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Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files. However, the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Plasmodium falciparum , an apicomplexan parasite, is one of the leading causes of global mortality and morbidity arising from an infectious disease [ 1 ].
The clinical symptoms of malaria consist of cyclical waves of fevers and chills, which coincide with the synchronous rupture of infected red blood cells RBCs. This results in the release of parasite progeny termed merozoites that are capable of invading and proliferating within new host RBCs.
Significant efforts have been made to understand the process by which P. Since the rupture of infected RBCs is critical for the propagation of the parasite, understanding how parasites mediate egress from the RBC is important as it may lead to the identification of new strategies to block parasite amplification within the blood.
Certainly, the discovery of new anti-malarial drug targets is an urgent priority given the emergence of P. While egress is known to be mediated by a highly regulated protease cascade [ 8 ], mechanistic insight into this process has been lacking.
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Forward chemical genetic approaches have implicated the proteases subtilisin 1 SUB1 and dipeptidyl aminopeptidase 3 DPAP3 as key players in egress [ 9 , 10 ]. SUB1 is one of three subtilisin-like proteases found in P. SUB1 acts on members of the serine repeat antigen SERAs family of papain-like proteins present in the parasitophorous vacuole PV in which the parasite replicates [ 9 , 10 ], resulting in destabilisation of the encasing PV membrane PVM [ 11 , 12 ]. SUB1 also plays an important role in the proteolytic maturation of the abundant merozoite surface protein 1 MSP1 , which is critical for interaction with the host RBC cytoskeleton to facilitate egress [ 5 , 13 ].
Bioinformatic and proteomic approaches have identified other proteins that are substrates or potential substrates of SUB1, including proteins that localise to the rhoptry bulb such as rhoptry associated protein 1 RAP1 , rhoptry associated membrane antigen RAMA and RhopH3 [ 14 ]. Thus SUB1, directly or through its action on its substrates, mediates development of invasive merozoites and their release from the host cell for the next round of invasion. In addition to identifying the SUB1 inhibitor JCP, the small molecule screen by Arastu-Kapur et al [ 10 ] identified a dipeptide vinyl sulfone inhibitor that specifically blocked parasite egress.
With the aid of a biotin-labelled activity-based probe closely related to the cysteine protease inhibitors, the authors identified an unanticipated target— P. DPAP3 is one of three dipeptidyl aminopeptidases encoded in the Plasmodium genome, which cleave dipeptides from the N termini of their substrates.
DPAP1 is an essential food vacuole cysteine exopeptidase instrumental in haemoglobin digestion [ 15 , 16 ], while DPAP2 is a gametocyte stage-specific protease [ 17 ]. The localisation of DPAP3 has yet to be determined but development of a DPAP3-specific inhibitor SAK1 that has minimal cross-reactivity with other proteases, produces a dose-dependent accumulation of mature, unruptured schizonts, providing further support for DPAP3 being critical for egress of asexual stage parasites [ 10 ]. This has led to the proposition that DPAP3 sits on top of the proteolytic cascade that leads to egress.
Inhibition of DPAP3 also blocked the production and localisation of apical membrane antigen 1 AMA1 , a micronemal protein, and thus DPAP3 may regulate parasite egress by also regulating the maturation of secretory proteins required for this process.
Dipeptidyl aminopeptidase-like protein 6
It is, therefore, important that the findings of SAK1 pharmacological blockade studies are validated in P. Thus to substantiate the role of DPAP3 in parasite egress and to assess whether this protease plays an important role in the maturation and trafficking of proteins to the apical organelles, we have examined the localisation of DPAP3 within the parasite and conditionally regulated its expression in P. Blood-stage P. This plasmid was adapted from pPTEXHAglmS [ 23 ] and incorporated the last kilobase of dpap3 coding sequence in place of the ptex coding sequence.
