The p47 pfsub-1 gene product localizes to dense granules within the apical domain of free P. falciparum merozoites. Top, air-dried smears of naturally released merozoites were fixed with acetone and then probed with anti-PfSUB-1m antibodies. Bound antibody was detected with a Texas Red conjugated anti-mouse IgG. The preparations were finally incubated briefly with an FITC-conjugated monoclonal antibody (2F10) reactive with MSP-1 and examined by confocal microscopy. The plate consists of an overlaid z series of 12 images recorded at 400-nm intervals, projected as a single image, with Texas Red fluorescence and FITC fluorescence shown overlaid. Bottom, thin sections of resin embedded merozoites (A) or mature schizonts (B and C) were probed with the anti-PfSUB-1m antiserum, and then bound antibodies were detected using a gold-labeled anti-mouse IgG antibody. Dense granules (D), rhoptries (R), and micronemes (Mi) are indicated, as is the cytoplasm of the infected erythrocyte (E). Bar, 0.5 mm.Blackman MJ, Fujioka H, Stafford WH, Sajid M, Clough B, Fleck SL, Aikawa M, Grainger M, Hackett F. A subtilisin-like protein in secretory organelles of Plasmodium falciparum merozoites. J Biol Chem. 1998 273:23398-409.

(A) Schizonts of 3D7SUB1HA3 clone C10 dual-labeled with the anti-HA mAb 3F10 (aHA; green) plus mAb 61.3 (aRhopH2), mAb 4G2 (aPfAMA1; micronemes, or mAb 28/2 (aRESA; dense granules. Nuclei were stained with DAPI (blue). Merged images (no DAPI) show that PfSUB1 localizes with none of the other markers. The scale bar represents 5 mm. Identical results were obtained with 3D7SUB1HA3 clone F7 or by using mAbs specific for RAP2 or EBA-175 as markers for rhoptries and micronemes respectively (data not shown). (B–G) Immunoelectron microscopic localization of PfSUB1 in P. falciparum schizonts. (B and C) Labeling of an elongated organelle, arrowed in (B) and at higher magnification in (C), by anti-PfSUB1 antibodies labeled with 10 nm immunogold. (D and E) Two more examples of PfSUB1 positive organelles are shown, also labeled with 10 nm immunogold. These are less elongate than in (B) and (C), reflecting some variation in organelle shape; however, they are typically ellipsoidal, in contrast with the rounded dense granules. (F and G) Staining for RESA with mAb 28/2 and 5 nm immunogold. In (F), an unlabeled microneme is also shown for size comparison. In (G) double staining for PfSUB1 and RESA with different sizes of immunogold shows that the two proteins are in different organelles.Yeoh S, O'Donnell RA, Koussis K, Dluzewski AR, Ansell KH, Osborne SA, Hackett F, Withers-Martinez C, Mitchell GH, Bannister LH, Bryans JS, Kettleborough CA, Blackman MJ. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell. 2007 131:1072-83.

Fluorescent microscopy images of localization of PfSUB1 in schizonts and merozoites stained with anti-PfSUB1 mouse sera and anti-EBA-175 rabbit sera. Nuclear DNA was counterstained with DAPI. PfSUB1 showed punctuate apical staining in schizonts and merozoites, distinct from staining of microneme marker EBA-175.Agarwal S, Singh MK, Garg S, Chitnis CE, Singh S. Ca2+ Mediated Exocytosis of Subtilisin-like Protease 1: A Key Step in Egress of P. falciparum Merozoites. Cell Microbiol. 2013 15(6):910-21

Indirect immunofluorescence assays on air-dried P.falciparum 3D7 segmented schizonts (upper), individual merozoites (lower). Merozoite nuclei are labeled with Hoescht 33342. The scale bar represents 3μm. A typical punctate pattern is seen with partial superposition with the signal produced by the anti-AMA1 mAb 28G2. This is in line with the subcellular localization of PfSUB1 and AMA1 in the exonemes and micronemes, respectively. Bouillon A, Giganti D, Benedet C, Gorgette O, Pêtres S, Crublet E, Girard-Blanc C, Witkowski B, Ménard D, Nilges M, Mercereau-Puijalon O, Stoven V, Barale JC. In silico screening on the 3D-model of the Plasmodium vivax SUB1 protease leads to the validation of a novel anti-parasite compound. J Biol Chem. 2013 288(25):18561-73

