Plasmodium parasitophorous vacuole membrane protein Pfs16 promotes malaria transmission by silencing mosquito immunity
2023; Elsevier BV; Volume: 299; Issue: 6 Linguagem: Inglês
10.1016/j.jbc.2023.104824
ISSN1083-351X
AutoresJulian Ramelow, Yacob Keleta, Guodong Niu, Xiaohong Wang, Jun Li,
Tópico(s)Insect Resistance and Genetics
ResumoWith rising cases for the first time in years, malaria remains a significant public health burden. The sexual stage of the malaria parasite infects mosquitoes to transmit malaria from host to host. Hence, an infected mosquito plays an essential role in malaria transmission. Plasmodium falciparum is the most dominant and dangerous malaria pathogen. Previous studies identified a sexual stage-specific protein 16 (Pfs16) localized to the parasitophorous vacuole membrane. Here, we elucidate the function of Pfs16 during malaria transmission. Our structural analysis identified Pfs16 as an alpha-helical integral membrane protein with one transmembrane domain connecting to two regions across parasitophorous vacuole membrane. ELISA assays showed that insect cell-expressed recombinant Pfs16 (rPfs16) interacted with Anopheles gambiae midguts, and microscopy found that rPfs16 was bound to midgut epithelial cells. Transmission-blocking assays demonstrated that polyclonal antibodies against Pfs16 significantly reduced the number of oocysts in mosquito midguts. However, on the contrary, feeding rPfs16 increased the number of oocysts. Further analysis revealed that Pfs16 reduced the activity of mosquito midgut caspase 3/7, a key enzyme in the mosquito Jun-N-terminal kinase immune pathway. We conclude that Pfs16 facilitates parasites to invade mosquito midguts by actively silencing the mosquito's innate immunity through its interaction with the midgut epithelial cells. Therefore, Pfs16 is a potential target to control malaria transmission. With rising cases for the first time in years, malaria remains a significant public health burden. The sexual stage of the malaria parasite infects mosquitoes to transmit malaria from host to host. Hence, an infected mosquito plays an essential role in malaria transmission. Plasmodium falciparum is the most dominant and dangerous malaria pathogen. Previous studies identified a sexual stage-specific protein 16 (Pfs16) localized to the parasitophorous vacuole membrane. Here, we elucidate the function of Pfs16 during malaria transmission. Our structural analysis identified Pfs16 as an alpha-helical integral membrane protein with one transmembrane domain connecting to two regions across parasitophorous vacuole membrane. ELISA assays showed that insect cell-expressed recombinant Pfs16 (rPfs16) interacted with Anopheles gambiae midguts, and microscopy found that rPfs16 was bound to midgut epithelial cells. Transmission-blocking assays demonstrated that polyclonal antibodies against Pfs16 significantly reduced the number of oocysts in mosquito midguts. However, on the contrary, feeding rPfs16 increased the number of oocysts. Further analysis revealed that Pfs16 reduced the activity of mosquito midgut caspase 3/7, a key enzyme in the mosquito Jun-N-terminal kinase immune pathway. We conclude that Pfs16 facilitates parasites to invade mosquito midguts by actively silencing the mosquito's innate immunity through its interaction with the midgut epithelial cells. Therefore, Pfs16 is a potential target to control malaria transmission. Malaria remains a significant global disease. In 2021, malaria case rates increased by 6% compared to the 2020 report (https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2021), and Africa remains the epicenter of malaria occurrence, reporting 94% of cases and deaths. About 241 million cases and 627,000 deaths were officially reported; the undocumented numbers are presumably higher. Therefore, a better understanding of the pathogenesis of this devastating disease is still needed. Several Plasmodium species, including Plasmodium falciparum, Plasmodium vivax, Plasmodium malaria, Plasmodium ovale, and Plasmodium knowlesi, are capable of infecting humans and causing the disease. P. falciparum accounts for approximately 99.7% of malaria cases in Africa and is the deadliest parasite (1Cowman A.F. Tonkin C.J. Tham W.H. Duraisingh M.T. The molecular basis of erythrocyte invasion by malaria parasites.Cell Host Microbe. 