Table1_The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes.xlsx

The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite’s asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation.</p

Enhanced Antimalarial and Antisequestration Activity of Methoxybenzenesulfonate-Modified Biopolymers and Nanoparticles for Tackling Severe Malaria

Severe malaria is a life-threatening condition that is associated with a high mortality. Severe Plasmodium falciparum infections are mediated primarily by high parasitemia and binding of infected red blood cells (iRBCs) to the blood vessel endothelial layer, a process known as sequestration. Here, we show that including the 5-amino-2-methoxybenzenesulfonate (AMBS) chemical modification in soluble biopolymers (polyglutamic acid and heparin) and poly(acrylic acid)-exposing nanoparticles serves as a universal tool to introduce a potent parasite invasion inhibitory function in these materials. Importantly, the modification did not add or eliminated (for heparin) undesired anticoagulation activity. The materials protected RBCs from invasion by various parasite strains, employing both major entry pathways. Two further P. falciparum strains, which either expose ligands for chondroitin sulfate A (CSA) or intercellular adhesion molecule 1 (ICAM-1) on iRBCs, were tested in antisequestration assays due to their relevance in placental and cerebral malaria, respectively. Antisequestration activity was found to be more efficacious with nanoparticles vs gold-standard soluble biopolymers (CSA and heparin) against both strains, when tested on receptor-coated dishes. The nanoparticles also efficiently inhibited and reversed the sequestration of iRBCs on endothelial cells. First, the materials described herein have the potential to reduce the parasite burden by acting at the key multiplication stage of reinvasion. Second, the antisequestration ability could help remove iRBCs from the blood vessel endothelium, which could otherwise cause vessel obstruction, which in turn can lead to multiple organ failure in severe malaria infections. This approach represents a further step toward creation of adjunctive therapies for this devastating condition to reduce morbidity and mortality

Targeted Disruption of <i>PfRh3</i> Shows It Is Not Essential for the 3D7ΔRh2b Chymotrypsin-Resistant Pathway

<div><p>(A) Disruption of the <i>PfRh3</i> gene in 3D7. The pCC4-<i>Rh3</i> plasmid contains the blasticidin-S deaminase selectable marker, a negative selectable marker (A. G. Maier and A. F. Cowman, unpublished data), and 5′ and 3′ <i>Rh3</i> regions. <i>PfRh3</i> is shown with homologous target sequences (shaded regions). The double-crossover integration events are shown for 3D7, resulting in the deletion of a 5′ region of the gene. Restriction enzymes are C (ClaI) and X (XbaI), with fragment sizes shown for a C/X digestion. RT-PCR amplification target is shown as a black bar.</p><p>(B) Southern blot of genomic DNA from parasites shown digested with ClaI and XbaI and probed with the 5′ <i>Rh3</i> flank from the pCC4-<i>Rh3</i> vector.</p><p>(C) RT-PCR of <i>PfRh2a</i> and <i>PfRh3</i> from 3D7 and three knockout lines.</p><p>(D) Invasion into enzyme-treated erythrocytes expressed as a percentage of that into untreated erythrocytes for 3D7 and three knockout lines. Values above each column indicate the mean % invasion, with error bars representing the 95% CI from three independent assays.</p></div

Comparison of Transcription of Key Invasion Genes in 3D7, 3D7ΔRh2b, and D10

<div><p>(A and B). Expression levels of <i>EBA</i> and <i>PfRh</i> genes controlled by the relative expression of either <i>actin</i> and <i>histone 2b</i> (two constitutively expressed genes) or <i>msp2</i> (a gene expressed later on in the life cycle). Error bars represent the 95% confidence interval (CI) of values from two to three independent amplifications.</p><p>(C and D) Affymetrix microarray comparison of log gene expression intensity (as measured by MOID) between cRNA isolated from late-schizont 3D7 and (C) 3D7ΔRh2b or (D) D10 parasites. Data shown are restricted to 548 genes whose expression profile matches clusters 4, 13, 14, 15 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010037#ppat-0010037-b034" target="_blank">34</a>], including genes that encode proteins known to be involved in invasion. Color represents significance of the change in the level of gene expression as measured by the <i>t</i>-statistic (<2 black, >2 red). The control genes <i>PfRh2b</i> (knocked out) and <i>EBA-140</i> (deleted in D10) and upregulation of <i>PfRh3</i> are shown.</p></div

Changes in Protein Levels Do Not Underlie the 3D7ΔRh2b Chymotrypsin-Resistant Pathway

<div><p>(A) Western blot of parasite culture supernatant material probed with rabbit polyclonal antibodies against the functional EBA and PfRh proteins. SERA5 is used as a loading control. Antibodies marked with an asterisk (*) indicate the same gel stripped and reprobed with a different antibody.</p><p>(B) Invasion into chymotrypsin-treated erythrocytes for five parasite lines represented as the percentage of invasion to that into untreated erythrocytes.</p><p>(C) Invasion into either untreated or chymotrypsin-treated erythrocytes in the presence of protein-G purified polyclonal rabbit antiserum raised against recombinant EBA-140 and EBA-175 (or both together). Values are represented as the percentage of invasion in the presence of NRS. Error bars represent the 95% CI from three independent assays.</p></div

Comparison of the different parasite strains in the infectivity of mosquitoes and the <i>in vitro</i> speed of salivary gland sporozoites.

