Use of an ABP to identify a DPAP1-selective substrate in parasite lysates.

<p><b>A.</b> Structure and reaction mechanism of the (Pro-Arg)₂-Rho substrate. <b>B.</b> Measurement of (Pro-Arg)₂-Rho apparent <i>K</i>_m in trophozoite lysates (circles) and with recombinant DPAP1 (triangle). Turnover rates at increasing concentrations of substrate were fitted to a Michaelis-Menten equation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011985#s4" target="_blank">methods</a> section. <b>C.</b> Labeling of DPAP1 activity in parasite lysates with FY01. Trophozoite lysates were incubated for 1 h with increasing concentrations of FY01. Labeling was stopped by boiling the sample in SDS-PAGE loading buffer. DPAP1 activity was measured using a flatbed fluorescent scanner. <b>D.</b> DPAP1 labeling correlates with substrate turnover inhibition. An aliquot of the samples treated for 1 h with FY01 was diluted in assay buffer containing 10 µM of (Pro-Arg)₂-Rho, and the initial turnover rate was measured in a 96-well plate (circles). This turnover rate is plotted with the labeling quantified in C.</p

Development of a DPAP1-specific HTS assay.

<p><b>A.</b> Continuous assay. The assay was carried out in 384-well plates using 1% of parasite lysates. Substrate turnover was continuously measured for 5 min. JCP410 (10 µM) was used as a positive inhibition control. Z’ factor, S/N, and % CV of the negative control are shown. <b>B.</b> End-point assay for HTS. The reaction described in A was quenched after 10 min by addition of 0.5 M acetic acid. The final concentration of rhodamine product was quantified by fluorescence.</p

Cat C-specific fluorogenic assay in rat liver lysates.

<p><b>A.</b> Labeling of Cat C with FY01. Rat liver extract extracts were treated with increasing concentrations of FY01 for 1 h and labeled proteins analyzed by SDS-PAGE followed by scanning of the gel using a flatbed laser scanner. The location of labeled Cat C is indicated. <b>B.</b> Inhibition of substrate turnover specifically correlates with Cat C labeling. The cleavage of (Pro-Arg)₂-Rho substrate was measured prior to analysis of FY01 labeling shown in part A. Quantification of the indicated labeled proteins relative to DMSO control is shown. <b>C.</b> Cat C-specific HTS assay in rat liver extracts. Rat liver lysates were treated for 30 min with either DMSO or JCP410 (10 µM) followed by the addition of 10 µM of (Pro-Arg)₂-Rho. The turnover rate was continuously measured for 5 min in a 384-well plate. Z’ factor, S/N, and % CV of the negative control are shown.</p

A Coupled Protein and Probe Engineering Approach for Selective Inhibition and Activity-Based Probe Labeling of the Caspases

Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation

DPAP3 has proteolytic activity.

<p>(<b>A</b>) Analysis of purified rDPAP3. Two main bands are detected by silver stain, both of which are strongly labelled by FY01 and recognized by the anti-Nt-DPAP3 and anti-Ct-DPAP3 antibodies. All other minor bands in the silver stain are also recognized by DPAP3 antibodies and represent degradation products that could not be separated during purification. (<b>B</b>) Measurement of VR-ACC turnover and FY01 labelling for WT and C504S MUT rDPAP3. Silver stain analysis shows equivalent amounts of protein were obtained from the purification of WT and MUT rDPAP3. (<b>C</b>) pH dependence of rDPAP3 activity measured at 10 μM VR-ACC (n = 3).</p

DPAP3 is secreted at the time of egress.

