scholarly journals Knockdown of the translocon protein EXP2, reduces growth and protein export in malaria parasites

2018 ◽  
Author(s):  
Sarah C. Charnaud ◽  
Rasika Kumarasingha ◽  
Hayley E. Bullen ◽  
Brendan S. Crabb ◽  
Paul R. Gilson
Keyword(s):  
Parasite Growth ◽  
Protein Export ◽  
Cell Protein ◽  
Host Cell Protein ◽  
Growth Reduction ◽  
Malaria Parasites ◽  
Asexual Blood Stage ◽  
Parasite Survival ◽  
Core Components ◽  
Exported Protein

AbstractMalaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effectors proteins exported from the parasites into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.

scholarly journals Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V

2018 ◽  
Author(s):  
Alexander J. Polino ◽  
S. Nasamu Armiyaw ◽  
Jacquin C. Niles ◽  
Daniel E. Goldberg
Keyword(s):  
Plasmodium Falciparum ◽  
Host Cell ◽  
Drug Targets ◽  
Parasite Growth ◽  
Aspartic Protease ◽  
Protein Export ◽  
New Drugs ◽  
Functional Studies ◽  
Parasite Survival ◽  
Post Transcriptional Regulation

AbstractUpon infection of a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This program of protein export is essential for parasite survival, hence there is interest in export-related proteins as potential drug targets. One proposed target is plasmepsin V (PMV), an aspartic protease that cleaves export-destined proteins in the parasite ER at a motif called the Plasmodium export element (PEXEL). This cleavage is essential for effector export across the vacuolar membrane. Despite long-standing interest in PMV, functional studies have been hindered by the failure of current technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a facile system for stringent post-transcriptional regulation, allowing a tightly controlled, tunable knockdown of PMV. Under maximal knockdown conditions, parasite growth was arrested, validating PMV as essential for parasite survival in RBCs. We found that PMV levels had to be dramatically depleted to affect parasite growth, suggesting that the parasite maintains this enzyme in substantial excess. This has important implications for antimalarial development. Additionally, we found that PMV-depleted parasites arrest immediately after invasion of the host cell, suggesting that PMV has an unappreciated role in early development that is distinct from its previously reported role in protein export in later-stage parasites.ImportanceMalaria is endemic to large swaths of the developing world, causing nearly 500,000 deaths each year. While infection can be treated with antimalarial drugs, resistance continues to emerge to frontline antimalarials, spurring calls for new drugs and targets to feed the drug development pipeline. One proposed target is the aspartic protease plasmepsin V (PMV) that processes exported proteins, enabling the export program that remodels the host cell. This work uses facile genetic tools to produce lethal depletion of PMV, validating it as a drug target and showing that PMV is made in substantial excess in blood-stage parasites. Unexpectedly, PMV depletion leads to parasite death immediately after invasion of RBCs, distinct from other disruptions of the export pathway. This suggests that PMV inhibitors could lead to relatively rapid parasite death, and that PMV has additional unexplored role(s) during RBC infection.

scholarly journals Using Lipoamidase as a Novel Probe To Interrogate the Importance of Lipoylation in Plasmodium falciparum

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Hugo Jhun ◽  
Maroya S. Walters ◽  
Sean T. Prigge
Keyword(s):  
Plasmodium Falciparum ◽  
Parasite Growth ◽  
Malaria Parasites ◽  
Subcellular Compartment ◽  
Content Type ◽  
Growth And Survival ◽  
Bacterial Enzyme ◽  
Parasite Survival ◽  
Key Aspects ◽  
Insight Into

