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Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ∼35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48–61) large-scale proteolysis of the cytoskeleton during rupture ∼48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release. Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ∼35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48–61) large-scale proteolysis of the cytoskeleton during rupture ∼48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release. Malaria is a global disease causing at least 500 million new clinical cases per year, which result in more than 1 million deaths (1Hay S.I. Guerra C.A. Gething P.W. Patil A.P. Tatem A.J. Noor A.M. Kabaria C.W. Manh B.H. Elyazar I.R. Brooker S. Smith D.L. Moyeed R.A. Snow R.W. A world malaria map: Plasmodium falciparum endemicity in 2007.PLoS Med. 2009; 6: e1000048Crossref PubMed Scopus (491) Google Scholar). Efforts to control malaria include small molecule-based prophylaxis and therapy, and insect vector elimination. However, the emergence of multidrug resistant Plasmodium falciparum, the cause of most malaria-associated deaths in humans, necessitates the discovery of novel antimalarials. P. falciparum traverses a complex life cycle involving sexual (mosquito) stages and multiple rounds of asexual division within the human host. The asexual blood stage begins when extracellular merozoites (initially released from the liver) invade red blood cells (RBCs) 1The abbreviations used are:RBCred blood celliRBCinfected red blood cellPVparasitophorous vacuolehpihours post-infectionAFMatomic force microscopyRPMIRoswell Park Memorial InstitutePBSphosphate-buffered salineSERAserine repeat antigen. 1The abbreviations used are:RBCred blood celliRBCinfected red blood cellPVparasitophorous vacuolehpihours post-infectionAFMatomic force microscopyRPMIRoswell Park Memorial InstitutePBSphosphate-buffered salineSERAserine repeat antigen. in the blood stream. Over the next 48 h, intracellular parasites differentiate into the “ring ” stage, metabolize hemoglobin (trophozoite stage), and replicate (schizont stage) to produce expanded populations of invasive merozoites that are released into the circulating blood after rupture of their host cell. red blood cell infected red blood cell parasitophorous vacuole hours post-infection atomic force microscopy Roswell Park Memorial Institute phosphate-buffered saline serine repeat antigen. red blood cell infected red blood cell parasitophorous vacuole hours post-infection atomic force microscopy Roswell Park Memorial Institute phosphate-buffered saline serine repeat antigen. During this erythrocytic cycle, the malaria parasite causes major changes to its host cell. P. falciparum infection reduces deformability (2Maier A.G. Rug M. O'Neill M.T. Brown M. Chakravorty S. Szestak T. Chesson J. Wu Y. Hughes K. Coppel R.L. Newbold C. Beeson J.G. Craig A. Crabb B.S. Cowman A.F. Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes.Cell. 2008; 134: 48-61Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) and changes the adhesive properties of the host RBC, because of the appearance of knoblike protrusions on the surface (3Sherman I.W. Crandall I. Smith H. Membrane proteins involved in the adherence of Plasmodium falciparum-infected erythrocytes to the endothelium.Biol. Cell. 1992; 74: 161-178Crossref PubMed Scopus (39) Google Scholar, 4Smith H. Crandall I. Prudhomme J. Sherman I.W. Optimization and inhibition of the adherent ability of Plasmodium falciparum-infected erythrocytes.Mem. Inst. Oswaldo Cruz. 1992; 87: 303-312Crossref PubMed Scopus (6) Google Scholar, 5Crabb B.S. Cooke B.M. Reeder J.C. Waller R.F. Caruana S.R. Davern K.M. Wickham M.E. Brown G.V. Coppel R.L. Cowman A.F. Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress.Cell. 1997; 89: 287-296Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar) and specialized secretion of parasite proteins into the host cell (6Marti M. Baum J. Rug M. Tilley L. Cowman A.F. Signal-mediated export of proteins from the malaria parasite to the host erythrocyte.J. Cell Biol. 2005; 171: 587-592Crossref PubMed Scopus (104) Google Scholar, 7Hiller N.L. Bhattacharjee S. van Ooij C. Liolios K. Harrison T. Lopez-Estraño C. Haldar K. A host-targeting signal in virulence proteins reveals a secretome in malarial infection.Science. 2004; 306: 1934-1937Crossref PubMed Scopus (658) Google Scholar). Yet perhaps the most profound change to the RBC is its complete collapse at the end of the parasite erythrocytic cycle, as the newly replicated merozoites are released (8Glushakova S. Yin D. Li T. Zimmerberg J. Membrane transformation during malaria parasite release from human red blood cells.Curr. Biol. 2005; 15: 1645-1650Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). For many years, parasite proteases have been thought to be important for rupture of malaria parasites from host cells (9Hadley T. Aikawa M. Miller L.H. Plasmodium knowlesi: studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors.Exp. Parasitol. 