. They find that the chaperone is essential for protease stability and that together they function to maintain organelle integrity and segregation into daughter cells.
The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of ...the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterised ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localises to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.
The literature on non-small cell lung cancer (NSCLC) brain metastases (BMs) managed using stereotactic radiosurgery (SRS) relies mainly on single-institution studies or randomized controlled trials ...(RCTs). There is a literature gap on clinical and radiological outcomes of SRS for NSCLC metastases in real-world practice. The objective of this study was to benchmark mortality and progression outcomes in patients undergoing SRS for NSCLC BMs and identify risk factors for these outcomes using a national quality registry.
The SRS Registry of the NeuroPoint Alliance was used for this study. This registry included patients from 16 enrolling sites who underwent SRS from 2017 to 2022. Data are prospectively collected without a prespecified research purpose. The main outcomes of this analysis were overall survival (OS), out-of-field recurrence, local progression, and intracranial progression. All time-to-event investigations included Kaplan-Meier analyses and multivariable Cox regressions.
Two hundred sixty-four patients were identified, with a mean age of 66.7 years and a female proportion of 48.5%. Most patients (84.5%) had a Karnofsky Performance Status (KPS) score of 80-100, and the mean baseline EQ-5D score was 0.539 quality-adjusted life years. A single lesion was present in 53.4% of the patients, and 29.1% of patients had 3 or more lesions. The median OS was 28.1 months, and independent predictors of mortality included no control of primary tumor (hazard ratio HR 2.1), KPS of 80 (HR 2.4) or lower (HR 2.4), coronary artery disease (HR 2.8), and 5 or more lesions present at the time of SRS treatment (HR 2.3). The median out-of-field progression-free survival (PFS) was 24.8 months, and the median local PFS was unreached. Intralesional hemorrhage was an independent risk factor of local progression, with an HR of 6.0. The median intracranial PFS was 14.0 months and was predicted by the number of lesions at the time of SRS (3-4 lesions, HR 2.2; 5-14 lesions, HR 2.5).
In this real-world prospective study, the authors used a national quality registry and found favorable OS in patients with NSCLC BMs undergoing SRS compared with results from previously published RCTs. The intracranial PFS was mainly driven by the emergence of new lesions rather than local progression. A greater number of lesions at baseline was associated with out-of-field progression, while intralesional hemorrhage at baseline was associated with local progression.
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