The dpap3 targeting region was amplified from P. Transgenic parasites were selected with 2. Following three rounds of drug cycling in the presence or absence of WR to select for integrants, PCR was used to verify integration had occurred using the oligonucleotides indicated in Fig 1. The parasite population was then cloned out by limiting dilution and clones that were positive for integration by PCR were analysed further by Western blot and immunofluorescence analysis. The endogenous dpap3 locus was modified to incorporate a triple haemagglutinin HA and streptavidin tag to the C-terminus of DPAP3, which is followed by the P.
In the absence of glucosamine GlcN , translation proceeds as normal. However, addition of GlcN activates the ribozyme, leading to cleavage and subsequent degradation of the mRNA molecule. SM, selectable marker.
Dipeptidyl Aminopeptidases in Health and Disease | Martin Hildebrandt | Springer
The position of oligonucleotides denoted a-f used for diagnostic PCRs is indicated. Western blot analysis showing DPAP3 is expressed in schizonts and merozoites and is also released into the culture medium. EXP2 serves as the loading control. Parasites pellets obtained after centrifugation were washed with PBS containing cOmplete proteases inhibitors Roche.
All antibody incubations were performed in 0. Primary antibodies for P. Magnetically-purified P. Fixed cells were pelleted 3, g for 1 min and washed 3 times in ice-cold PBS, equilibrated into water, deposited into low melting point agarose plugs for ease of handling, then dehydrated in series of increasing ethanol concentrations. Parasites were harvested at schizont stage in the same cycle or in the following cycle.
Experiments were performed on three separate occasions. After invasion, the synchronized cultures of P. Parasitemias in Giemsa-stained smears were determined by counting a minimum of RBCs. Following thawing, lysis buffer 20 mM Tris pH 7. After incubation in the dark for 1 h, fluorescence activity was measured on a plate reader using excitation and emission wavelengths of nm and nm, respectively. All data were corrected for the background fluorescence of uninfected RBCs. When the parasites developed into schizonts, 2. The cultures were then washed three times with RPMI media to remove Compound 1 and incubated with fresh RBCs under shaking conditions rpm for 30 min to facilitate invasion.
Pf18SrRNA was used as a control to allow comparison of gene expression between parasite lines. Control reactions in which reverse transcriptase or cDNA template had been excluded were performed with each experiment to exclude genomic DNA contamination.
All reactions were performed on three separate RNA harvests, with reactions performed in triplicate. In this study, we attempted to explore DPAP3 function using a reverse genetic approach. Considering the haploid nature of the genome of Plasmodium and the previous described role for DPAP3 that indicated a conventional knockout of DPAP3 may be lethal [ 10 ], we directly opted to generate a transgenic P. The degree of ribozyme self-cleavage of the chimeric mRNA and thus the level of gene knockdown can be controlled by adding various concentrations of glucosamine GlcN to the parasite culture medium.
Genomic DNA extracted from transgenic parasites were analysed by PCR, which confirmed the parasite population had integrated the targeting construct as expected Fig 1B. The transgenic parasites were cloned and western blot of parasite lysates using anti-HA antibodies validated that the endogenous DPAP3 had been successfully epitope-tagged Fig 1C.
These results revealed that DPAP3 is expressed late in the cell cycle in schizont stages as well as in free merozoites, the latter indicating the protease is not completely secreted before merozoite egress Fig 1D. DPAP3 was also released into the culture supernatant during parasite egress from the host erythrocyte. An additional band of apparent mass of 85 kDa was also observed Fig 1D.
However, given this 85 kDa band was only observed in the occasional western blot, it is suspected that this band represents a protein degradation product during parasite lysis rather than it being a product of proteolytic cleavage of the full-length protein per se. No bands were observed in parental 3D7 parasites with the anti-HA antibody as expected. The results show a strong punctate pattern in the schizont stages, characteristic of apical localisation.