2017; 22: 232-245Google Scholar, 2Alonso P.L. Brown G. Arevalo-Herrera M. Binka F. Chitnis C. Collins F. et al.A research agenda to underpin malaria eradication.PLoS Med. 2011; 8e1000406Google Scholar). When a malaria parasite invades an erythrocyte, a portion of the host cell membrane will be invaginated to form a parasitophorous vacuole membrane (PVM) around the parasite. The PVM is modified by the parasite and integrated by parasite proteins through secretory organelles during parasite development (3Goldberg D.E. Zimmerberg J. Hardly vacuous: the parasitophorous vacuolar membrane of malaria parasites.Trends Parasitol. 2020; 36: 138-146Google Scholar). During the parasite development at the intraerythrocytic stage, some trophozoites experience sexual differentiation and develop into male and female gametocytes, which can infect anopheline mosquitoes, definitive hosts for parasite sexual reproduction (4Carter R. Mendis K.N. Miller L.H. Molineaux L. Saul A. Malaria transmission-blocking vaccines--how can their development be supported?.Nat. Med. 2000; 6: 241-244Google Scholar, 5McCoy K.D. Weldon C.T. Ansumana R. Lamin J.M. Stenger D.A. Ryan S.J. et al.Are malaria transmission-blocking vaccines acceptable to high burden communities? Results from a mixed methods study in Bo, Sierra Leone.Malar. J. 2021; 20: 183Google Scholar, 6Carter R. Transmission blocking malaria vaccines.Vaccine. 2001; 19: 2309-2314Google Scholar). After a mosquito takes infected blood, gametocyte egress comprises a set of sequential events including rapturing the PVM and releasing gametes and PVM pieces (7Andreadaki M. Hanssen E. Deligianni E. Claudet C. Wengelnik K. Mollard V. et al.Sequential membrane rupture and vesiculation during Plasmodium berghei gametocyte egress from the red blood cell.Sci. Rep. 2018; 8: 3543Google Scholar). Female and male gametes form ookinetes to infect mosquitoes. Many factors inside mosquito midguts affect parasite transmission (8Keleta Y. Ramelow J. Cui L. Li J. Molecular interactions between parasite and mosquito during midgut invasion as targets to block malaria transmission.NPJ Vaccines. 2021; 6: 140Google Scholar). In particular, several interactive mosquito or parasite proteins have been identified and linked to be crucial for parasite transmission to mosquitoes in either anchoring parasites (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar, 10Cui Y. Niu G. Li V.L. Wang X. Li J. 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Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar). The expression of Pfs16 is induced immediately following the invasion of a red blood cell in sexually committed ring-stage parasites and continues throughout gametocytogenesis (13Dechering K.J. Thompson J. Dodemont H.J. Eling W. Konings R.N. Developmentally regulated expression of pfs16, a marker for sexual differentiation of the human malaria parasite Plasmodium falciparum.Mol. Biochem. Parasitol. 1997; 89: 235-244Google Scholar). Notably, it is located primarily on the PVM of P. falciparum gametocytes (14Baker D.A. Daramola O. McCrossan M.V. Harmer J. Targett G.A. Subcellular localization of Pfs16, a Plasmodium falciparum gametocyte antigen.Parasitology. 1994; 108: 129-137Google Scholar). The Pfs16 gene knockout parasites showed a 4- to 5-fold reduction in gametocytes generated (15Kongkasuriyachai D. Fujioka H. Kumar N. Functional analysis of Plasmodium falciparum parasitophorous vacuole membrane protein (Pfs16) during gametocytogenesis and gametogenesis by targeted gene disruption.Mol. Biochem. Parasitol. 