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Journal of Cell Science Oct 2023The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including...
The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.
Topics: Animals; Humans; Biotinylation; Erythrocytes; Malaria; Plasmodium falciparum; Porins; Protein Transport; Protozoan Proteins
PubMed: 37772444
DOI: 10.1242/jcs.260506 -
Nature Communications Jul 2023Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids....
Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment.
Topics: Animals; Humans; Glutamate-Ammonia Ligase; Asparagine; Amino Acids; Glutamine; Plasmodium falciparum; Artemisinins; Parasites
PubMed: 37452051
DOI: 10.1038/s41467-023-39670-4 -
PLoS Pathogens Jul 2023A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the...
A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.
Topics: Animals; Plasmodium falciparum; Vacuoles; Protein Transport; Protozoan Proteins; Erythrocytes; Parasites; Peptide Hydrolases
PubMed: 37523385
DOI: 10.1371/journal.ppat.1011006 -
MBio Jun 2021Artemisinin and its semisynthetic derivatives (ART) are fast acting, potent antimalarials; however, their use in malaria treatment is frequently confounded by...
Artemisinin and its semisynthetic derivatives (ART) are fast acting, potent antimalarials; however, their use in malaria treatment is frequently confounded by recrudescences from bloodstream parasites that enter into and later reactivate from a dormant persister state. Here, we provide evidence that the mitochondria of dihydroartemisinin (DHA)-exposed persisters are dramatically altered and enlarged relative to the mitochondria of young, actively replicating ring forms. Restructured mitochondrial-nuclear associations and an altered metabolic state are consistent with stress from reactive oxygen species. New contacts between the mitochondria and nuclei may support communication pathways of mitochondrial retrograde signaling, resulting in transcriptional changes in the nucleus as a survival response. Further characterization of the organelle communication and metabolic dependencies of persisters may suggest strategies to combat recrudescences of malaria after treatment. The major first-line treatment for malaria, especially the deadliest form caused by Plasmodium falciparum, is combination therapy with an artemisinin-based drug (ART) plus a partner drug to assure complete cure. Without an effective partner drug, ART administration alone can fail because of the ability of small populations of blood-stage malaria parasites to enter into a dormant state and survive repeated treatments for a week or more. Understanding the nature of parasites in dormancy (persisters) and their ability to wake and reestablish actively propagating parasitemias (recrudesce) after ART exposure may suggest strategies to improve treatment outcomes and counter the threats posed by parasites that develop resistance to partner drugs. Here, we show that persisters have dramatically altered mitochondria and mitochondrial-nuclear interactions associated with features of metabolic quiescence. Restructured associations between the mitochondria and nuclei may support signaling pathways that enable the ART survival responses of dormancy.
Topics: Antimalarials; Artemisinins; Cell Nucleus; Erythrocytes; Humans; Malaria, Falciparum; Mitochondria; Plasmodium falciparum
PubMed: 34044591
DOI: 10.1128/mBio.00753-21 -
Malaria Journal Dec 2019Malaria continues to be endemic in the coast and Amazon regions of Ecuador. Clarifying current Plasmodium falciparum resistance in the country will support malaria...
BACKGROUND
Malaria continues to be endemic in the coast and Amazon regions of Ecuador. Clarifying current Plasmodium falciparum resistance in the country will support malaria elimination efforts. In this study, Ecuadorian P. falciparum parasites were analysed to determine their drug resistance genotypes and phenotypes.
METHODS
Molecular analyses were performed to search for mutations in known resistance markers (Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, k13). Pfmdr1 copy number was determined by qPCR. PFMDR1 transporter activity was characterized in live parasites using live cell imaging in combination with the Fluo-4 transport assay. Chloroquine, quinine, lumefantrine, mefloquine, dihydroartemisinin, and artemether sensitivities were measured by in vitro assays.
RESULTS
The majority of samples from this study presented the CVMNT genotype for Pfcrt (72-26), NEDF SDFD mutations in Pfmdr1 and wild type genotypes for Pfdhfr, Pfdhps and k13. The Ecuadorian P. falciparum strain ESM-2013 showed in vitro resistance to chloroquine, but sensitivity to quinine, lumefantrine, mefloquine, dihydroartemisinin and artemether. In addition, transport of the fluorochrome Fluo-4 from the cytosol into the digestive vacuole (DV) of the ESM-2013 strain was minimally detected in the DV. All analysed samples revealed one copy of Pfmdr1.
CONCLUSION
This study indicates that Ecuadorian parasites presented the genotype and phenotype for chloroquine resistance and were found to be sensitive to SP, artemether-lumefantrine, quinine, mefloquine, and dihydroartemisinin. The results suggest that the current malaria treatment employed in the country remains effective. This study clarifies the status of anti-malarial resistance in Ecuador and informs the P. falciparum elimination campaigns in the country.
Topics: Antimalarials; Drug Resistance; Ecuador; Genotype; Humans; Malaria, Falciparum; Parasitic Sensitivity Tests; Phenotype; Plasmodium falciparum; Protozoan Proteins
PubMed: 31822269
DOI: 10.1186/s12936-019-3044-z -
PLoS Pathogens Apr 2021Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our... (Review)
Review
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.
Topics: Animals; Antimalarials; Erythrocytes; Humans; Life Cycle Stages; Malaria, Falciparum; Plasmodium falciparum; Protozoan Proteins
PubMed: 33886685
DOI: 10.1371/journal.ppat.1009442 -
Cell Host & Microbe Dec 2021Amino acid deprivation from reduced hemoglobin degradation in Pfkelch13 artemisinin-resistant parasites reduces fitness. In this issue of Cell Host & Microbe,...
