Revisiting gametocyte biology in malaria parasites

Gametocytes are the only form of the malaria parasite that is transmissible to the mosquito vector. They are present at low levels in blood circulation and significant knowledge gaps exist in their biology. Recent reductions in the global malaria burden have brought the possibility of elimination and eradication, with renewed focus on malaria transmission biology as a basis for interventions. This review discusses recent insights into gametocyte biology in the major human malaria parasite, Plasmodium falciparum and related species

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence

Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity

Protein export is central for the survival and virulence of intracellular P. falciparum blood stage parasites. To reach the host cell, exported proteins cross the parasite plasma membrane (PPM) and the parasite-enclosing parasitophorous vacuole membrane (PVM), a process that requires unfolding, suggestive of protein translocation. Components of a proposed translocon at the PVM termed PTEX are essential in this phase of export but translocation activity has not been shown for the complex and questions have been raised about its proposed membrane pore component EXP2 for which no functional data is available in P. falciparum. It is also unclear how PTEX mediates trafficking of both, soluble as well as transmembrane proteins. Taking advantage of conditionally foldable domains, we here dissected the translocation events in the parasite periphery, showing that two successive translocation steps are needed for the export of transmembrane proteins, one at the PPM and one at the PVM. Our data provide evidence that, depending on the length of the C-terminus of the exported substrate, these steps occur by transient interaction of the PPM and PVM translocon, similar to the situation for protein transport across the mitochondrial membranes. Remarkably, we obtained constructs of exported proteins that remained arrested in the process of being translocated across the PVM. This clogged the translocation pore, prevented the export of all types of exported proteins and, as a result, inhibited parasite growth. The substrates stuck in translocation were found in a complex with the proposed PTEX membrane pore component EXP2, suggesting a role of this protein in translocation. These data for the first time provide evidence for EXP2 to be part of a translocating entity, suggesting that PTEX has translocation activity and provide a mechanistic framework for the transport of soluble as well as transmembrane proteins from the parasite boundary into the host cell

Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale.

Resistance against all available antimalarial drugs calls for novel compounds that hit unexploited targets in the parasite. Here, we show that the recently discovered Plasmodium falciparum lactate/proton symporter, PfFNT, is a valid druggable target, and describe a new class of fluoroalkyl vinylogous acids that potently block PfFNT and kill cultured parasites. The original compound, MMV007839, is derived from the malaria box collection of potent antimalarials with unknown targets and contains a unique internal prodrug principle that reversibly switches between a lipophilic transport form and a polar, substrate-analogous active form. Resistance selection of cultured P. falciparum parasites with sub-lethal concentrations of MMV007839 produced a single nucleotide exchange in the PfFNT gene; this, and functional characterization of the resulting PfFNT G107S validated PfFNT as a novel antimalarial target. From quantitative structure function relations we established the compound binding mode and the pharmacophore. The pharmacophore largely circumvents the resistance mutation and provides the basis for a medicinal chemistry program that targets lactate and proton transport as a new mode of antimalarial action

Interactome analysis reveals endocytosis and membrane recycling of EpCAM during differentiation of embryonic stem cells and carcinoma cells

Summary: Transmembrane epithelial cell adhesion molecule (EpCAM) is expressed in epithelia, carcinoma, teratoma, and embryonic stem cells (ESCs). EpCAM displays spatiotemporal patterning during embryogenesis, tissue morphogenesis, cell differentiation, and epithelial-to-mesenchymal transition (EMT) in carcinomas. Potential interactors of EpCAM were identified in murine F9 teratoma cells using a stable isotope labeling with amino acids in cell culture-based proteomic approach (n = 77, enrichment factor >3, p value ≤ 0.05). Kyoto Encyclopedia of Genes and Genomes and gene ontology terms revealed interactions with regulators of endosomal trafficking and membrane recycling, which were further validated for Rab5, Rab7, and Rab11. Endocytosis and membrane recycling of EpCAM were confirmed in mF9 cells, E14TG2α ESC, and Kyse30 carcinoma cells. Reduction of EpCAM during mesodermal differentiation and TGFβ-induced EMT correlated with enhanced endocytosis and block or reduction of recycling in ESCs and esophageal carcinoma cells. Hence, endocytosis and membrane recycling are means of regulation of EpCAM protein levels during differentiation of ESC and EMT induction in carcinoma cells

Image_1_Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.png

IntroductionHead and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches.MethodsIn the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. ResultsIn vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor.DiscussionHence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.</p

Image_2_Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.png

IntroductionHead and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches.MethodsIn the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. ResultsIn vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor.DiscussionHence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.</p

PEXEL TM proteins require translocation for export and some PEXEL proteins can also jam the translocon.