2004; 133: 275-285Google Scholar). Pfs16 KO parasites failed to infect mosquitoes when compared to WT parasites (15Kongkasuriyachai D. Fujioka H. Kumar N. Functional analysis of Plasmodium falciparum parasitophorous vacuole membrane protein (Pfs16) during gametocytogenesis and gametogenesis by targeted gene disruption.Mol. Biochem. Parasitol. 2004; 133: 275-285Google Scholar). Anopheles mosquitoes can deploy several effective antiplasmodial responses by activating innate immune signaling cascades (16Frolet C. Thoma M. Blandin S. Hoffmann J.A. Levashina E.A. Boosting NF-kappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei.Immunity. 2006; 25: 677-685Google Scholar, 17Garver L.S. Dong Y. Dimopoulos G. 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The JNK pathway also interplays with other immune pathways including the immune deficiency and Toll pathways by sharing key proteins, such as transforming growth factor-β-activated kinase 1 (25Park J.M. Brady H. Ruocco M.G. Sun H. Williams D. Lee S.J. et al.Targeting of TAK1 by the NF-kappa B protein Relish regulates the JNK-mediated immune response in Drosophila.Genes Dev. 2004; 18: 584-594Google Scholar, 26Silverman N. Zhou R. Erlich R.L. Hunter M. Bernstein E. Schneider D. et al.Immune activation of NF-kappaB and JNK requires Drosophila TAK1.J. Biol. Chem. 2003; 278: 48928-48934Google Scholar). Immune deficiency and Toll pathways mediate ookinete lysis (27Garver L.S. Bahia A.C. Das S. Souza-Neto J.A. Shiao J. Dong Y. et al.Anopheles Imd pathway factors and effectors in infection intensity-dependent anti-Plasmodium action.PLoS Pathog. 2012; 8e1002737Google Scholar). Previous studies showed antibodies against Pfs16-inhibited P. falciparum transmission to mosquitoes (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar). However, its specific role remains unknown. Several potential mechanisms exist where Pfs16 can exhibit its effects in malaria transmission to mosquitoes: (1) direct interaction to position parasites for midgut invasion, (2) enzymatic digestion to disrupt midgut physical barriers, (3) direct or indirect inhibition of the mosquito's innate immunity to promote transmission, among others. In this work, we found that Pfs16 facilitated malaria transmission by negatively regulating the JNK response in the mosquito immune activation pathway, increasing parasite oocysts in mosquito midguts. The findings provide insightful mechanisms of newly discovered parasite-mosquito interplay during malaria transmission and present Pfs16 as a prime candidate for transmission-blocking development. Pfs16 is 157 amino acid residues in length. SignalP (28Teufel F. Almagro Armenteros J.J. Johansen A.R. Gislason M.H. Pihl S.I. Tsirigos K.D. et al.SignalP 6.0 predicts all five types of signal peptides using protein language models.Nat. Biotechnol. 2022; 40: 1023-1025Google Scholar) and TMHMM (29Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Google Scholar) predicted that Pfs16 consists of a signal peptide from residue 1 to 25 for protein-membrane trafficking and a transmembrane domain from residue 105 to 125 (89–100 in the mature protein form) for PVM integration. AlphaFold2 (30Pakhrin S.C. Shrestha B. Adhikari B. Kc D.B. Deep learning-based advances in protein structure prediction.Int. J. Mol. Sci. 2021; 22: 5553Google Scholar) predicted the structure of the mature Pfs16 that contains one long α-helix from residue 54 to 100. The Pfs16 protein is synthesized by the parasite and integrated into PVM with the help of translocon machinery (31Beck J.R. Ho C.M. Transport mechanisms at the malaria parasite-host cell interface.PLoS Pathog. 