ABSTRACT Lipoate is a redox active cofactor that is covalently bound to key enzymes of oxidative metabolism. Plasmodium falciparum is auxotrophic for lipoate during the intraerythrocytic stages, but it is not known whether lipoate attachment to protein is required or whether attachment is required in a specific subcellular compartment of the parasite. To address these questions, we used an enzyme called lipoamidase (Lpa) as a probe of lipoate metabolism. Lpa was first described in Enterococcus faecalis, and it specifically cleaves protein-bound lipoate, inactivating enzymes requiring this cofactor. Enzymatically active Lpa could be expressed in the cytosol of P. falciparum without any effect on protein lipoylation or parasite growth. Similarly, Lpa could be expressed in the apicoplast, and although protein lipoylation was reduced, parasite growth was not inhibited. By contrast, while an inactive mutant of Lpa could be expressed in the mitochondrion, the active enzyme could not. We designed an attenuated mutant of Lpa and found that this enzyme could be expressed in the parasite mitochondrion, but only in conjunction with a chemical bypass system. These studies suggest that acetyl-CoA production and a cryptic function of the H protein are both required for parasite survival. Our study validates Lpa as a novel probe of metabolism that can be used in other systems and provides new insight into key aspects of mitochondrial metabolism that are responsible for lipoate auxotrophy in malaria parasites. IMPORTANCE Lipoate is an essential cofactor for a small number of enzymes that are important for central metabolism. Malaria parasites require lipoate scavenged from the human host for growth and survival; however, it is not known why this cofactor is so important. To address this question, we designed a probe of lipoate activity based on the bacterial enzyme lipoamidase (Lpa). Expression of this probe in different subcellular locations allowed us to define the mitochondrion as the compartment housing essential lipoate metabolism. To gain further insight into the specific uses of lipoate in the mitochondrion, we designed a series of catalytically attenuated probes and employed the probes in conjunction with a chemical bypass system. These studies suggest that two lipoylated proteins are required for parasite survival. We were able to express Lpa with different catalytic abilities in different subcellular compartments and driven by different promoters, demonstrating the versatility of this tool and suggesting that it can be used as a probe of lipoate metabolism in other organisms.

scholarly journals The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicholas M. Negretti ◽  
Christopher R. Gourley ◽  
Prabhat K. Talukdar ◽  
Geremy Clair ◽  
Courtney M. Klappenbach ◽  
...  
Keyword(s):  
Host Cell ◽  
Campylobacter Jejuni ◽  
Foodborne Pathogen ◽  
Small Gtpase ◽  
Host Cells ◽  
Cell Protein ◽  
Host Cell Protein ◽  
Cell Binding ◽  
Actin Reorganization ◽  
Cell Cytosol

AbstractCampylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.

Evaluating Immunogenicity Risk Due to Host Cell Protein Impurities in Antibody-Based Biotherapeutics

2016 ◽  
Vol 18 (6) ◽  
pp. 1439-1452 ◽  
Author(s):  
Vibha Jawa ◽  
Marisa K. Joubert ◽  
Qingchun Zhang ◽  
Meghana Deshpande ◽  
Suminda Hapuarachchi ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Protein ◽  
Host Cell Protein

scholarly journals Host cell protein platform assay development for therapeutic mAb bioprocessing using mammalian cells

2015 ◽  
Vol 9 (S9) ◽  
Author(s):  
Nadine Kochanowski ◽  
Gaetan Siriez ◽  
Larissa Mukankurayija ◽  
Aurélie Delangle ◽  
Alex Murray-Smith ◽  
...  
Keyword(s):  
Host Cell ◽  
Mammalian Cells ◽  
Assay Development ◽  
Cell Protein ◽  
Host Cell Protein

scholarly journals Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors

Biologicals ◽  
2018 ◽  
Vol 52 ◽  
pp. 59-66 ◽  
Author(s):  
Sabine Johnson ◽  
Jun X. Wheeler ◽  
Robin Thorpe ◽  
Mary Collins ◽  
Yasuhiro Takeuchi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Host Cell ◽  
Lentiviral Vectors ◽  
Mass Spectrometry Analysis ◽  
Cell Protein ◽  
Protein Species ◽  
Host Cell Protein ◽  
Spectrometry Analysis

Poliovirus-induced inhibition of host-cell protein synthesis

Cell ◽  
1982 ◽  
Vol 28 (3) ◽  
pp. 435-436 ◽  
Author(s):  
Ellie Ehrenfeld
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Cell Protein ◽  
Host Cell Protein

New insights into protein export in malaria parasites

2010 ◽  
Vol 12 (5) ◽  
pp. 580-587 ◽  
Author(s):  
Silvia Haase ◽  
Tania F. de Koning-Ward
Keyword(s):  
Protein Export ◽  
Malaria Parasites
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