With the help of standard cloning procedures, several transgenic parasite lines were generated which showed the localisation of this protein at the parasite’s surface. Moreover, distinct amino acids at the very C-terminus of LIMP were found in having an important role for the adhesion/invasion of the mosquito’s salivary gland. To identify protein-protein interacting partners of LIMP, biotinylation of nearby proteins in ookinetes and pulldown approaches in sporozoites were performed. Thus, during the financing period several transgenic Plasmodium berghei parasite lines were generated to study the localisation of LIMP, the function of distinct amino acids and interacting proteins.
In order to get more insights into the role of LIMP, different mutated versions of this protein were generated during the reporting period. Before the starting period the protein was tagged endogenously at the C-terminus with a GFP tag. Due to the size difference of tag (27 kDA) and protein of interest (13 kDa), different tagged versions were generated to assure correct localisation of LIMP. We chose to use a small myc tag at the C-terminus and a mCherry tag at the N-terminus which was placed after the signal peptide to guarantee its correct cleavage. All 3 tagged lines showed the same localisation at the parasite's Surface and intracellular in speckle like structures in salivary gland sporozoites. Interestingly only limp::GFP sporozoites showed the previously observed transient curvature changes (limping) during gliding motility. Modelling of the three tagged lines revealed that the GFP tag is folded onto the C-terminal alpha helical domain which could lead to a hindrance of protein-protein interactions. Another hint that the C-terminus might play an important role for adhesion and gliding motility are transgenic lines generated during the financing period that differ in their amino acid composition at the very end of the C-terminus. Depending on the mutation the sporozoites either showed the previously observed limping phenotype or a reduction in salivary gland Invasion. Hence, we could identify specific amino acids important for adhesion and/or salivary gland invasion. In summary, during the reporting period the mentioned tasks of objective 1 were reasonable fulfilled. Furthermore, milestone 1 (generation of vectors) and milestone 2 (generation of transgenic parasite lines) were achieved within the suggested period. The most efficient way to tag this protein was identified which is important for Objective 2. Secondly, distinct amino acids important for salivary invasion and motility were identified at the very C-terminus and thus milestone 3 was completed.
Two strategies were used to find interacting partners of LIMP in sporozoites and ookinetes to gather insights whether LIMP has similar or different protein-protein interacting partners in these 2 motile Plasmodium stages. Proximity-dependent biotin identification (BioID) was used to identify proteins in close proximity to LIMP in the ookinete stage. The establishment of a working protocol for this method was achieved as an important deliverable. Successful biotinylation was documented via Western Blot. However, not enough material could be detected after purification with streptavidin beads. Nevertheless, the approach itself worked efficiently only the purification step requires settle optimisation for reliable mass spec analysis.
Unfortunately, we discovered BioID is no feasible technique to study proteins in sporozoites, because unspecific binding of streptavidin to the parasite’s surface occurred. For this reason, Co-Immunoprecipitation, a well established technique to capture novel interacting proteins using agarose beads coupled with antibodies specific for the target protein, was performed to identify novel proteins involved in gliding motility and adhesion in sporozoites. The pulldown could successfully be achieved and the samples were send to the Core Facility for Mass Spectrometry & Proteomics (CFMP) at the ZMBH in Heidelberg. The analysis of potential hits was still under execution when the funding period finished. Because the initially used parasite number was too low to gather enough protein material, a 5 to 10 times higher parasite amount had to be used (5-10 million sporozoites). The collection of sporozoites is carried out through dissection of mosquito salivary glands and due to the high number of sporozoites required several mosquito cages had to be dissected, which was more time consuming than expected. Therefore, milestone 4 is not completely fulfilled yet, but based on the now relatively high protein amount it seems promising.
Concluding, working protocols for BioID and pulldown experiments were established and samples generated ready to be analysed by mass spectrometry.
Moreover parts of the above mentioned results were presented at the following conferences: poster presentation at 27th Annual Meeting of the German Society for Parasitology (2016) in Göttingen, Germany and talk at BioMalPar XII: Biology and Pathology of the Malaria Parasite (2016) in Heidelberg, Germany.
Additionally, the publication of several generated/characterised LIMP mutants was achieved in a peer reviewed open access journal:
Santos JM, Egarter S, Zuzarte-Luís V, et al. Malaria parasite LIMP protein regulates sporozoite gliding motility and infectivity in mosquito and mammalian hosts.. eLife. 2017;6:e24109. doi:10.7554/eLife.24109.