2021; 17e1009394Google Scholar). Taking together, the mature Pfs16 is an integrated membrane protein with the N-terminal inside parasitophorous vacuole and the C-terminal in erythrocyte cytoplasm, respectively (Fig. 1A). To investigate the functional role of the Pfs16 protein, we previously generated rabbit polyclonal antibodies against Escherichia coli–expressed Pfs16 (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar). Using the antibodies, we determined the endogenous Pfs16 in cellular fraction using this polyclonal antibody by Western blotting assays. In brief, P. falciparum gametocytes were lysed with Tris buffer to obtain the soluble fraction. After centrifugation, the supernatant contained soluble protein, and the insoluble pellet was extracted with Tris–Triton to extract the membrane proteins. The soluble and membrane proteins were separated by SDS-PAGE and analyzed by Western blotting. The result showed that endogenous Pfs16 protein was detected only in the membrane fraction of the parasite lysate and not in the soluble fraction (Fig. 1B), confirming that Pfs16 is a membrane protein. We expressed recombinant Pfs16 protein (rPfs16) using the baculovirus expression system in High Five (Hi5) cells. The Pfs16 was cloned into pFastBac (Fig. 2A), which generated a recombinant baculovirus that expressed rPfs16 in Hi5 cells. The cells were harvested at different times by centrifugation. Proteins in cells were extracted by native cell lysis buffer that contains detergent and protease inhibitors. The amount of rPfs16 in the culture medium and cells was quantified with anti-His mAb by ELISA. The results showed that rPfs16 is highly expressed in Hi5 cells at 72 h and 96 h (Fig. 2B) but not in the culture medium, which is consistent with endogenous Pfs16. The rPfs16 protein was isolated from the cells via FPLC chromatography using HisTrap affinity column to a single major band on SDS-PAGE (Fig. 2C). We examined the effect of Pfs16 on P. falciparum infection in mosquito midguts by feeding rPfs16. To keep the rPfs16 protein in its native membrane forms and mimic PVM whorls, we mixed about 1 × 105 rPfs16-expressing Hi5 cells with 300 μl P. falciparum–infected blood containing about 1 × 105 stage V gametocytes and performed standard membrane-feeding assays (SMFAs). The same number of Hi5 cells containing empty vectors was used as the control. The results showed that the number of oocysts per midgut significantly increased when compared to the control group. We observed the same results in two replicates, with one having higher infection prevalence than the other (Fig. 3). In addition, we examined the difference in infection prevalence and found that in the low and high infection experiments, oocyst prevalence significantly increased from 39% to 60% and from 57% to 90%, respectively (p < 0.05). Since our previous report identified that antibodies against Pfs16 inhibited P. falciparum transmission to mosquitoes (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar), our initial hypothesis is that Pfs16 plays a similar role as FREP1, which anchors (32Zhang G. Niu G. Franca C.M. Dong Y. Wang X. Butler N.S. et al.Anopheles midgut FREP1 mediates Plasmodium invasion.J. Biol. Chem. 2015; 290: 16490-16501Google Scholar) and orientates parasites (33Zhang G. Niu G. Hooker D. Shabani S. Ramelow J. Wang X. et al.Targeting Plasmodium α-tubulin-1 to block malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2023; 131132647Google Scholar). If the initial hypothesis is true, the results from rPfs16 feeding assays were unexpected. Thus, we reexamined the effects of Pfs16-specific antibody-blocking assays. The purified rabbit polyclonal antibodies against Pfs16 were mixed with P. falciparum–infected blood and fed to mosquitoes to analyze antibody effects on malaria transmission. The purified rabbit nonspecific polyclonal antibodies were added to keep the total protein concentration at 0.4 mg/ml. Results showed that anti-Pfs16 antibodies significantly decreased the number of oocysts and infection prevalence in SMFAs (Fig. 4). We observed a dose-dependent decrease in oocyst load per midgut analyzed. The highest concentration had the highest inhibition. All concentrations of 0.1 mg/ml, 0.2 mg/ml, and 0.4 mg/ml were able to reduce oocyst load significantly (p < 0.001). The infection prevalence was calculated to be 88%, 72%, 58%, and 36% for the control, 0.1 mg/ml, 0.2 mg/ml, and 0.4 mg/ml groups, respectively. The prevalence decreased significantly compared to the control (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), calculated by the chi-square test. These results confirmed the previous report that antibodies against Pfs16 blocked malaria transmission (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar). Therefore, Pfs16 must function beyond simple anchoring (33Zhang G. Niu G. Hooker D. Shabani S. Ramelow J. Wang X. et al.Targeting Plasmodium α-tubulin-1 to block malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2023; 131132647Google Scholar). Since anti-Pfs16 antibodies and rPfs16 protein showed opposite effects on P. falciparum transmission to mosquitoes, we hypothesized that Pfs16 might inhibit mosquito innate immunity. Therefore, we examined the effects of anti-Pfs16 antibodies and rPfs16 protein on the activities of caspase 3/7, key enzymes in JNK pathway, in Anopheles gambiae midguts of uninfected blood-fed, P. falciparum–infected blood-fed, and naïve mosquitoes. Bovine serum albumin (BSA) in PBS was used as a control. We used the uninfected blood-fed mosquito midguts as a baseline to investigate the caspase 3/7 activity. A nonrelated protein, BSA in PBS, and nonspecific rabbit IgG were used as a protein and antibody control, respectively. Mosquito midguts were collected 18 h after treatments, and the activities of caspase 3/7 in midguts were measured using Apo-ONE. The relative fluorescence units were measured every 30 min for 5 h to optimize the incubation time. For the uninfected blood-fed mosquitoes, blood-feeding increased caspase activities compared with naïve mosquito midguts. The caspase 3/7 activities in rPfs16-fed mosquito midguts were lower than those in the controls, for example, BSA and nonspecific antibody (Ab)-fed mosquitoes (Fig. 5A). There was no significant difference for the caspase 3/7 activities among anti-Pfs16 Ab-fed mosquito midguts, BSA, and nonspecific Ab controls, consistent to the fact that there was no Pfs16 in uninfected blood. In the Plasmodium-infected A. gambiae midguts (Fig. 5B), we observed that rPfs16 reduced caspase activities compared with those in BSA and nonspecific Ab-fed controls. Different from noninfected blood, the infected blood contained endogenous Pfs16. Results show that upon adding anti-Pfs16 Ab, which neutralized endogenous Pfs16, the caspase activity in anti-Pfs16 Ab-treated midguts was the highest of all (Fig. 5B). Statistical analyses of all the treated mosquito midguts showed that rPfs16 significantly reduced caspase activities (p < 0.0001) compared with the controls, for example, BSA and nonspecific Ab-fed mosquitoes. Anti-Pfs16 significantly increased caspase activities for the infected blood-fed mosquitoes but not the uninfected blood-fed mosquitoes. Analyzing the infected blood-fed and the uninfected blood-fed A. gambiae midguts, by comparing Figure 5, A and B, also revealed that P. falciparum–infected blood significantly increased caspase activities in mosquito midguts, compared with noninfected blood, which is consistent with the fact that P. falciparum invasion stimulates mosquito immunity. Collectively, P. falciparum Pfs16 inhibits the activity of caspases, components of mosquito immune response. Since Pfs16 inhibits caspase activities of midgut cells, it should interact with epithelial cells. This is different from the previous report that suggested Pfs16 interacts with peritrophic matrix (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar). We reexamined this interaction with ELISA. We fed 3- to 5-day-old mosquitoes with blood and removed the blood bolus from mosquito midguts 24 h post blood meal. The isolated naïve and blood-fed mosquito midguts were homogenized, and supernatants (1 mg/ml proteins) were used to coat ELISA plates, followed by incubation with rPfs16. The retained rPfs16 was detected by rabbit polyclonal anti-Pfs16 antibody. The wells coated with BSA were used as the control. ELISA assays showed that the bound rPfs16 in both naïve and blood-fed midgut lysate-coated wells was significantly higher than the control (Fig. 