1983; 55: 306-311Crossref PubMed Scopus (109) Google Scholar): from initial inhibitor-based data, a two-step model of parasite rupture was proposed, requiring the activity of a cysteine and possibly a serine protease, as the general cysteine protease inhibitor E64 and the cysteine and serine protease inhibitors leupeptin and chymostatin inhibited PV and RBC plasma membrane rupture, respectively (10Salmon B.L. Oksman A. Goldberg D.E. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 271-276Crossref PubMed Scopus (195) Google Scholar, 11Wickham M.E. Culvenor J.G. Cowman A.F. Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte.J. Biol. Chem. 2003; 278: 37658-37663Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 12Glushakova 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: 95-105Crossref PubMed Scopus (51) Google Scholar). More recently, several reports have implicated the involvement of three proteases in the rupture process: the parasite-derived proteases PfDPAP3 (13Arastu-Kapur S. Ponder E.L. Fonović U.P. Yeoh S. Yuan F. Fonović M. Grainger M. Phillips C.I. Powers J.C. Bogyo M. 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Scholar), we that the parasite host RBC proteins and to the host to exit from the host cell. we in a more of changes to the infected red blood cells membrane and cytoskeleton as the parasites the host cell for rupture and calpain-1 and from and by was from and the and from was from human calpain-1 was from P. falciparum was under in human with in Roswell Park Memorial Institute malaria parasites in PubMed Scopus Google Scholar). parasites h invasion from a and in the of as by the was with protease inhibitor and by at for to remove uninfected and infected This was by at for to remove extracellular RBC membrane including small vesicles and from rupture of parasites from was then from the by at for h at The RBC membrane was then three with phosphate-buffered saline The RBC vesicles with and for a on of uninfected as as from stages of P. falciparum as of the and first in a AFM was on a in the at a of with a of then by the vesicles by a vesicles to to the surface of with a a that of for 1 h at as F. J. H. A. and of erythrocyte cytoskeleton with the atomic force 2003; PubMed Scopus Google Scholar). The then with and used for AFM the a The parasites then with in for at and at The was at This and parasitophorous vacuole was then for h at to The membrane and cytoskeletal was three with and to a by M. P. M. analysis of the membrane and proteome of red blood PubMed Scopus Google Scholar) for to with protease inhibitors at uninfected erythrocyte as M. P. M. analysis of the membrane and proteome of red blood PubMed Scopus Google Scholar). with and to in protease inhibitors for in by of the for h at The the RBC membrane was the as the membrane from and from in under on a with For of from the of the for of with and in from on a for in and by an a at A was the next the to at a of to a and in the was used as the and was used to the most the of the most was then to of for and for was We used the from for from The a human the Plasmodium The and with two of cysteine and of for the The and by as an to to of the was the with a of which for to a discovery and was for most was and proteins with at least three by as to and the was used for from the from analysis to the of proteins in the three in by and to and with to and of with was as (15Chandramohanadas C. parasites host to their from infected 2009; PubMed Scopus Google Scholar). of erythrocyte membrane with of calpain-1 in and for at by RBC with to the of in under and and by A from was on to membrane and for and and was by the of for was the in which it was an of from and postrupture RBC membrane of proteins in that to the the three of in of the in the postrupture proteome and a of this in uninfected and infected in the from from the at the of the for several proteins that parasites in for 1 h at in and in for at in in for 1 h with then with at in three in then with at in three in and on a We used AFM to at changes to the surface and cytoskeleton during parasite development and egress. AFM major changes in and appearance of the plasma membrane as by the knoblike which are to to of cells However, during early large holes in the cytoskeleton, with a in of the cytoskeletal This that changes and perhaps of the host cytoskeleton as early as ∼35 than the of parasite egress. been that at the of parasite the membrane and postrupture vesicles the of an RBC (8Glushakova S. Yin D. Li T. Zimmerberg J. Membrane transformation during malaria parasite release from human red blood cells.Curr. Biol. 2005; 15: 1645-1650Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). We postrupture vesicles by and by AFM The to to a with for AFM The postrupture vesicles with from to in A of to be as to