Loss of SERA5 results in accelerated schizont rupture but defective egress.

<p>(A) Stills from time-lapse DIC microscopic imaging showing stages in rupture of mock (DMSO)-treated (control) and RAP-treated (ΔSERA5) schizonts of <i>P</i>. <i>falciparum</i> clone floxSERA5-1B6. Movies were started exactly 4.5 min following removal of the reversible PKG inhibitor compound 2 (time following washing away the inhibitor is indicated). ΔSERA5 parasites underwent accelerated membrane rupture which had often already started even at the commencement of imaging (white arrows) with only gradual dispersal of the merozoites, unlike the ‘explosive’ egress and merozoite scattering typical of control parasites. Egress of the control parasites did not commence in this experiment until ~9 min (an example is indicated, black arrow). Scale bar, 20 μm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s001" target="_blank">S1 Movie</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s002" target="_blank">S2 Fig</a>. (B) Quantitative profiling of the timing of membrane rupture in the control and ΔSERA5 parasites. Whereas egress in the control parasites only rarely took place before 9 min, much of the membrane rupture evident in the ΔSERA5 parasites had already occurred by that point. Data were collated from visual examination of frames from 3 separate videos each of mock and RAP-treated clone floxSERA5-1B6 (total number of egress events: mock-treated, 178, RAP-treated, 179;). Time of egress is recorded to the nearest 0.5 min. Note that because imaging began 4.5 min following washing, all rupture events in the RAP-treated population that had already taken place at the beginning of the video microscopy are recorded as occurring at 4.5 min. Time to egress statistics were calculated for the RAP-treated parasites (mean 7.9 min, SD 3.2 min) and for the control parasites (mean 13.7 min, SD 3.3. min), with a two-tailed unpaired <i>t</i>-test revealing the difference to be extremely significant (t = 16.8268, d.f. = 355, p <0.0001). (C) Stills from time-lapse DIC microscopic images of individual control and ΔSERA5 schizonts, showing the very different characteristics of merozoite dispersal in the period immediately following the point of schizont rupture (the first still was imaged 10 seconds prior to rupture). Scale bar, 10 μm. (D) Coomassie-stained, SDS PAGE-fractionated and Western blot analysis of supernatants from mock and RAP-treated (ΔSERA5) floxSERA5-1B6 schizonts following release from a compound 2 block, showing more rapid release of schizont soluble proteins, including proteolytically processed forms of SUB1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref059" target="_blank">59</a>] from the ΔSERA parasites. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s003" target="_blank">S3 Fig</a>.</p

Model for role of SERA5 in regulating a lag phase that enables efficient bounding membrane disruption at egress.

<p>Schematic model of the role of SERA5 based on the results of this and previous studies. The erythrocyte and PV membranes of mature schizonts are indicated in red and green respectively. SUB1 in exonemes of mature segmented schizonts is depicted as blue dots, whilst full-length SERA5 is shown as black strands within the PV. Evidence that the PVM becomes porous prior to PKG activation, that PVM disruption results in vesiculated ‘rolls’ of membrane and that erythrocyte membrane rounding up and/or poration is associated with rapid collapse of the host cell cytoskeleton is from earlier work [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref026" target="_blank">26</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref032" target="_blank">32</a>]. Timelines shown to egress are approximate minima and are based on reversible inhibition of egress by compound 2. The final step of host cell membrane rupture is sensitive to E64 in both wild type and ΔSERA5 parasites. However, in the presence of E64 the time to PVM rupture is similar for wild type and ΔSERA5 mutants (not indicated schematically). RBC, red blood cell.</p

ΔSERA5 parasite clones show a replication defect but SERA5 is not essential for asexual blood stage growth <i>in vitro</i>.