<p>Significant differences are indicated with an asterisk. Note that sporozoites from the R24A, R28A mutant, despite lower numbers in the salivary gland moved with comparable speed to the coronin-mCherry parasites. Note that coronin-mCherry parasites are significantly slower than WT parasites.</p

Speculative working model on coronin function at the interface between calcium and cAMP signaling during motility (red Arab numbers) and invasion (orange Roman numbers).

<p>Upon activation of a trans-membrane receptor by extracellular ligands on the salivary gland or in the skin (1), phospholipase C is activated and converts PIP2 into IP3 (2). This leads to the release of calcium from intracellular stores (3) and subsequent exocytosis of micronemes, which bring more receptors to the plasma membrane (5). These reinforce signaling and activate actin polymerization (6). Actin filaments are organized by surface receptors and relocalization of coronin from peripheral membranes (either PM or IMC) to actin filaments (7). This leads to efficient adhesion and force production essential for motility in 2D. Coronin (crn) relocalizes from actin filaments as these are disassembled through the action of PKA, which is possibly activated by cytosolic adenylate cyclase (AC). Upon stimulation of membrane bound adenylate cyclase more cAMP is produced leading to higher PKA activity and further calcium release from intracellular stores. The additionally released receptors then mediate invasion.</p

Comparison of different parasite lines with different coronin mutants for the percentage of sporozoites showing localization of coronin-mCherry and different movement patterns.

<p>The localization is classified as rear, peripheral and cytoplasmic, while the movement pattern is classified as moving, partially moving, attached and drifting.</p

cAMP signaling downstream of coronin relocalization modulates motility.

<p>(A) Sporozoites were incubated in RPMI + 3% BSA with the indicated kinase inhibitors at the indicated concentrations, imaged and their movement pattern quantified as gliding, waving or attached. The inhibitors stopped sporozoite motility in a dose-dependent fashion. Over 150 sporozoites were examined for each experiment. Significant differences (Fisher’s exact test) from the controls show both concentrations of H89 tested (p<0.0001) as well as 500 μM SQ22536 (SQ) (p = 0.01; p = 0.46 for 200 μM SQ22536). (B) Forskolin partially stimulates sporozoite motility when added to sporozoites in RPMI. Over 300 sporozoites were examined and their motility pattern quantified as in A. The shown difference is significant (p<0.0001; Fisher’s exact test). (C) Coronin-mCherry expressing sporozoites were investigated under various kinase inhibitors (each at 0,1 mM) with a fluorescence microscope to determine coronin-mCherry localization. The fluorescence stays at the rear in activated sporozoites arrested with PKA inhibitors. Coronin-mCherry relocalizes to the periphery when additional cytochalasin D is applied. Addition of forskolin to non-activated sporozoites leads to the localization of coronin-mCherry to the rear. Scale bar: 5 μm. (D) Table showing percentages of motile and non-motile sporozoites and the associated localization patterns (rear versus non-rear) of coronin-mCherry under the indicated conditions; low and high concentrations of SQ22536 (SQ) were 0,1 and 0,5 mM, respectively. Between 105 and 120 sporozoites were examined per condition. Statistical differences determined by Fisher’s exact test was p<0.0001 from the respective controls listed in the table of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005710#ppat.1005710.g004" target="_blank">Fig 4A</a>. (E) Examples of FRAP of motile sporozoites with coronin-mCherry localized to the rear. Scale bar: 5 μm. Circle indicates location of the bleach spot. (F) Quantitative analysis of FRAP. Coronin-mCherry recovers as fast in motile (>0.25 μm/s) as in non-motile (<0.25 μm/s) sporozoites if pooled across all conditions. There is also no difference in recovery time depending on the localization of coronin-mCherry [top graphs]. Quantitative analysis of FRAP data over a range of conditions [bottom graph]. Average values (+/- S.D.) are indicated above the graph. Bars show significant differences (* p<0.05; ** p<0.01), non-significance (ns) or p-values (Students t-test). Coronin-mCherry recovers significantly faster in non-motile sporozoites incubated in RPMI than in any other condition. With all other conditions there is no difference from each other with the exception of H89, where coronin-mCherry recovers significantly slower when compared to controls, 1 μM Cytochalasin D and 100 nM Jasplakinolide [bottom].</p

Mutant coronins reveal distinct binding to membranes and actin filaments.

<p>(A) Multiple sequence alignment shows that amino acids found to be important for actin binding in yeast are conserved between mouse, yeast and apicomplexans and are marked in red. (B) Fluorescence microscopy of a parasite line overexpressing coronin-mCherry from the uis3 promoter. Note the peripheral fluorescence in non-motile (RPMI) and motile (RPMI+BSA) salivary gland derived sporozoites. Numbers indicate time in seconds. Scale bar: 5 μm. (C) Localization of a number of overexpressed mutant coronin-mCherry fusion proteins. Note the three different types of localizations: cytoplasmic (coronin-8mut; K283A/E, D285A/R), peripheral (R24E, R28E; R349A/E, K350A/E) and polarized (WT, R24A, R28A). Scale bar: 5 μm. (D) Graph showing a quantitative assessment of the front versus rear ratio from 20 images of the various parasite lines overexpressing mutated coronin-mCherry as indicated on the x-axis. (E) The peripheral localization of R349A/E, K350A/E mutants is not altered when sporozoites are incubated with cytochalasin (100 nM), jasplakinolide (100 nM), ionomycin (1000 nM) or cytochalasin D (100 nM) + ionomycin (500 nM) Scale bar: 5 μm.</p