No transient localisation characteristic of protein trafficking to the apical end of the parasite was observed for DPAP3 in earlier stage parasites and prior to when RAP1 is expressed. Rather it showed partial co-localisation with rhoptry and microneme markers but no co-localisation with the dense granule marker RESA. This indicates that DPAP3 may not be housed in any of these three Plasmodium apical organelles, but potentially in secretory vesicles in close proximity to the rhoptries and micronemes.
Whilst no substantial labeling was observed in wildtype parasites S1 Fig , DPAP3 was observed at multiple locations in the parasites, including towards the periphery of rhoptry bulb and neck, in other apical regions and also at the parasitophorous vacuole membrane PVM Fig 2B. Although the HA labelling seen in the immuno-EM was not obviously contained in membranous structures indicative of secretory vesicles, this is not surprising as the immuno-EM process used in this circumstance yields relatively poor membrane preservation.
Protein samples were prepared from late schizont cultures within the same cycle Cycle 1 or the following replication cycle Cycle 2.
Western blot analysis showing dose-dependent PfDPAP3-HA protein expression in two independent clones after treatment with glucosamine GlcN for one upper panel or two lower panel cell cycles Cyc1 and Cyc2, respectively. We also analysed the localisation of RhopH3, another rhoptry bulb protein predicted to be a SUB1 substrates but it has yet to be investigated whether processing of bulb proteins is critical for correct trafficking to this organelle.
IFA showed that irrespective of the levels of expression of DPAP3, each of these apical proteins, as well the rhoptry neck protein RON6 and dense granule protein RESA included as controls could all traffic normally to their expected destination Fig 4. This indicates that DPAP3 does not contribute to the trafficking of these proteins to their respective locations.
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Moreover, given that knockdown of DPAP3 expression did not impact on the ability or timing of the parasites to mature from rings into schizonts to perform this analysis, these results also indicate that DPAP3 is not required for the normal maturation of schizonts. In the case of RAP1, N-terminal truncation of the kDa precursor form gives rise to an kDa form, a kDa intermediate form and a final processed product of 67 kDa. The last processing step is mediated by SUB1 and occurs late in schizont maturation [ 32 , 33 ].
It has been hypothesised that processing of RAP1 is required to release it from a complex that contains RAMA once it is trafficked to the rhoptries [ 34 ]. Interestingly the kDa RAMA protein is processed into a kDa form, and both an in silico prediction tool and proteomic analysis have flagged this protein as being processed by SUB1 [ 14 ]. Then, following heparin and C1 washout, egress was monitored and quantitated both by Giemsa stained blood smears and flow cytometry see Fig 6A for overview of methodology. The conversion of schizonts to ring was also analysed Fig 6C and whilst knockdown of DPAP3 appeared to lead to a greater lag in invasion as indicated by fewer rings at the earlier time points compared to parasite lines expressing DPAP3, this was not statistically significant.
The number of rings that had converted from the schizont populations by 6 h was also comparable between the lines. Overview of egress and growth experiments. For both experiments, P.
Dipeptidyl Aminopeptidases in Health and Disease
Heparin was washed out to allow invasion to occur, after which 2. For the egress experiments heparin was also added back to the culture medium red dotted line , followed by the addition of Compound 1 for 4 hours red line when parasites reached schizogony. Egress was monitored at the indicated time points after heparin and Compound 1 were washed out. For the growth experiments, infected erythrocytes were harvested mid-cycle for analysis by microscopy and SYBR Green I assay.
Analysis of parasite growth by microscopy. The parasitemia of cultures harvested approximately mid-cycle were measured by microscopy of Giemsa-stained blood smears and SYBR Green I-based assays that measure nuclear content were also conducted to measure parasite growth. While no significant reduction in parasitemia was observed over time when DPAP3 expression was knocked down Fig 6D , the more sensitive SYBR Green I-based assay indicated that despite treatment with GlcN having some impact on parasite growth, including on the parental 3D7 line, knockdown of DPAP3 did ultimately have a significant effect on the parsaites after h in culture Fig 6E.
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