<p>(A) Scatter plots showing plaque numbers and distribution of plaque sizes obtained following treatment of the parental 1G5DC clone or conditional knockout clones floxSERA5-1B6 and floxSERA5-3B6 with DMSO (control, mock-treated) or RAP to induce disruption of the <i>SERA5</i> gene. In each case plaque numbers were counted from a total of 24 microwells. Note that plaque numbers were lower in the RAP-treated floxSERA5-1B6 and floxSERA5-3B6 clones (n = 60 and 20 respectively) than in the mock-treated samples (n = 94 and 86 respectively). Plaque dimensions in scanned images of the plates were quantified using the Lasso tool of Photoshop CS6 (Adobe). Horizontal bars, mean plaque area ±1 SD. Brackets and asterisks indicate parasite populations for which statistically different plaque sizes were obtained (Student’s two-tailed <i>t</i>-test). The difference in plaque size for the +/- RAP-treated parental 1G5DC parasites were not significant (p-value = 0.1564). (B) Western blot confirmation of ΔSERA5 parasite clones obtained from plaque assay wells containing a single plaque. Clone 2F8_ΔSERA5 (derived from RAP-treated clone floxSERA5-1B6) and C6_ΔSERA5 (derived from RAP-treated clone floxSERA5-3B6) were expanded, then schizont extracts probed with antibodies to SERA3, SERA4, SERA5 and SERA6. (C) Growth assay showing relative replication rates of ΔSERA5 <i>P</i>. <i>falciparum</i> clones 2F8_ΔSERA5 and C6_ΔSERA5 compared to their parental floxSERA5-1B6 and floxSERA5-3B6 clones (not RAP-treated), over 3 erythrocytic cycles. Cultures were fed daily by replacing the medium, but were not passaged by addition of fresh erythrocytes. Parasitaemia values were determined by FACS as described in Materials and Methods. Data are averaged from 3 independent biological replicate experiments. Error bars, ±SEM. Relative to the parental parasites, the ΔSERA5 clones showed a mean reduction in replication rate per cycle of 28±3%.</p

Simultaneous disruption of both the <i>SERA4</i> and <i>SERA5</i> genes results in a replication defect similar to that produced by <i>SERA5</i> disruption alone.

<p>(A) Western blot analysis of the C10_ΔSERA4/5 and G8_ΔSERA4/5 <i>P</i>. <i>falciparum</i> clones (as well as the 1G5DC parental clone as a control), confirming loss of expression of both SERA4 and SERA5 in the double knockout parasites (note that the 1G5DC loading control blots are the same as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.g003" target="_blank">Fig 3</a>, since all the Western blots were performed at the same time). (B) Growth assay showing relative replication rates of the ΔSERA4/5 <i>P</i>. <i>falciparum</i> clones compared to the single knockout 2F8_ΔSERA5 and C6_ΔSERA5 clones, over 3 erythrocytic cycles. Cultures were fed daily by replacing the medium, but were not passaged by addition of fresh erythrocytes. Parasitaemia values were determined by FACS. Data are averaged from 5 independent biological replicate experiments. Error bars, ±SEM. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s005" target="_blank">S5 Fig</a>.</p

Genetic complementation experiments show that proteolytic processing of SERA5 is important for its function.