<p>(<b>A</b>) C2-arrested schizonts (DPAP3-HA) were either left on C2, treated with E64 after C2 wash out, or allowed to egress for 1 h in the presence of FY01. Parasite pellets from free merozoites and schizonts (insoluble fraction obtained after saponin lysis), proteins precipitated from the culture supernatant, and PV and RBC cytosol components (soluble saponin fraction), were run on a SDS-PAGE. The presence of DPAP3-HA in each fraction was visualized as a fluorescent band at around 130kDa, which correspond to the band identified by WB using an anti-HA antibody (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s008" target="_blank">S4A Fig</a>). Hsp70 and BiP antibodies were used as WB markers of intracellular proteins (cytosol and ER, respectively), and SERA5 as a PV marker. (<b>B</b>) Mature DPAP3-mCh schizonts were arrested with C2 for 3 h, and egress observed by live video microscopy after C2 wash out (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s016" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s018" target="_blank">S3</a> Videos). The representative still-frame pictures show DIC and mCherry signal (red) before or after PVM breakdown, and after RBCM rupture. (<b>C</b>) Quantification of mCherry signal measured on consecutive frames before and after PVM breakdown. Around 20% of the signal originates from the hemozoin autofluorescence (red line). As a bleaching control, the mCherry signal of schizonts that did not egress was quantified at the corresponding time frames. (<b>D</b>) IEM section obtained from DPAP3-GFP parasites. Close-up images of individual intracellular merozoites on the left show immunogold staining of DPAP3-GFP in close proximity to the rhoptries (green arrows) and at the apical end of merozoites (blue arrows). Images on the right show representative sections of schizonts with an intact (black arrows) or rupture PVM. Staining of extracellular DPAP3-GFP (white arrows) was only observed in schizonts lacking a PVM. Rhoptries (r), nuclei (n), and the RBCM (red arrows) are indicated. Rabbit anti-GFP and colloidal gold-conjugated anti-rabbit antibodies were used. Bar graph = 200 nm. IEM images obtained on the 3D7 control line are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s007" target="_blank">S3D Fig</a>, and the uncropped IEM images for the DPAP3-GFP line in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s007" target="_blank">S3E Fig</a>.</p

Generation of complementation lines.

<p>(<b>A</b>) Quantification of excision efficiency for DPAP3cKO and complementation lines. Schizonts collected after DMSO or RAP treatment were stained with anti-mCherry and anti-SUB1 antibodies. SUB1 staining was used as a marker of schizont maturity. The amount of mCherry positive schizonts was quantified in relation to the total amount of mature schizonts. In DMSO treated parasites, all mature schizonts were mCherry positive (100%). The amount of non-excised parasites after RAP treatment was <5% in all biological replicates for all the cKO and complementation lines tested. Each circle corresponds to a different biological replicate (>100 schizonts were analyzed per biological isolate). Filled circles correspond to F8cKO and its complementation lines, and empty ones to the A1cKO line. (<b>B</b>) IFA analysis of the complementation lines showing colocalization of chromosomal DPAP3-mCh and episomal DPAP3-HA expressed under the <i>dpap3</i> or <i>ama1</i> promoters. Mature schizonts from the F8cKO+WT_dpap3, F8cKO+MUT_dpap3, F8cKO+WT_ama1, and F8cKO+MUT_ama1 were fixed and stained with anti-HA (red) and anti-mCherry (green). DNA was stained with DAPI (blue); scale bar: 5μm. Single coloured images are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s010" target="_blank">S6C Fig</a>. (<b>C</b>) Quantification of the amount of DPAP3-HA positive schizonts in the complementation lines. Schizonts were fixed at 48 h.p.i. and stained with anti-HA and anti-SUB1 antibodies. Only 60–80% of mature schizonts show positive HA staining with no difference between DMSO and RAP treated parasites. More than 100 schizonts per biological replicate were analyzed. (<b>D</b>) FY01 labelling of cKO and complementation lines. After DMSO or RAP treatment at ring stage, C2-arrested schizonts were collected, lysed, and labelled with FY01. Labelling of chromosomal DPAP3-mCh is clearly visible as a band around 150kDa along with some post-lysis degradation products indicated by asterisks. The loss of this 150kDa upon RAP treatment is observed in all lines except the E7ctr. Episomal WT DPAP3-HA is labelled by FY01 independently of RAP treatment and co-migrates with one of the degradation products of DPAP3-mCh at 125kDa. No labelling of MUT DPAP3-HA was observed. DPAP1 labelling by FY01 is shown as a loading control.</p

Poisson analysis of plaque formation shows that each plaque derives from a single parasite-infected erythrocyte.

