The Aspartyl Protease Ddi1 Is Essential for Erythrocyte Invasion by the Malaria Parasite

Malaria pathology is caused by the exponential replication of Plasmodium parasites in the blood stream. The bottleneck of the parasite life cycle is the invasion of erythrocytes immediately after parasites egress from infected red blood cells. DNA damage-inducible protein 1 (Ddi1) is a conserved eukaryotic proteasome shuttle protein containing an internal retroviral-like protease domain. Using conditional genetics, we now show that the proteolytic activity of the P. falciparum homologue, PfDdi1, is critically required for invasion of red blood cells. Furthermore, PfDdi1 disruption results in the accumulation of highly polyubiquitinated proteins that can be processed by purified PfDdi1 or distant eukaryotic homologues. We also show that PfDdi1 interacts with multiple components of the ubiquitin-proteasome system and that parasites lacking PfDdi1 are more sensitive to proteasome inhibition. Overall, this study establishes PfDdi1 as a key component of the eukaryotic ubiquitin-proteasome system and as a promising antimalarial target.


Introduction 35
Malaria remains one of the most devastating infectious diseases worldwide 36 with more than 200 million clinical cases per year and close to half a million deaths 37 (WHO, 2018). Over the last 15 years, the implementation of global vector control 38 programs and artemisinin-combination therapies have led to a very significant 39 reduction in malaria incidence and mortality (Bhatt et al., 2015). However, the rise of

172
The conditional truncation (cKO) strategy for PfDdi1 is outlined in Figure 1B

253
We also monitored the progression of each PfDdi1 conditional line population 254 over 5 cycles after RAP treatment by flow cytometry. One clone of each conditional 255 line was RAP treated, and a sample of mature schizonts fixed and analyzed at the end 256 of each cycle. The population of excised vs non-excised parasites was quantified 257 based on the GFP signal ( Figure 2D). As previously described, the excision rate was 258 13 excellent at cycle 0 (> 96%). While the PfDdi1_cWT line experienced a 3.3-fold 259 increase in parasitaemia in the first cycle, the parasitaemia of KO and mutant cultures 260 dropped by ~50% each cycle, while the small proportion of non-excised parasites 261 increased exponentially. These results indicate that PfDdi1-deficient parasites 262 experience a profound and consistent replication defect. However, a very small 263 proportion of merozoites (1 in the ~40-60 merozoites that are produced per every two 264 schizonts) are able to invade and replicate each cycle. Nonetheless, these results

265
show that PfDdi1-deficient parasites are unable to propagate in culture, indicating that 266 PfDdi1 is essential, and that its aspartyl protease activity is required for asexual blood 267 stages.

PfDdi1 is essential for RBC invasion 269
To determine which development stage is affected by the loss of PfDdi1

280
To confirm these results, the invasion rate in PfDdi1_cKO, PfDdi1_cWT and

299
The invasion defect associated with the loss of PfDdi1 function can be 300 rescued by delaying parasite egress 301 We were surprised to observe that PfDdi1-deficient parasites show a 302 pronounced invasion defect rather than a delay in intracellular parasite development.

303
In other organisms, Ddi1 mainly localizes to the cytoplasm and its function is closely

PfDdi1-deficient parasites show a delay in cytokinesis 360
To further explore this potential delay in merozoite maturation, PfDdi1_cMUT showed no overall significant difference between DMSO and RAP treated parasites.

370
These results clearly show that loss of PfDdi1 activity has no effect in nuclear division                Figure 6D).

PfDdi1 cleaves HMWUPs 577
We recently showed that hDdi2, but not its catalytically dead counterpart, is

585
Remarkably, we also found that recombinant leishmania LmDdi1 is able to cleave 586 HMWUPS present in PfDdi1 KO lysates ( Figure 6F), and that FLAG-PfDdi1 cleaves 587 HMWUPs present in human MRC5 hDdi2 KO cell lysates ( Figure 6G). Overall, these 588 results show that PfDdi1 has proteolytic activity directed towards a variety of proteins 589 with long ubiquitin chains and that its biochemical function is conserved across 590 eukaryotes.

592
In this study, we identify Ddi1 as an essential aspartyl protease in the 593 replication of P. falciparum asexual blood stage parasites. Loss of PfDdi1 and its 594 activity results in a profound invasion defect. PfDdi1 is cytoplasmic and interacts with 595 multiple components of the UPS. Indeed, we find that its loss sensitizes P. falciparum 596 blood-stage parasites to proteasomal inhibition and that PfDdi1 is required for the 597 turnover of hyperubiquitinated proteins.

The proteolytic activity of PfDdi1 is critical for erythrocyte invasion 600
PfDdi1 has an essential function in the replication of the malaria parasite during

690
The main challenge to protease drug discovery is achieving target specificity 691 as proteases of the same families have conserved mechanism of actions. The RVP 692 domain is rare and may mean that Ddi1 can be targeted over other aspartyl proteases.

693
Indeed, HIV proteñase, a close homologue of Ddi1, is the target of a multitude of highly 694 specific drugs (Ghosh et al., 2016). If PfDdi1 has ubiquitin-dependent activity then this 695 novel mechanism may make it possible to target it in a highly specific manner. Although

Generation of constructs for conditional modification of PfDdi1 707
All primers used for PCR in this study are listed in Table supplement

716
To generate the pT2A_PfDdi1_cWT construct, a RR2 (recodonised to P.

Fluorescence microscopy 834
Thin smears of infected erythrocytes were air dried and were fixed in 4% 835 formaldehyde for 30 min and permeabilized in 0.1% Triton X-100 (Sigma) for 10 min.

836
The slides were blocked in 3% BSA in PBS for at least 1 h. All antibodies used in IFA 837 were diluted with 3% BSA in PBS. Slides were probed with primary antibodies followed 838 by AlexaFluor antibody (Invitrogen). In the case of anti-HA probing, a 3-step approach 839 was taken using a biotinylated secondary antibody and a final incubation step with a

847
Fluorescence microscopy analysis of samples stain with the RNA dye 132A 848 was performed as previously described (Bell, et. al, 2020). Briefly, parasite cultures

932
The SUMO tag can be cleaved using the Ulp1 SUMO protease. PfDdi1 was expressed 933 in E. coli with an N-terminal His6, SUMO and FLAG tags, and purified using a 4-step 934 approach including a step to remove the N-terminal SUMO tag. A catalytically inactive 935 D262N mutant was also expressed in this way.

936
The entire Ddi1 expression construct was ordered from GenScript and

973
Native mass spectrometry and circular dichroism were used to assess the protein 974 quality ( Figure S5).               PfDdi1 is mainly homodimeric. Note that a contaminant (black squares) was detected in the FLAG-