Mutant/mutation
Different mutants are described that express mutated (chimeric) forms of profilin. We designed the profilin acidic loop chimeras such that a P. falciparum profilin contained the loop of either P. berghei (Pf PfnPb loop) or T. gondii (Pf PfnTg loop) and also included a P. berghei profilin with a loop of P. falciparum (Pb PfnPf loop).

Protein (function)
Profilins are ubiquitous and essential actin monomer binding proteins, known to interact with proline-rich regions in a variety of proteins and to regulate actin filament formation. For fast actin polymerization at selected sites, profilin is an essential control element by recruiting actin monomers in a polymerizable form to actin polymerization machineries. Profilins are usually defined by their shared, highly conserved structure and by their ability to bind actin, proline-rich sequences.

Compared to human actin, Plasmodium profilin contains several additional motifs, including a β-hairpin arm motif and an acidic loop. Comparison between different Apicomplexan species shows that there is little difference in the arm motif while T. gondii profilin has only a very short acidic loop compared with the profilins in Plasmodium spp. The regions bordering the acidic loop are highly conserved between P. falciparum and P. berghei profilin (100% over the 15 amino acid residues following the loop and only one A to E difference in the 15 amino acid residues preceding the loop). In contrast, 6 of the 8 amino acid residues of the acidic loop are not conserved. The T. gondii profilin loop sequence is more divergent, consisting of just four aspartic acid residues.

Phenotype
To investigate a potential role of the acidic loop in stabilizing the overall
structure of profilin and in actin binding, we performed molecular dynamics simulations on a series of chimeric profilins similar to those described for the arm-motif mutations . We designed the profilin acidic loop chimeras such that a P. falciparum profilin contained the loop of either P. berghei (Pf PfnPb loop) or T. gondii (Pf PfnTg loop) and also included a P. berghei profilin with a loop of P. falciparum (Pb PfnPf loop).
To test the chimeras in vivo, we generated a series of P. berghei strain ANKA parasite lines expressing the chimeras in place of the endogenous profilin. As the absence of  introns did not influence life cycle progression or motility of parasites we introduced intron-free genes.
As we previously also showed that a C-terminal fluorescent protein tag could slow down parasites, we further opted for exchanging the genes without such a tag. After isolating clones from the different lines, we next investigated the progression of these along the life cycle.

We first compared the blood stage growth rates of the chimeric parasite lines to those of wild type P. berghei and the previously reported P. berghei line expressing P. falciparum profilin. This showed that the parasites expressing the P. berghei profilin with the P. falciparum loop grew as fast as wild type P. berghei. Those lines expressing P. falciparum profilin and P. falciparum profilin with the P. berghei profilin loop grew somewhat faster but at comparable rates (Table 1). Intriguingly, expression of the P. falciparum chimera featuring the T. gondii loop slowed blood stage growth indicating that this chimeric profilin might not perform as efficiently in vivo. 

Infection of Anopheles stephensi mosquitoes however revealed similar infection rates and numbers of sporozoites.

Infections of mice by mosquito bite showed a mild reduction in infectivity of the parasite lines expressing P. falciparum profilins containing the loops of P. berghei or T. gondii.

P. berghei sporozoites expressing P. falciparum profilin migrated faster than wild type P. berghei sporozoites although fewer parasites were gliding.

Examination of the parasite lines expressing loop chimeras showed that sporozoites of all chimeras moved in the typical circular fashion of wild type parasites. About the same percentage of sporozoites were gliding, but curiously, those expressing P. falciparum profilin containing the loop of T. gondii showed a higher percentage of persistently moving sporozoites than those just expressing the P. falciparum profilin. The quantification of their average and instantaneous speeds showed however, that all sporozoites expressing chimeras were over 50% slower than their respective control parasite lines. 

Additional information
From the Abstract: 
' Here we show that different mutations in profilin, not affecting actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow-down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.' 

Other mutants