IFA of WT schizonts with glycophorin A (GA) labeled erythrocytes, localized SUB1 in an apical punctate pattern similar to SERA5. EBA-175 was detected in the apical ends of merozoites. GAP45 appeared submembrane. DIC, differential interference contrast; Hoechst = nucleic acid stain.Balu B, Maher SP, Pance A, Chauhan C, Naumov AV, Andrews RM, Ellis PD, Khan SM, Lin JW, Janse CJ, Rayner JC, Adams JH. CCR4-associated factor-1 coordinates expression of Plasmodium falciparum egress and invasion proteins. Eukaryot Cell. 2011 10(9):1257-63

D) Segmented schizonts of 3D7SUB1HA3clone C10 dual-labeled with mAb 3F10 (aHA; green) and mAb 1E1 (aMSP119; red). Merged images (no DAPI) show partial colocalization of PfSUB1HA3 and MSP1. Dual labeling suggestisthat near the end of schizogony PfSUB1 can translocate from its previous organellar location into the PV. (E) A ‘‘cloud’’ of PfSUB1HA3 around a bursting schizont of 3D7SUB1HA3 clone C10 dual-labeled with mAb 3F10 (aHA; green) and mAb 61.3 (aRhopH2). The right-hand panel shows the merged image. The scale bar represents 5 mm. Where bursting schizonts were visible, the free merozoites were surrounded by a ‘‘cloud’’ of anti-HA signal, suggesting that at the point of egress PfSUB1 discharge had already begun.Yeoh S, O'Donnell RA, Koussis K, Dluzewski AR, Ansell KH, Osborne SA, Hackett F, Withers-Martinez C, Mitchell GH, Bannister LH, Bryans JS, Kettleborough CA, Blackman MJ. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell. 2007 131:1072-83.

Localization of PfAEP in the exonemes of merozoites (A) Co-localization of PfAEP was studied with micronemal resident protein AMA-1 in bursting schizonts. PfAEP (rabbit serum; red) does not co-localize with AMA-1 (green). The staining for PfAEP co-localized with exonemal marker PfSUB1. Scale bar 2 mm. Three-dimensional reconstruction of the confocal z-stack images of schizonts is shown in panel below Scale bar 1.5mm (B) 2D-structured illumination microscopy (SIM) showed co-localization between the staining of PfAEP and PfSUB1 indicating its localization in the exonemes of P. falciparum merozoites. (C) Young rings were immunostained with anti PfAEP antibody and the antibody against MSP119 as a positive control. No staining was observed for PfAEP in young rings whereas MSP119 showed typical disk like staining around the nucleus.Hans N, Relan U, Dubey N, Gaur D, Chauhan VS. Identification and localization of a Novel Invasin of Plasmodium falciparum. Mol Biochem Parasitol. 2015 Sep 29. [Epub ahead of print]

C1 and C2 do not affect expression, trafficking or maturation of PfSUB1. (A) IFA of mature wt 3D7 parasites following treatment for 6 h with DMSO only (1% v/v) or 2.5 mM C1, probed with the anti-PfSUB1 mAb NIMP.M7 or a rabbit anti-PfAMA1 serum. The PfPKG inhibitor had no effect on localisation or expression of PfSUB1 or PfAMA1. For clarity, the anti-PfSUB1 and anti-PfAMA1 signals are shown merged in addition to a final merge with the DAPI signal. Similar results were obtained following treatment with 1.5 mM C2 (not shown). Note that treatment with the PKG inhibitors also had no effect on schizont replication as determined by counts of the average number of nuclei per schizont (data not shown, but compare DAPI staining of untreated and treated schizonts). Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 2013 9(5):e1003344.

PfPKG inhibitors block discharge of exonemes and micronemes. (A) IFA of wt 3D7 parasites allowed to develop beyond the point of egress in the presence of E64 only (50 mM). 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. (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).Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 2013 9(5):e1003344.