6A). When comparing blood-fed versus sugar-fed midgut lysates, there was no significant difference. Since sugar-fed mosquitoes do not have a peritrophic matrix while blood-fed mosquitoes do, this result suggests that rPfs16 binds to molecules from epithelial. Blood-fed mosquito midgut sections were incubated with rPfs16 and detected with anti-Pfs16 antibody using indirect immunofluorescence assay. Substitution of rPfs16 with BSA was the negative control. Results showed that the rPfs16 protein (red color) bound to broad areas inside the A. gambiae midgut when compared to the BSA control (Fig. 6B). Specifically, the fluorescence intensity of epithelial cells is significantly stronger than that in the peritrophic matrix (Fig. 6C). This result is consistent with the ELISA result and our previous actual data (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar), i.e., Pfs16 bound to molecules at the epithelial cell layer, although we cannot exclude interactive molecules, which may also exist in the peritrophic matrix. Malaria remains a leading disease. Besides regular vaccines to prevent parasites from infecting humans, many studies over the last decade also focused on vaccine development to block malaria transmission, for example, transmission-blocking vaccines, and discovered some key targets such as Pfs25, Pfs47, and α-tubulin-1 from the parasite (12Molina-Cruz A. Canepa G.E. Alves E.S.T.L. Williams A.E. Nagyal S. Yenkoidiok-Douti L. et al.Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 2597-2605Google Scholar, 33Zhang G. Niu G. Hooker D. Shabani S. Ramelow J. Wang X. et al.Targeting Plasmodium α-tubulin-1 to block malaria transmission to mosquitoes.Front. Cell. Infect. 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Blocking malaria transmission by small molecules is also under investigation, and several fungal secondary metabolites have been identified (40Niu G. Annamalai T. Wang X. Li S. Munga S. Niu G. et al.A diverse global fungal library for drug discovery.PeerJ. 2020; 8e10392Google Scholar, 41Niu G. Hao Y. Wang X. Gao J.M. Li J. Fungal metabolite asperaculane B inhibits malaria infection and transmission.Molecules. 2020; 25: 3018Google Scholar, 42Niu G. Wang X. Hao Y. Kandel S. Niu G. Raptis R.G. et al.A novel fungal metabolite inhibits Plasmodium falciparum transmission and infection.Parasit. Vectors. 2021; 14: 177Google Scholar). The molecular mechanisms of parasite invasion inside mosquito midguts remain elusive. Recently, Pfs16 was reported to interact with mosquito midguts (9Niu G. Cui Y. Wang X. Keleta Y. Li J. Studies of the parasite-midgut interaction reveal Plasmodium proteins important for malaria transmission to mosquitoes.Front. Cell. Infect. Microbiol. 2021; 11654216Google Scholar). Pfs16 is localized to the PVM with two regions across the PVM, consistent with a previous report (43Eksi S. Williamson K.C. Protein targeting to the parasitophorous vacuole membrane of Plasmodium falciparum.Eukaryot. Cell. 2011; 10: 744-752Google Scholar). After egress, most Pfs16 protein found in the membrane remains termed multilaminated whorls (14Baker D.A. Daramola O. McCrossan M.V. Harmer J. Targett G.A. Subcellular localization of Pfs16, a Plasmodium falciparum gametocyte antigen.Parasitology. 1994; 108: 129-137Google Scholar), which ruptures into multilaminar vesicles (44Hale V.L. Watermeyer J.M. Hackett F. Vizcay-Barrena G. van Ooij C. Thomas J.A. et al.Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 3439-3444Google Scholar, 45Glushakova S. Mazar J. Hohmann-Marriott M.F. Hama E. Zimmerberg J. Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes.Cell. Microbiol. 2009; 11: 95-105Google Scholar). Since protozoan parasites are known to be resourceful to be effective invaders/evaders, it is plausible that the sexual stage parasites use the remaining multilaminated whorls as a type of extracellular vesicles or exosomes for communication, pathogenesis, and/or immune regulation (46Co
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