<p>(<b>A-C</b>) Representative images of live <i>P</i>. <i>falciparum</i> parasites expressing the constructs shown schematically above each panel (features as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005618#ppat.1005618.g001" target="_blank">Fig 1</a>, numbers refer to the length of the amino acids sequence between TM and blocking domain). DIC, differential interference contrast. Size bars: 5 μm. A schematic of the co-block (<b>B</b>) and failure to co-block (<b>C</b>) are shown to the right (features as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005618#ppat.1005618.g002" target="_blank">Fig 2</a>).</p

Translocation intermediates are in a complex with EXP2.

<p>(<b>A-B</b>) Representative live fluorescence (<b>A</b>) or IFA (<b>B</b>) images of the cell line expressing endogenous EXP2 fused to 3xHA (EXP2-3xHA^endo) and SBP1-mDHFR-GFP (episomal) grown with (+WR) and without WR (control). DIC, differential interference contrast. Size bars: 5 μm. (<b>C</b>) IP of DSP-treated EXP2-3xHA^endo parasites (+) using HA binding beads compared to 3D7 parasites (-). Released crosslink eluates (ReCLIP) and NaOH eluates (Eluate2) were separated by SDS-PAGE and silver stained. Asterisk, shows EXP2-3HA; arrowheads show antibody chains. The table shows the 20 top hits after mass spectrometry analysis of a ReCLIP and an Eluate 2. Peptides/control show peptide counts of the indicated protein over 3D7 (ReCLIP and Eluate 2 peptide counts pooled). SP, signal peptide; TM transmembrane domain. (<b>D</b>) Western blots of a representative IP experiment using HA binding beads show co-purification of SBP1-mDHFR-GFP (asterisk) in parasites grown with WR but not in untreated controls. Input, total lysate before IP; post, lysate after IP. (<b>E</b>) Western blots of a representative IP experiment as in (<b>D</b>) but using GFP binding beads to bind SBP-mDHFR-GFP. Asterisk: enrichment of co-purifying EXP2-3xHA in parasites grown with (+WR) compared to control (no WR). (<b>F</b>) Quantification of the signal intensity of the IPed EXP2-3xHA and the co-purifying SBP1-mDHFR-GFP (left, asterisk comparing SBP1-mDHFR-GFP in WR+ over its control: p = 0,0288; paired, two-tailed t test; n = 3) or the IPed SBP1-mDHFR-GFP and the co-purifying EXP2-3xHA (right, asterisk comparing EXP2-3xHA in WR+ over its control: p = 0,035; paired, two-tailed t test. Asterisk comparing EXP2-3xHA enrichment with SBP1-mDHFR-GFP enrichment: p = 0,047; unpaired, two-tailed t test; n = 3) in parasites grown with (WR+) or without (control) WR. Error bars show S.D., n.s., not significant. (<b>G</b>) Western blots of a representative IP experiment with REX2-mDHFR-GFP expressed in EXP2-3xHA^endo parasites. REX2-mDHFR-GFP is not enriched in parasites grown with WR over untreated controls. Input, total lysate before IP; post, lysate after IP.</p

The distance between blocking domain and TM is important for co-blocking and BPTI sensitivity.

<p>(<b>A-E</b>) Representative images of live <i>P</i>. <i>falciparum</i> parasites expressing the constructs shown schematically above each panel (features as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005618#ppat.1005618.g001" target="_blank">Fig 1</a>, numbers refer to the length of the amino acids sequence between TM and blocking domain). (<b>A-C</b>) Parasites grown in the presence (+WR) or absence of WR (control). DIC, differential interference contrast. Size bars: 5 μm. (<b>F</b>) Table showing the properties of the different types of constructs.</p