FIKK 1, FIKK10.1 and FIKK10.2 co-localised with the Maurers Cleft marker MAHRP1, whereas FIKK4.1, FIKK4.2 and FIKK11 localised to the RBC periphery, as predicted previously for FIKK4.137 (Figure 2D and Extended Data Figure 5). which is usually trafficked via membranous vacuoles in the RBC cytosol called Maurers clefts and inserted into protrusions around the RBC surface called knobs 5. Only one of the ~60 PfEMP1 variants is expressed at a given time, determining adhesion to a specific host receptor. For example, the gene encodes a PfEMP1 variant that binds to CSA around the Rabbit polyclonal to CNTF placenta ADU-S100 ammonium salt 6. Although other human-infecting Plasmodium species may cytoadhere7, they do not express PfEMP1, and the symptoms of contamination are usually much milder in patients infected by these parasites. The FIKK kinases are a family of 18-26 serine/threonine kinases which are exported into the host cell by parasites of the clade, which includes and other great ape infecting species 8C12. Other species possess just one ancestral FIKK kinase, named FIKK8 in substrates remain unknown. Both RBC components and exported parasite proteins are phosphorylated during contamination19C28; we hypothesise that this FIKK kinase family expanded to mediate these changes. Open in a separate window Physique 1 Contamination of RBCs with or induces species-specific changes to the phosphoproteome of RBC proteins independently of blood type. (a) Maximum likelihood phylogenetic tree of species, with ADU-S100 ammonium salt clades grouped together. Silhouettes show host specificity. Divergence was calculated around the sequences of FIKK8 from each species. Figures in white circles are the quantity of active FIKKs, black circles are pseudogenes. (b) Experimental workflow for phosphoproteomics of and (i) or (ii). A positive L2FC indicates that a protein residue is more phosphorylated in iRBCs than uRBCs. y axis = log10 or in different blood types. (f) Comparison of the phosphoproteome of three different blood types, A+ (x axis), AB+ (y axis) and O+ (colour scale). Left C uRBC phosphorylation intensity, Right C L2FC in RBC protein phosphosite intensity between uRBCs and and infected RBCs (iRBCs). Deep phosphoproteome profiling of knockout lines for all those FIKK kinases predicted to be exported provides a detailed map of phosphorylation events controlled by each kinase, confirming that this FIKK kinases are key regulators of but not in other human-infecting species suggests that FIKK kinase activity plays a species-specific role in modulating RBC properties (Physique 1A). To test this, we compared the phosphoproteome of RBCs ADU-S100 ammonium salt infected with to that of a strain adapted for culture in human RBCs 29 (Physique 1B). As a control, we also decided the phosphoproteome of the uninfected RBCs (uRBCs) cultured under the same conditions. We purified late stage iRBCs at ~44 hours post contamination (hpi) for and ~24hpi for and iRBCs and uRBCs combined from both cultures, as well as one uRBC sample from your culture ADU-S100 ammonium salt only. We observed a substantial increase in ser/thr phosphorylation of RBC proteins in iRBCs compared to uRBCs (Physique 1C and Table S1). Using a threshold based on the log2 fold change (L2FC) and the and 6 phosphosites showed a decrease in phosphorylation (Physique 1C panel i, and Table S3). Upon contamination with and in a concordance-discordance (DISCO) plot (Physique 1D). This illustrates that most residues are phosphorylated upon contamination with only (Physique 1D, Table S3E). Notably, 53% of the residues phosphorylated by only have not been observed in uninfected RBC phosphorylation datasets previously (PhosphoSitePlus), indicating that these may be phosphorylated by FIKK kinases. To test whether phosphorylation of RBC proteins is usually affected by different host environments, we compared the phosphoproteome of cultured for one cycle in blood from three different donors with different blood types, A+, AB+ or O+ (Physique 1E). To ensure the parasites were collected at the same time points, we designed a magnetic purification stand with capacity for 5 medium-sized magnetic columns (MACS CS), based on a design by Kim iRBCs. To identify the substrates of the FIKK kinases, we generated conditional gene knockouts (cKO) for all those 19 FIKKs (excluding the pseudogenes FIKK7.2 and FIKK14). We targeted 12 FIKK kinases individually (FIKK1, 3, 4.1,.