<p>Poisson analysis of plaque formation shows that each plaque derives from a single parasite-infected erythrocyte.</p

Role of DPAP3 in RBC invasion.

<p>(<b>A</b>) Representative FACS plot (forward light scattering vs. Hoechst staining) showing a decrease in invasion of the A1cKO upon RAP treatment. The populations of uRBCs, rings, and schizonts (Sch) are indicated. (<b>B</b>) Analysis of invasion efficiency of DPAP3 cKO and complementation lines. Schizonts collected 45 h after DMSO or RAP treatment were incubated with fresh erythrocytes for 8–14 h, fixed, stained with Hoechst, and analyzed by FACS. Shown is the ratio in invasion efficiency between RAP- and DMSO-treated parasites. Filled and empty circles represent individual biological replicates for the F8cKO and A1cKO, respectively and their corresponding complementation lines. Student’s t test significance values between cell lines are shown above the lines, or above each bar when comparing to the E7ctr. Only significant p-values are shown. (<b>C</b>) FACS analysis of extracellular merozoites. C2-arrested A1cKO schizonts pretreated with DMSO or RAP were incubated with fresh RBCs after C2 removal. Samples were collected at the indicated time points, fixed, and stained with Hoechst and WGA-Alexa647. The FACS plot and histogram show samples collected 20 min after C2 washout. Free merozoites (Mrz) show positive staining for DNA but negative for WGA-Alexa647. Quantification of the different parasite stage populations over time is shown on the bar graph; biological replicates are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s013" target="_blank">S9 Fig</a>. (<b>D</b>) Quantification of attached merozoites by flow cytometry. Samples collected 15 and 20 min after C2 washout during invasion assays (performed as in <b>C</b>), were stained with Hoechst and anti-MSP1 antibody (anti-mouse Alexa488 as secondary antibody). Because MSP1 is shed during invasion, merozoites attached to the RBCM (Att Mrz) can be differentiated from intracellular parasites as the cell population positive for DNA and MSP1 staining. FACS plots compare anti-mouse Alexa488 staining in samples treated with or without the anti-MSP1 antibody. MSP1 staining (green) of attached merozoites under these conditions was confirmed by microscopy (central panel). Quantification of the population of attached merozoites relative to the ring population is shown on the bar graph. Circles represent different biological replicates: filled for F8cKO and empty for A1cKO. No significant difference was observed between DMSO and RAP treatment.</p

Rapid phenotypic characterization of a lethal conditional <i>P</i>. <i>falciparum</i> mutant using the plaque assay.

<p>(A) Left hand-side; schematic of the results of plaque analysis of RAP-treated and DMSO (mock)-treated SERA6:loxP parasites. Microplate wells coloured green indicate those that contained plaques 14 days following plating out the parasites at a theoretical 10 parasites/well. White wells contained no plaques (wells shown in grey were not used for the cloning). Whereas plaques were present in every well of the mock-treated culture, only a single plaque appeared in one well (well D8) of the RAP-treated culture. Right hand-side; example wells from the RAP-treated and control plates (green channel only of the scanned image shown to enhance plaque visibility, displayed as a grayscale image; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157873#sec008" target="_blank">Materials and Methods</a> for details). (B) Diagnostic PCR analysis of either the bulk SERA6:loxP parasite population immediately following RAP or DMSO-treatment (before plaque assay), or parasites expanded from well D8 of the +RAP plate. RAP-treatment significantly reduced the intact-<i>SERA6</i> locus-specific signal in the parasite population and resulted in appearance of a signal specific for the excised locus. Parasites rescued from well D8 of the RAP-treated parasites displayed a non-excised genomic architecture. The results strongly suggest that excision of the <i>SERA6</i> gene is lethal. Arrow-heads indicate the oligonucleotide primers used for PR analysis: blue, SERA6-34; yellow, JTS5synthF; brown, JTPbDT3’R (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157873#sec008" target="_blank">Materials and Methods</a> for primer sequences and PCR parameters). Expected sizes of the PCR amplicons are indicated.</p