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

Mutant coronins exhibit defects in sporozoite motility.

<p>Time-lapse images and randomly selected tracks of different parasite lines expressing endogenous coronin fused to mCherry with the indicated mutations in their putative actin-binding sites. Note that R24A, R28A moves in a similar manner to WT, while the other mutants move differently and exhibit bending movements so far not seen in any wild type or mutated sporozoite, most strikingly seen in the time lapse of a sporozoites expressing the R349E, K350E mutation. A small but discernable fraction of sporozoite expressing the K283A, D285A mutation in coronin can move in persistent circles. Scale bars: 5 μm (images), 10 μm (tracks).</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

Inter-subunit interactions drive divergent dynamics in mammalian and <i>Plasmodium</i> actin filaments

<div><p>Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using <i>Plasmodium</i>, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of <i>Plasmodium</i> actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit–subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.</p></div

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

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

Comparison of the different parasite strains for their growth within cultured liver cells and their infectivity for mice.

<p>Note that all parasites with a lowered number of sporozoites in their salivary glands (all but WT and coronin-mCherry) failed to infect all mice following mosquito bites. While 100% (24 out of 24) of the mice bitten by mosquitoes infected with either wild type or coronin-mCherry expressing parasites were infected, only 78% (53 out of 68) of the mice bitten by mosquitoes infected with the various mutants developed a blood stage infection. As the motility was similar between WT, coronin-mCherry and the R24A/R28A mutant on one hand and between all the other mutants on the other hand (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005710#ppat.1005710.t001" target="_blank">Table 1</a>), we further analyzed the time to infection of mice successfully infected by mosquitoes. Hence, the first group showed an average time to blood stage infection of 3.4 days and the second group of 3.9 days for i.v. injected parasites (p<0.001, one way ANOVA) and 3.5 versus 4.1 days (p<0.01, one way ANOVA) for mice receiving parasites by mosquito bites.</p

Coronin displays reduced binding, yet overexpression rescues the PbS4Oc phenotype.

<p>(A) Overexpression of actin binding proteins specifically in the sporozoite stage of the life cycle. (B) Coronin localises at the back of motile wild-type but along the cell periphery in PbS4Oc sporozoites or sporozoites treated with CD, suggesting reduced efficiency of coronin binding. Red arrowheads indicate the front of the sporozoites, scale bar: 5 μm. (C–E) Profilin and ADF2 overexpression were unable to restore organ penetration (c), motility (d), and reduce pausing (e), while coronin overexpression was able to partially rescue the phenotype observed in the PbS4Oc line. In (C) data represented by mean ± standard error. In (D) black, grey, and white bars: efficiently, partially, and non-motile but attached sporozoites, respectively. Fisher’s exact test comparing moving groups in [D] and 0 pause groups in [E]; <i>n</i> is the total numbers of parasites counted per group. A coronin actin binding mutant (K349E,R350E) and a coronin construct lacking the C-terminal region (yet having the intact actin binding domain, ΔC) were unable to rescue the phenotype, suggesting an interplay between N- and C-terminal regions to mediate rescue. Please note that the wild-type and PbS4Oc data are the same as from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005345#pbio.2005345.g002" target="_blank">Fig 2C, 2F and 2H</a>. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005345#pbio.2005345.s016" target="_blank">S1 Data</a>. ADF2, actin depolymerising factor 2; CD, cytochalasin D; ΔC, coronin construct lacking C-terminal region; K349E,R350E, full length coronin actin binding mutant; mCh, mCherry.</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

Intracellular calcium release precedes the relocalization of coronin and motility.

<p>(A) Activated and non-activated sporozoites overexpressing coronin-mCherry were treated with or without BAPTA-AM to investigate effects on motility and coronin-mCherry localization with fluorescence microscopy. Note that BAPTA-AM ceases motility and relocalizes coronin from the periphery to the cytoplasm in activated salivary gland sporozoites (panel 1–2, top; p < 0.0001). Treatment with BAPTA-AM of non-activated salivary gland sporozoites (panel 3–4, top; p < 0.0002) and midgut sporozoites (panel 5–6, top; p < 0.0001) relocalizes coronin to the cytoplasm. Scale bar: 5 μm. The table shows the percentage of motile and non-motile sporozoites as determined from imaging at least 100 sporozoites and their associated localization pattern of coronin-mCherry [bottom]. The statistical differences are calculated by Fisher’s exact test. (B) Addition of ethanol or of the ionophore ionomycin to non-activated sporozoites leads to coronin-mCherry relocalization to the rear and motility. Scale bar: 5 μm. (C) Model of the role of calcium mediated receptor secretion on sporozoite progression from mosquito to the liver. In the absence of cytosolic calcium coronin (red) localizes to the sporozoite cytoplasm. At low calcium concentrations coronin localizes to the periphery and few TRAP family adhesins are released onto the sporozoite surface. At medium calcium concentrations, coronin localizes to the rear and more adhesins are released onto the sporozoite surface. Signaling associated with these events leads to optimal motility. Finally at very high calcium concentrations massive secretion leads a further increase of adhesins on the surface, which leads to invasion [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005710#ppat.1005710.ref049" target="_blank">49</a>].</p