<p>(A) Left-hand side: transgene constructs for genetic complementation of the ΔSERA5 parasite clone 2F8_ΔSERA5. All constructs were designed for constitutive expression of cytoplasmic mCherry, driven by the <i>P</i>. <i>falciparum</i> CAM promoter. Constructs pDC2_mC_sgS5 and pDC2_mC_sgS5mut additionally incorporated cassettes for expression of a synthetic recodonised <i>SERA5</i> gene (under control of the <i>SERA5</i> promoter) modified by inclusion of an internal mini TAP tag which includes the HA3 epitope [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref006" target="_blank">6</a>]. Positions of mutations (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s006" target="_blank">S6 Fig</a> for details of mutations) introduced into pDC2_mC_sgS5mut to prevent processing by SUB1 and protease X [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref006" target="_blank">6</a>] are indicated (arrowheads). <i>bsd</i>, blasticidin deaminase drug resistance cassette. Right-hand side: IFA of the blasticidin-resistant parasite lines selected following transfection of the SERA5-null parasite clone 2F8_ΔSERA5 with the constructs. Reactivity with the anti-HA3 mAb 3F10 was observed only in mature schizonts of the 2F8_ΔSERA5:SERA5wt and 2F8_ΔSERA5:SERA5mut lines, in a pattern typical of PV localisation. For clarity, the merged images do not include the DAPI signal. Scale bar, 10 μm. (B) Western blot analysis of purified mature schizonts of the transfected parasite lines, either following incubation for 4 h in the presence of the PKG inhibitor compound 2 (C2 blocked) or 40 min after washing away of compound 2 to allow egress of the majority of the schizonts (egressed). This involves discharge of SUB1 and proteolytic processing of SERA5 and the merozoite surface protein MSP1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref018" target="_blank">18</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref027" target="_blank">27</a>]. HA3-tagged SERA5 was expressed only in the 2F8_ΔSERA5:SERA5wt and 2F8_ΔSERA5:SERA5mut lines. As expected, proteolytic processing of the protein was defective in the 2F8_ΔSERA5:SERA5mut line, leading to a species (asterisked) that was much larger than the HA3-tagged processing product of wild-type SERA5 (indicated and labelled). In contrast correct SUB1-mediated processing of MSP1 to form the MSP1₈₃ processing product [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.ref027" target="_blank">27</a>] occurred similarly in all three parasite lines. (C) Quantitative profiling plot of the timing of egress in the 2F8_ΔSERA5:SERA5wt and 2F8_ΔSERA5:SERA5mut parasite lines. In each case, time to egress of mCherry-positive schizonts (red) following washing away of a compound 2 block is shown compared with that of mCherry-negative parasites (grey) in the same fields. The mCherry-positive schizonts were defined as those with a mean fluorescence intensity value of ≥30 under standardised fluorescence imaging conditions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#sec010" target="_blank">Materials and Methods</a>). Time-lapse imaging began precisely 4.5 min following washing, and data were collated from visual examination of frames from 3 separate videos each of the two lines. Time to egress statistics were calculated for the mCherry-positive 2F8_ΔSERA5:SERA5wt parasites (mean 17.08 min, SD 4.52 min) and for the mCherry-negative 2F8_ΔSERA5:SERA5wt parasites (mean 12.0 min, SD 6.42 min), with a two-tailed unpaired <i>t</i>-test revealing the difference to be extremely significant (t = 3.5985, d.f. = 59, p <0.001). In contrast, similar analysis of the time to egress statistics for the mCherry-positive 2F8_ΔSERA5:SERA5mut parasites (mean 13.28 min, SD 5.91 min) and mCherry-negative 2F8_ΔSERA5:SERA5mut parasites (13.69 min, SD 5.57 min) revealed the difference to be non-significant (t = 0.2397, d.f. = 45, p = 0.812). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s015" target="_blank">S9 Movie</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s016" target="_blank">S10 Movie</a>.</p

DiCre-mediated conditional disruption of <i>P</i>. <i>falciparum</i> SERA5 expression.

