Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Burda, Paul‐Christian; Crosskey, Thomas; Lauk, Katharina; Zurborg, Aimo; Söhnchen, Christoph; Liffner, Benjamin; Wilcke, Louisa; Pietsch, Emma; Strauss, Jan; Jeffries, Cy M.; Svergun, Dmitri I.; Wilson, Danny W.; Wilmanns, Matthias; Gilberger, Tim-Wolf

doi:10.1016/j.celrep.2020.107817

Cited by 26 publications

(30 citation statements)

References 84 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For generating the PhIL1‐BirA‐GFP construct, a homology region of 669 bp was amplified using 3D7 gDNA and cloned into pSLI‐TGD (Birnbaum et al, 2017) via NotI and AvrII restriction sites, next the BirA sequence was amplified from pSP‐GFP‐BirA (Khosh‐Naucke et al, 2018) and inserted via the AvrII/MluI restriction site, resulting in pSLI‐PhIL1‐BirA‐GFP. For endogenous tagging using the SLI system (Birnbaum et al, 2017), a homology region of 426–1087 bp (1001 bp for Pf 3D7_1229300, 1087 bp for Pf 3D7_0822900, 1056 bp for Pf 3D7_0415800, 866 bp for Pf 3D7_0623800, 1028 bp for Pf 3D7_0203000, 1041 bp for Pf 3D7_0508900, 917 bp for Pf 3D7_1318700, 426 bp for Pf 3D7_1310700 and 1038 bp for Pf 3D7_1312800 respectively) was amplified using 3D7 gDNA and cloned into pSLI‐GFP‐glmS (Burda et al, 2020) (derived from pSLI‐GFP (Birnbaum et al, 2017)) using the NotI/MluI restriction site. For PF3D7_0308300, a 650 bp homology region was amplified from 3D7 gDNA and cloned into pSLI‐sandwich (Birnbaum et al, 2017) via NotI and AvrII restriction sites.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

Wichers

Wunderlich

Heincke

et al. 2021

Cellular Microbiology

Self Cite

View full text Add to dashboard Cite

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single‐celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity‐dependent biotin identification (BioID)‐based proteomics approach, using the established IMC marker protein Photosensitized INA‐Labelled protein 1 (PhIL1) as bait in asexual blood‐stage parasites. Subsequent mass spectrometry‐based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP‐tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood‐stage parasites.

show abstract

Section: Methodsmentioning

confidence: 99%

“…GlmS‐based knockdown assay was adapted from previously published assays (Burda et al, 2020; Prommana et al, 2013). To induce knockdown, highly synchronous early rings stage parasites were split into two dishes and 2.5 mM glucosamine was added to one of them and parasite growth was measured by flow cytometry over 5 days as described above.…”

Section: Methodsmentioning

confidence: 99%

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

Wichers

Wunderlich

Heincke

et al. 2021

Cellular Microbiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…50,51 Further support comes from experiments in which lipocalin-like protein PV5 (PF3D7_0925900, PfLCN) was knocked down. These PV5-deficient parasites are less efficient in polymerizing toxic heme to hemozoin 78,79 and were hypersensitive to artesunate, chloroquine, and atovaquone, 78 suggesting that increased levels of heme result in increased susceptibility to artesunate and other drugs related to hemoglobin digestion. However, it is worth noting that decreased hemozoin formation also leads to an increased level of free radicals, which might lead to additional cellular stress.…”

Section: Inactivation Of K13 and K13 Compartment Proteins Causes Art Resistancementioning

confidence: 99%

The newly discovered role of endocytosis in artemisinin resistance

Behrens

Schmidt

Spielmann

2021

Medicinal Research Reviews

View full text Add to dashboard Cite

Artemisinin and its derivatives (ART) are the cornerstone of malaria treatment as part of artemisinin combination therapy (ACT). However, reduced susceptibility to artemisinin as well as its partner drugs threatens the usefulness of ACTs. Single point mutations in the parasite protein Kelch13 (K13) are necessary and sufficient for the reduced sensitivity of malaria parasites to ART but several alternative mechanisms for this resistance have been proposed.Recent work found that K13 is involved in the endocytosis of host cell cytosol and indicated that this is the process responsible for resistance in parasites with mutated K13.These studies also identified a series of further proteins that act together with K13 in the same pathway, including previously suspected resistance proteins such as UBP1 and AP-2μ. Here, we give a brief overview of artemisinin resistance, present the recent evidence of the role of endocytosis in ART resistance and discuss previous hypotheses in light of this new evidence. We also give an outlook on how the new insights might affect future research.

show abstract

“…For all analyses, medium was changed daily, and fresh glucosamine was added every day. GlmS-based knockdown assay was adapted from previously published assays 40,71 . To induce knockdown, highly synchronous early ring stage parasites were split into two dishes, 2.5 mM glucosamine was added to one of them and parasite growth was measured by FACS after two and four parasite replication cycles.…”

Section: Methodsmentioning

confidence: 99%

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 10, 2021. ; https://doi.org/10.1101/2021.09.08.459553 doi: bioRxiv preprint analyses, medium was changed daily, and fresh glucosamine was added every day. GlmSbased knockdown assay was adapted from previously published assays 40,71 . To induce knockdown, highly synchronous early ring stage parasites were split into two dishes, 2.5 mM glucosamine was added to one of them and parasite growth was measured by FACS after two and four parasite replication cycles.…”

Section: Glms-based Knockdownmentioning

confidence: 99%

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

Wichers

Gelder

Fuchs

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

During the symptomatic human blood phase malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and the blood plasma. Amino acids are delivered to the parasite through the parasite surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). In contrast, amino acid transport across the parasite plasma membrane (PPM) is not well characterized to date. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the Pf ApiATs at the PPM using endogenous GFP-tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites.

show abstract

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Cited by 26 publications

References 84 publications

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

The newly discovered role of endocytosis in artemisinin resistance

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

Contact Info

Product

Resources

About