with an erythrocyte that a AFM of disruption of the cytoskeleton as vesicles it was to more We a approach to the for the large changes in the cytoskeleton and highlighted AFM spectrometry-based the plasma membrane proteome from uninfected the membrane proteome from to parasite egress and the membrane proteome from postrupture be important during parasite changes in the proteome by thus we proteins in the membrane We a to three host membrane RBC by by from from uninfected A. Grainger M. J. J. Bogyo M. A for the protease 1 in host cell invasion by the human malaria PubMed Scopus Google Scholar), then with which at the the parasite plasma membrane (15Chandramohanadas C. parasites host to their from infected 2009; PubMed Scopus Google Scholar). The then by to remove parasite cells and by as (15Chandramohanadas C. parasites host to their from infected 2009; PubMed Scopus Google Scholar). postrupture from of and to egress in merozoites then from postrupture membrane by membrane vesicles then from the by at for For we of uninfected parasite after rupture that uninfected and postrupture vesicles as as released merozoites with and postrupture vesicles from the after parasite egress We parasite in the membrane the for postrupture that we of membrane we the of the RBC membrane and the loss of from a parasite and proteins from three we an approach to the approach used by the A. H. J. M. for of proteins and PubMed Scopus Google Scholar), in which first by then the and to The and first by with for 1 h at which the of in the analysis M. P. M. analysis of the membrane and proteome of red blood PubMed Scopus Google Scholar). 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We that erythrocyte and from the membrane which was striking as proteins have been to be important for the of the cytoskeleton S. Y. regulate the and deformability of the erythrocyte PubMed Scopus Google Scholar). loss of the loss of been to and S. Y. regulate the and deformability of the erythrocyte PubMed Scopus Google Scholar) leading to and in in R.F. Targeted of in and and in 2008; PubMed Scopus Google Scholar, L. C. Targeted disruption of the gene causes red blood cell in Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). analysis of membrane that is the first of the life cycle is from the host cytoskeleton at ∼35 This was which that is to the plasma membrane of uninfected and early stage to an of which is in infected cells possibly because of parasite of host We that loss from the cytoskeleton is on as into and loss from the membrane of by analysis The loss of as as and after that cytoskeletal in for egress begins than that are in postrupture of proteins that in the uninfected RBC and in the and postrupture membrane 1 blood 1 1 in a new the of proteases in parasite we next analysis of the on proteins that to be during egress. we for proteins that at than in the postrupture membrane proteome with the and uninfected RBC as proteins during parasite egress. We used several to determine proteins that in for their in the to their in the for which a to progressive changes in the three cytoskeletal proteins as and at their in the proteins at of the in the postrupture membrane that been is that been to be by parasite P. Y. S. M. A cysteine protease activity from Plasmodium falciparum human erythrocyte Parasitol. PubMed Scopus Google Scholar). However, the of as and as by the of This that several proteins from the postrupture membrane proteome than their the of proteins during rupture and at their in three proteome Using this we the of proteins in three We a in the of proteins to from uninfected to membrane the of proteolysis was in the proteome that proteolysis then into the of proteins of the cytoskeleton for a complete most of cytoskeletal proteins have been to be in uninfected during P. falciparum we analysis of a the parasite life cycle from in to uninfected and postrupture vesicles of several proteins at the of rupture that to be from the including the cytoskeletal proteins and as in the that in to in the analysis it that membrane proteins as more resistant to proteolysis which be because of their than to the cytoskeleton, to proteins including the from and as for the of the in We the presence of several proteases in the proteome was host calpain-1 that was in the and postrupture and its to the membrane are important for malaria parasite as we (15Chandramohanadas C. parasites host to their from infected 2009; PubMed Scopus Google Scholar). We activity for the of of the cytoskeletal proteolysis during parasite with RBC membrane in the presence of for We in calpain-1 a of cytoskeletal of to in and of postrupture vesicles to parasite-derived which have been to have a in in and postrupture membrane (14Yeoh S. O'Donnell R.A. Koussis K. Dluzewski A.R. Ansell K.H. Osborne S.A. Hackett F. Withers-Martinez C. Mitchell G.H. Bannister L.H. Bryans J.S. Kettleborough C.A. Blackman M.J. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes.Cell. 2007; 131: 1072-1083Abstract Full Text Full Text PDF PubMed Scopus (251) Google from and postrupture membrane A of proteases with more from infected and postrupture membrane are in the from from postrupture RBC activity been for activity been for activity been for in a new P. falciparum egress is thought to in at the end of the erythrocytic cycle the complete of the host This process is thought to be mediated by parasite proteins as proteases (10Salmon B.L. Oksman A. Goldberg D.E. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 271-276Crossref PubMed Scopus (195) Google Scholar, 11Wickham M.E. Culvenor J.G. Cowman A.F. Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte.J. Biol. Chem. 2003; 278: 37658-37663Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 12Glushakova 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: 95-105Crossref PubMed Scopus (51) Google Scholar, S. Ponder E.L. Fonović U.P. Yeoh S. Yuan F. Fonović M. Grainger M. Phillips C.I. Powers J.C. Bogyo M. Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum.Nat. Chem. Biol. 2008; 4: 203-213Crossref PubMed Scopus (203) Google Scholar), a I. S. J.C. membrane disruption by a parasite exit from host 2009; PubMed Scopus Google Scholar), and from the parasites (8Glushakova S. Yin D. Li T. Zimmerberg J. Membrane transformation during malaria parasite release from human red blood cells.Curr. Biol. 2005; 15: 1645-1650Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, L. H. M. T. by malaria a to host cell 2004; PubMed Scopus Google Scholar). that parasites human (15Chandramohanadas C. parasites host to their from infected 2009; PubMed Scopus Google Scholar), we that host is used to the RBC this we to a more global of changes that occur to the host RBC membrane proteome during parasite egress. The appearance of large holes in the from AFM of infected and of postrupture vesicles of cytoskeletal at least two steps the host cytoskeleton during parasite egress. that several host including most from the plasma membrane at the that large holes in the host RBC Targeted disruption of been to result of the RBC membrane that to and in R.F. Targeted of in and and in 2008; PubMed Scopus Google Scholar, L. C. Targeted disruption of the gene causes red blood cell in Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, H. F. J. Li of and a novel effect on the leading to erythrocyte and Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). this initial of the cytoskeleton the loss of the the loss of and as be required for the of membrane to parasite We that loss from the RBC cytoskeleton is temporally and distinct from the changes on the host cell at the of egress by host calpain-1. This a new mechanism used by malaria h to parasite egress in to the for This process be mediated by parasite to host proteins during egress because of cytoskeletal proteins been to result in progressive changes to the cytoskeletal P.W. M. of its for Biol. Chem. Full Text PDF PubMed Google Scholar, P.W. M. of and with PubMed Scopus Google Scholar, S. Y. K. of erythrocyte membrane by and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). parasite have been to be into the host cell A. P. a from Plasmodium falciparum, is to the host erythrocyte and is involved in hemoglobin PubMed Scopus Google Scholar, C. A. A novel in Plasmodium falciparum is and to of the host Microbiol. 2007; PubMed Scopus Google Scholar), a parasite activity and cytoskeletal changes during egress to be to the loss of proteomics that a of RBC cytoskeletal proteins parasite egress. in postrupture the of to of that in uninfected RBC more than of the proteins that to be with the host cytoskeleton including most the and that global and proteolysis of the host cytoskeleton during the parasite egress process proteases with the and postrupture including host an that parasites for egress (15Chandramohanadas C. parasites host to their from infected 2009; PubMed Scopus Google Scholar). We that calpain-1 most cytoskeletal proteins during which for the striking of the host RBC membrane the calpain-1 is of many of the cytoskeletal it is that parasite proteases as proteases this to be in on and postrupture is the activity of proteins for their during parasite egress from the RBC, it is that to the egress process of the host membrane a that parasite egress occur as a at the end of the life cycle. More the distinct of cytoskeletal which we a for egress that at least h in which the parasite the cytoskeleton to for on this of parasite proteins parasite into the host PV RBC be for during egress. M. M. P. falciparum and with of C. P. falciparum and proteomics and with of A. P. A. the H. S. AFM C. D. microscopy and parasite C. T. L. and the AFM D. C. the in and the We I. for to and D. and P. for in is We the for and at the of for the use of the with
Millholland et al. (Fri,) studied this question.
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