<p>(A) Strategy for conditional deletion of the <i>SERA5</i> gene. Transgenic <i>P</i>. <i>falciparum</i> clone 1G5DC contains a wild-type <i>SERA4</i> gene (white boxes, introns indicated as gaps) upstream of a fully functional chimeric <i>SERA5</i> gene (recodonised sequence, hatched). This is followed by a single <i>loxP</i> site (black arrow head) and the DiCre expression cassette (grey box). The targeting constructs contained ~1.0 kb (pSERA4loxPa) or ~1.4 kb (pSERA4loxPb) of 3´ <i>SERA4</i> sequence to drive integration of the entire construct by single-crossover homologous recombination. In both cases the targeting sequence extended to and included the <i>SERA4</i> stop codon and was followed by the 3′ UTR of the <i>P</i>. <i>berghei</i> dihydrofolate reductase (P<i>bdt</i>) gene to ensure correct regulation of the modified <i>SERA4</i> gene. Correct integration was expected to reconstitute the <i>SERA4</i> gene whilst introducing a second <i>loxP</i> site downstream of the introduced human dihydrofolate reductase-thymidylate synthase (<i>hdhfr</i>) selection cassette which confers resistance to the antifolate WR99210. DiCre-mediated recombination was predicted to excise the entire sequence between the <i>loxP</i> sites, including the <i>SERA5</i> gene. Positions of hybridisation of primers used for diagnostic PCR analysis of integration and excision events are shown as coloured arrows. Primer identities are: a, S4_F4; b, S4_DS_R1; c, CAM5´_R3; d, hsp86_3´_R1 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s017" target="_blank">S1 Table</a> for sequences of primers used in this study). Insert, diagnostic PCR analysis of genomic DNA from the parental 1G5DC <i>P</i>. <i>falciparum</i> clone and integrant clones, confirming the predicted integration events. The expected sizes of the various PCR amplicons are indicated. (B) Diagnostic PCR analysis of genomic DNA from RAP-treated or control parental 1G5DC and integrant <i>P</i>. <i>falciparum</i> clones, confirming the predicted DiCre-mediated excision events. The expected sizes of the PCR amplicons specific for the intact or excised locus are indicated. (C) IFA of mature schizonts of control (DMSO-treated) and RAP-treated integrant clone floxSERA5-1B6 ~44 h following treatment. No SERA5-specific signal was detectable in the majority (~98%) of the RAP-treated population. Scale bar, 5 μm. (D) Western blot of mature schizonts of the integrant clones ~44 h following RAP-treatment or mock treatment, showing near complete loss of the SERA5 signal. As a loading control, blots were probed with antibodies to both SERA5 and the major merozoite surface protein MSP1. Quantitation of the residual SERA5 signal in the RAP-treated parasites (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s001" target="_blank">S1 Fig</a>) indicated that this was likely due to lack of DiCre-mediated gene excision in a small proportion (~2%) of the parasite population.</p

SERA5 does not play a role in host cell membrane poration or vesiculation at egress.

<p>(A) Stills from simultaneous time-lapse DIC and fluorescence microscopic imaging showing ingress of Alex Fluor 488-labelled phalloidin into intact mock-treated (control) and RAP-treated (ΔSERA5) schizonts of <i>P</i>. <i>falciparum</i> clone floxSERA5-3B6 in the presence of E64. Movies were started exactly 4.5 min following removal of compound 2 (time following washing away the inhibitor is indicated). Scale bar, 20 μm. (B) Quantitative profiling of the timing of phalloidin labelling in the control and ΔSERA5 schizonts. Data were collated from visual examination of frames from 3 separate videos each of mock and RAP-treated parasites (total number of phalloidin labelling events: RAP-treated, 56; mock-treated, 51). Time to labelling is indicated to the nearest 0.5 min. Statistics were calculated for the ΔSERA5 parasites (mean 23.7 min, SD 7.9 min) and the control parasites (mean 27.2 min, SD 7.6. min), with a two-tailed unpaired <i>t</i>-test revealing the difference to be just significant (t = 2.3260, d.f. = 105, p = 0.0219). (C) Stills from simultaneous time-lapse DIC and fluorescence microscopic imaging of control and ΔSERA5 floxSERA5-3B6 schizonts in the presence of fluorescent phalloidin but the absence of E64, showing vesiculation and phalloidin labelling of the disrupted host cell membranes following egress. Note that the released merozoites are slightly out of focus in these images in order to focus on the vesiculated membranes. Scale bar, 10 μm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006453#ppat.1006453.s004" target="_blank">S4 Fig</a>.</p

Structurally distinct PfPKG inhibitors block PfSUB1-mediated proteolytic processing in <i>P. falciparum</i>.

