Genetic validation ofPfFKBP35 as an antimalarial drug target

Abstract

Plasmodium falciparumis the most virulent malaria parasite and accounts for the majority of over 600'000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged, posing a serious threat to disease control efforts. The need for drugs with alternative modes of action is thus undoubted. The parasite's FKBP protein (PfFKBP35) has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506. Whilst there is considerable interest in targetingPfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing. As a consequence, the function ofPfFKBP35 in live parasites remains elusive. Here, we report the generation of inducible knock-out, knock-down and over-expression cell lines and show that blood stage parasites requirePfFKBP35 for maintaining ribosome function. LimitingPfFKBP35 levels are lethal toP. falciparumand result in a delayed-death phenotype that is characterized by defective ribosome homeostasis and protein translation. We furthermore show that, while FK506 is indeed binding toPfFKBP35, the drug exerts it parasiticidal activity in aPfFKBP35-independent manner and, using cellular thermal shift assays, we identify FK506-targets beyondPfFKBP35. In addition to revealing first functional insights on the role ofPfFKBP35 inP. falciparumparasites, our results show that current efforts to developPfFKBP35-targeting drugs need to be reconsidered urgently.

The Kelch13 compartment contains highly divergent vesicle trafficking proteins in malaria parasites

“…In order to do this we excluded proteins that (i) had previously been analysed, (ii) were either linked with or had been shown to localise to the inner membrane complex (IMC) or the basal complex (PF3D7_1345600 [ 60 ]; PF3D7_0109000 (PhIL1) [ 61 – 63 ]; PF3D7_0717600 (IMC32) [ 64 ]; PF3D7_0822900 (PIC2) [ 65 ], PF3D7_1018200 (PPP8) [ 65 – 67 ], (iii) are considered typical DiQ-BioID ‘contaminants’ (PF3D7_0708400 (HSP90) and PF3D7_1247400 (FKBP35) [ 29 , 68 ]), (iv) localised to the apical polar ring in P . berghei (PF3D7_1141300 (APR1) [ 69 ]), (v) localised to the nucleus PF3D7_1247400 (FKBP35) [ 70 , 71 ], (vi) were linked with the apicoplast (PF3D7_0721100 [ 72 ]) or (vi) were also present in BioID experiments using Clathrin heavy chain (CHC) as bait [ 29 ] (PF3D7_0408100). These selection criteria resulted in a candidate list of thirteen proteins (PF3D7_1438400 (MCA2), PF3D7_1243400, PF3D7_1365800, PF3D7_1447800, PF3D7_1142100, PF3D7_0103100 (VPS51), PF3D7_1329100 (MyoF), PF3D7_1329500, PF3D7_0405700 (UIS14), PF3D7_0907200, PF3D7_0204300, PF3D7_1117900 and PF3D7_1016200), of which ten were chosen for further characterization in this manuscript (Tables 1 and S1 ).…”

The Kelch13 compartment is a hub of highly divergent vesicle trafficking proteins in malaria parasites

“…To identify novel K13 compartment proteins we further exploited previously described proximity-dependent biotinylation experiments that had used K13 or the K13 compartment protein Eps15 as bait, selecting enriched proteins not characterized in our previous work [26] (Table S1). In order to do this we excluded proteins that (i) had previously been analysed (ii) were either linked with or had been shown to localise to the inner membrane complex (IMC) (PF3D7_1345600 [49]; PF3D7_0109000 (PhIL1) [50–52]; PF3D7_0717600 (IMC32) [53]; PF3D7_0822900 (PIC2) [54], PF3D7_1018200 (PPP8) [54,55], (iii) are considered typical DiQ-BioID ‘contaminants’ (PF3D7_0708400 (HSP90) and PF3D7_1247400 (FKBP35) [26,56]), (iv) localised to the apical polar ring in P. berghei (PF3D7_1141300 (APR1) [57]), (v) localised to the nucleus PF3D7_1247400 (FKBP35) [58,59], (vi) were linked with the apicoplast (PF3D7_0721100 [60]) or (vi) were also present in BioID experiments using Clathrin heavy chain (CHC) as bait [26] (PF3D7_0408100). These selection criteria resulted in a candidate list of thirteen proteins (PF3D7_1438400 (MCA2), PF3D7_1243400, PF3D7_1365800, PF3D7_1447800, PF3D7_1142100, PF3D7_0103100 (VPS51), PF3D7_1329100 (MyoF), PF3D7_1329500, PF3D7_0405700 (UIS14), PF3D7_0907200, PF3D7_0204300, PF3D7_1117900 and PF3D7_1016200), of which the top ten were chosen as putative K13 compartment proteins for further characterization in this manuscript (Table 1, Table S1).…”