Zaprinast (PKG inhibitor) induces rapid discharge of exonemes and micronemes. (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. Zaprinast-induced relocalisation of PfSUB1 and PfAMA1 was evident even in immature (non-segmented) schizonts, but here took the form of translocation of both proteins to a striking circumferential punctate pattern, perhaps indicating accumulation of immature micronemes and exonemes at the periphery of the schizont.Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 2013 9(5):e1003344.

Truncation of MSP1 Produces an Egress Defect. (C) RAP treatment produces a loss of mAb X509 reactivity and a shift in the IFA pattern of MSP1 to one typical of PV proteins, consistent with the predicted truncation. Numbers of DAPI-stained nuclei did not differ between control and RAP treated schizonts (mean values: 21.2 ± 3.4 and 20.6 ± 4.0 nuclei per schizont, respectively, n = 24). (D) IFA showing co-localization of truncated MSP1 with SERA5 indicating a PV location. The punctate localization of SUB1 and the microneme protein AMA1 indicates normal organelle biogenesis. (E) IFA showing lack of surface-bound MSP1 on merozoites of RAP-treated 3D7MSP1flox42C1 clone E3. Antibodies to AMA1 (which is expressed on free merozoites) were used as a control. Analysis of RAP-treated 3D7MSP1flox42C parasites showed highly efficient excision, resulting in exclusive expression of truncated MSP1 in mature schizonts at the end of the same erythrocytic cycle (C). No effects on merozoite development were discernible. The modified MSP1 was trafficked to the PV as expected for a non-membrane bound merozoite surface protein (C–D) but was not present on the surface of free merozoites (E)Das S, Hertrich N, Perrin AJ, Withers-Martinez C, Collins CR, Jones ML, Watermeyer JM, Fobes ET, Martin SR, Saibil HR, Wright GJ, Treeck M, Epp C, Blackman MJ. Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs. Cell Host Microbe. 2015 18(4):433-44. PMID: 26468747

In-depth analysis of protease localization. A) IFAs using polyclonal mouse antisera directed against PfSUB1, PfαβH and PfM16 were used to investigate the granular localization of these proteases in merozoite-containing mature schizonts (in green). The merozoites were visualized via immunolabelling with polyclonal rabbit antisera directed against PfMSP1 (in red). PfM16 localized to granular structures in schizonts, which did not coincide with the merozoites B) Immunolabeling of osmiophilic bodies using polyclonal rabbit antisera directed against Pfg377 (in red) was employed to demonstrate an accumulation of PfPM6, labeled with the respective mouse antisera (in green), in these organelles. Hoechst 33342 staining was used to highlight the parasite nuclei (in blue). Bar, 2 μm. Results are representative of two independent experiments. The localization of PfPM6 in female gametocytes was don by co-localization experiments were performed, using rabbit antibodies directed against the osmiophilic body protein Pfg377. The double-labelling assays demonstrated that PfPM6 is present in these organelles.Weißbach T, Golzmann A, Bennink S, Pradel G, Julius Ngwa C. Transcript and protein expression analysis of proteases in the blood stages of Plasmodium falciparum. Exp Parasitol. 2017 Mar 25. [Epub ahead of print]

Protein expression profile of selected P. falciparum proteases. IFAs using polyclonal mouse antisera directed against 12 of the proteases was used to demonstrate protein expression in the asexual blood stages (R, ring; T, trophozoite; iS, immature schizont; mS, mature schizont) and gametocytes (GC stages III, IV and V) as well as in macrogametes (MaG) at 20 min post-activation (in green). The asexual and the sexual blood sstages were visualized via immunolabelling with polyclonal rabbit antisera against PfMSP1 and Pfs230, respectively (in red). Hoechst 33342 staining was used to highlight the parasite nuclei (in blue). Bar, 5 μm. Results are representative of three to four independent experiments.Weißbach T, Golzmann A, Bennink S, Pradel G, Julius Ngwa C. Transcript and protein expression analysis of proteases in the blood stages of Plasmodium falciparum. Exp Parasitol. 2017 Mar 25. [Epub ahead of print]