<p>(A) Molecular structures of C1 and C2. (B–D) Mature wild-type (wt 3D7) or PfPKG_T618Q<i>P. falciparum</i> schizonts were supplemented with cysteine protease inhibitor E64 only (50 µM), C1 only (2.5 µM), C2 only (1.5 µM), or combinations indicated, or vehicle only (DMSO, 1% v/v). Cultures were sampled immediately (start) or after further incubation for 4 h. SDS extracts of the residual schizonts (B, C) or culture supernatants (D) were analysed by Western blot, probing with mAbs specific for MSP1 or SERA5. An antibody to erythrocyte spectrin was used as loading control for the schizont extracts. Processing of MSP1 by PfSUB1 converts it to 4 polypeptides that include the MSP1₄₂ fragment <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344-Koussis1" target="_blank">[4]</a>. In wt parasites, but not in the PfPKG_T618Q clone, both C1 and C2 inhibited PfSUB1-mediated conversion of MSP1 to MSP1₄₂ (B) or conversion of the SERA5 precursor P126 to the processing products P56 and P50 (C), although some background processing is evidence in the presence of the inhibitors, perhaps due to imperfect synchrony of the parasite cultures. The relatively low abundance of processed SERA5 in the PfPKG_T618Q schizont extracts in the presence of C1 or C2 only (C) are likely due to their loss from the schizonts at egress. Note that E64 inhibits conversion of P56 to P50, which is not mediated by PfSUB1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344-Yeoh1" target="_blank">[1]</a>. In (D), treatment of wt 3D7 with C1 or C2 resulted in release of only low levels of unprocessed SERA5 P126, whereas the normally-processed P50 form appeared in the PfPKG_T618Q supernatants irrespective of the presence of the PfPKG inhibitors. See also Figure S1 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a>.</p

PfPKG inhibitors block discharge of exonemes and micronemes.

<p>(A) IFA of wt 3D7 parasites allowed to develop beyond the point of egress in the presence of E64 only (50 µM). Top two rows: segmented schizonts displaying a merozoite surface localisation of PfAMA1 (arrowed; ∼45% of the schizont population) as a result of its discharge from micronemes, exhibit a weak PfSUB1 signal compared to less mature schizonts in which both the PfAMA1 and PfSUB1 signals remain punctate. The bottom row of images in (A) shows a schizont in which the PfAMA1 signal is intermediate between punctate and merozoite surface. (B) Surface translocation of PfAMA1 is associated with loss or redistribution of the PfSUB1-specific IFA signal. The PfSUB1-specific mean fluorescence intensity in individual schizonts relative to that of the PfAMA1-specific signal was acquired as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#s4" target="_blank">Materials and Methods</a>. Individual relative fluorescence intensity values (at least 17 schizonts per group) are shown plotted, with mean and SD indicated. Within the parasites treated with E64 only, values were significantly lower for the schizonts displaying the merozoite surface PfAMA1 phenotype (Student's unpaired <i>t</i>-test, two-tailed P value<0.0001). There was no significant difference between the E64-treated and E64 plus C1-treated values for schizonts with the punctate PfAMA1 phenotype (see panel C). (C) IFA of wt 3D7 parasites allowed to develop beyond the point of egress in the presence of both E64 and C1. No discharge or relocalisation of PfSUB1 or PfAMA1 was evident in counts of >5,000 schizonts from a total of 3 independent experiments. Identical results were obtained with E64 plus C2 (not shown). (D) Western blot of culture supernatants from synchronous wt 3D7 or PfPKG_T618Q schizonts allowed to develop beyond the point of egress in the presence of E64 only (50 µM), C1 only (2.5 µM), C2 only (1.5 µM), or indicated combinations. Cultures were sampled immediately (start) or after incubation for 4 h. The p54 and p47 forms of PfSUB1 usually observed following its maturation are arrowed. The relatively low levels of PfSUB1 in supernatants of the PfPKG_T618Q parasites in the presence of C1 or C2 plus E64 are likely due to the block in egress mediated by E64. See also Figure S3 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a>.</p

Zaprinast induces rapid discharge of exonemes and micronemes.

<p>(A) Zaprinast induces intracellular processing of MSP1 and SERA5. Western blot of schizonts treated as indicated in the additional presence of E64 to prevent egress. (B) Zaprinast treatment of schizonts (in the presence of E64) induces rapid relocalisation of PfAMA1 and loss of PfSUB1. Fixed, permeabilized schizonts were examined by IFA with the antibodies indicated. See also Figure S5 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a>.</p