Amino acid deprivation from reduced hemoglobin degradation in Pfkelch13 artemisinin-resistant parasites reduces fitness. In this issue of Cell Host & Microbe, Mesén-Ramírez et al. decipher the role of nutrient permeable channel activity within the parasitophorous vacuolar membrane to compensate for this fitness cost in asexual blood-stage Plasmodium falciparum parasites.
Topics: Antimalarials; Artemisinins; Drug Resistance; Mutation; Plasmodium falciparum; Protozoan Proteins
PubMed: 34883062
DOI: 10.1016/j.chom.2021.11.007 -
Malaria Journal Apr 2021Plasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) has historically posed a major threat to malaria control throughout the world....
BACKGROUND
Plasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) has historically posed a major threat to malaria control throughout the world. The country of Angola officially replaced CQ with artemisinin-based combination therapy (ACT) as a first-line treatment in 2006, but malaria cases and deaths have recently been rising. Many classic resistance mutations are relevant for the efficacy of currently available drugs, making it important to continue monitoring their frequency in Angola.
METHODS
Plasmodium falciparum DNA was sampled from the blood of 50 hospital patients in Cabinda, Angola from October-December of 2018. Each infection was genotyped for 13 alleles in the genes crt, mdr1, dhps, dhfr, and kelch13, which are collectively involved in resistance to six common anti-malarials. To compare frequency patterns over time, P. falciparum genotype data were also collated from studies published from across Angola in the last two decades.
RESULTS
The two most important alleles for CQ resistance, crt 76T and mdr1 86Y, were found at respective frequencies of 71.4% and 6.5%. Historical data suggest that mdr1 N86 has been steadily replacing 86Y throughout Angola in the last decade, while the frequency of crt 76T has been more variable across studies. Over a third of new samples from Cabinda were 'quintuple mutants' for SP resistance in dhfr/dhps, with a sixth mutation at dhps A581G present at 9.6% frequency. The markers dhfr 51I, dhfr 108N, and dhps 437G have been nearly fixed in Angola since the early 2000s, whereas dhfr 59R may have risen to high frequency more recently. Finally, no non-synonymous polymorphisms were detected in kelch13, which is involved in artemisinin resistance in Southeast Asia.
CONCLUSIONS
Genetic markers of P. falciparum resistance to CQ are likely declining in frequency in Angola, consistent with the official discontinuation of CQ in 2006. The high frequency of multiple genetic markers of SP resistance is consistent with the continued public and private use of SP. In the future, more complete haplotype data from mdr1, dhfr, and dhps will be critical for understanding the changing efficacy of multiple anti-malarial drugs. These data can be used to support effective drug policy decisions in Angola.
Topics: Adolescent; Adult; Angola; Antimalarials; Child; Child, Preschool; Drug Resistance; Epidemiological Monitoring; Genetic Markers; Humans; Infant; Malaria, Falciparum; Middle Aged; Plasmodium falciparum; Population Surveillance; Young Adult
PubMed: 33827587
DOI: 10.1186/s12936-021-03713-2 -
Indian Journal of Medical Microbiology 2020Prohibitins (PHBs) are evolutionarily conserved mitochondrial integral membrane proteins, shown to regulate mitochondrial structure and function, and can be classified...
Prohibitins (PHBs) are evolutionarily conserved mitochondrial integral membrane proteins, shown to regulate mitochondrial structure and function, and can be classified into PHB1 and PHB2. PHB1 and PHB2 have been shown to interact with each other, and form heterodimers in mitochondrial inner membrane. Plasmodium falciparum has orthologues of PHB1 and PHB2 in its genome, and their role is unclear. Here, by homology modelling and yeast two-hybrid analysis, we show that putative Plasmodium PHBs (Pf PHB1 and Pf PHB2) interact with each other, which suggests that they could form supercomplexes of heterodimers in Plasmodium, the functional form required for optimum mitochondrial function.
Topics: Membrane Proteins; Mitochondria; Models, Molecular; Plasmodium falciparum; Prohibitins; Protein Conformation; Protein Multimerization; Protozoan Proteins; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Two-Hybrid System Techniques
PubMed: 32883936
DOI: 10.4103/ijmm.IJMM_20_28 -
Revista Do Instituto de Medicina... 2021Malaria represents a serious public health problem, presenting with high rates of incidence, morbidity and mortality in tropical and subtropical regions of the world.... (Review)
Review
Malaria represents a serious public health problem, presenting with high rates of incidence, morbidity and mortality in tropical and subtropical regions of the world. According to the World Health Organization, in 2018 there were 228 million cases and 405 thousand deaths caused by this disease in the world, affecting mainly children and pregnant women in Africa. Despite the programs carried out to control this disease, drug resistance and invertebrate vector resistance to insecticides have generated difficulties. An efficient vaccine against malaria would be a strategy with a high impact on the eradication and control of this disease. Researches aimed at developing vaccines have focused on antigens of high importance for the survival of the parasite such as the Circumsporozoite Surface Protein, involved in the pre-erythrocytic cycle during parasites invasion in hepatocytes. Currently, RTS'S is the most promising vaccine for malaria and was constructed using CSP; its performance was evaluated using two types of adjuvants: AS01 and AS02. The purpose of this review was to provide a bibliographic survey of historical researches that led to the development of RTS'S and its performance analysis over the decade. The search for new adjuvants to be associated with this antigen seems to be a way to obtain higher percentages of protection for a future malaria vaccine.
Topics: Humans; Malaria; Malaria Vaccines; Membrane Proteins; Plasmodium falciparum; Protozoan Proteins
PubMed: 33533814
DOI: 10.1590/S1678-9946202163011