Schematic cartoon summarizing the complex interplay of factors leading to sporozoite motility.

<p>Actin modulators such as profilin (this study) or coronin [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006412#ppat.1006412.ref030" target="_blank">30</a>] influence the state of actin by either promoting or preventing filament formation, filament crosslinking and filament orientation. The actin state influences retrograde flow and force generation as revealed by the use of inhibitors or mutants. Motility is the outcome of a non-trivial interplay between retrograde flow and force as these are not perfectly inversely correlated. Hence we stipulate the existence of another unknown factor that influences the actin state to produce optimal motility. The system is robust within a certain range (red dashed line), while unbalancing one or several of these factors leads to aberrant motility.</p

MM-PBSA and MM-GBSA estimates of binding free energies of wild type and different mutant complexes.

<p>MM-PBSA and MM-GBSA estimates of binding free energies of wild type and different mutant complexes.</p

Aberrant motility of <i>P</i>. <i>berghei</i> sporozoites expressing <i>P</i>. <i>falciparum</i> ‘AAA profilin’.

<p>(A) Representative images of ookinetes from the indicated lines moving for 10 minutes. White lines indicate tracks after 10 min. (B) Average speeds of ookinetes with median (line) and mean (arrowheads) from different parasite lines. At least 40 parasites per line were tracked over 15 min; significance was tested with a one-way ANOVA; ** denotes p<0.01, *** p<0.001. Horizontal bar: median, arrowheads indicate mean. (C) 10 ookinetes with median average speeds were plotted for their instantaneous speeds (time lapse: 20 s); significance was tested with a Kruskal-Wallis test; *** denotes p<0.001. Horizontal bars: median, arrowheads: mean. (D-F) Assembly of 40 randomly selected sporozoite tracks with representative time-lapse images below expressing (D) wild type, (E) QNQ and (F) AAA <i>P</i>. <i>falciparum</i> profilin. Images below are 3 seconds apart. Scale bar: 10 μm. Note the different patterns of migration in the AAA mutant. (G) Movement patterns of sporozoites clustered within three groups: ‘persistently moving’ denotes parasites that showed gliding motility for at least 50 consecutive frames (recorded at 0.33 Hz) without their speed dropping below 0.2 μm/s for more then 10 frames; ‘partially moving‘ denotes parasites that showed some circular gliding motility but did not fulfill the ‘persistently moving‘ criteria and ‘attached/waving‘ [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006412#ppat.1006412.ref074" target="_blank">74</a>] denotes parasites that did not display any active circular gliding. (H) Average speeds of persistently moving sporozoites tracked between 50 and 100 frames (150–300 s) for at least 50 sporozoites, *** denotes p<0.001; Kruskal-Wallis test. Horizontal bars: median, arrowheads: mean. (I) 10 sporozoites with median average speeds were plotted for their instantaneous speeds (from one frame to the next = 3 s). Note the shift of AAA sporozoites to a high abundance of very slow speeds as well as some notably high speeds, *** denotes p<0.001; Kruskal-Wallis test. Horizontal bars: median, arrowheads: mean.</p

Fluorescent tagging and absence of introns can impact profilin function.

<p>(A) Quantification of profilin-mCherry fluorescence per volume in ring stages, trophozoites and sporozoites (red shading). Each dot represents the fluorescence intensity normalized to parasite volume for one parasite. Horizontal bar: median. Significances were tested with Mann-Whitney tests; * denotes p<0.05; *** p<0.001.(B) Quantification of total profilin-mCherry fluorescence intensity of ookinetes. Note that fluorescence was much stronger in the nucleus of ookinetes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006412#ppat.1006412.s003" target="_blank">S3B Fig</a>) and thus ookinetes were not directly comparable with other stages. Significance was tested with a Mann-Whitney test. (C, D) Average speed with median (line) and mean (arrowheads) of ookinetes (C) and sporozoites (D) from the different parasite lines; Wt: wild type; Pfn⁺ⁱ mCh: Profilin-mCherry with introns; Pfn^-i mCh: Profilin-mCherry without introns; Pfn^-i // mCh: Profilin-mCherry without introns and long linker, which is cleaved (see panel E). At least 50 parasites per line were tracked over at least half of the acquisition time (sporozoites: 5 min, ookinetes: 15 min); significance was tested with a Kruskal-Wallis test; *** denotes p<0.001. (E) Western blot showing a weak band for the profilin-mCherry fusion protein (arrowhead) and a major band for a cleaved mCherry of the parasite line expressing profilin-mCherry with a long linker (Pfn^-i // mCh) detected with an anti-mCherry antibody in blood stage schizont lysates (lane 1). Note that at least 75% of profilin-mCherry is cleaved. Lane 2: control line expressing only mCherry. Loading control: anti-HSP70.</p

Effect of <i>Pf</i> and mutant profilins on pig muscle α-actin polymerization.

<p>Polymerization curves and relative initial polymerization rates of 4 μM α-actin alone and in the presence of 4–20 μM <i>Pf</i> wild type (A), QNQ (B) and AAA (C) profilins. Initial polymerization rates are depicted below the curves and were determined for the time frame of 300–600 s.</p

Sporozoites expressing profilin mutants generate less force but faster retrograde flow rates.

<p>(A) Image series of a sporozoite pulling a polystyrene bead out of a laser trap. (i-iii) The bead is held in the trap and put in contact with the sporozoite surface; (iv) the sporozoite pulls the bead out of the trap; (v) the sporozoite transports the bead to the rear end. Scale bar: 10 μm. (B) Quantitative analysis of the capacity of <i>P</i>. <i>falciparum</i> expressing sporozoites to pull a bead out of the trap from > 200 sporozoites tested with two to three different counter forces over 2–3 separate days. Bars represent the percentage of sporozoites that managed to pull the bead out of the trap. Black and white dashed lines indicate the level of wild type sporozoites treated with 50 nM jasplakinolide or cytochalasin D, respectively as published in Quadt et al., 2016 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006412#ppat.1006412.ref020" target="_blank">20</a>]. Significance was determined by Fishers exact test. (C) Representative speed plots of four beads tracked on four different sporozoites expressing either wild type <i>P</i>. <i>berghei</i> or <i>P</i>. <i>falciparum</i> profilin or mutated versions of the latter (namely QNQ or AAA). (D) Retrograde flow speeds as measured from over 170 sporozoites of the indicated parasite lines; boxes represent 50% of data, horizontal bar: mean, whiskers with 10–90 percentiles. Black and white dashed lines indicate the percentage of wild type sporozoites treated with 50 nM jasplakinolide or cytochalasin D, respectively that managed to pull a bead out of the trap as published in Quadt et al., 2016 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006412#ppat.1006412.ref020" target="_blank">20</a>]. Significance was determined by unpaired t-test. Note the higher peak values in the mutants of up to 14 μm/s compared to around 7 μm/s for wild type sporozoites.</p

Comparative life cycle progression of the different parasite lines.

<p>Comparative life cycle progression of the different parasite lines.</p