The Malarial Serine Protease SUB1 Plays an Essential Role in Parasite Liver Stage Development


Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.


Vyšlo v časopise: The Malarial Serine Protease SUB1 Plays an Essential Role in Parasite Liver Stage Development. PLoS Pathog 9(12): e32767. doi:10.1371/journal.ppat.1003811
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003811

Souhrn

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.


Zdroje

1. EjigiriI, SinnisP (2009) Plasmodium sporozoite-host interactions from the dermis to the hepatocyte. Curr Opin Microbiol 12: 401–407.

2. LindnerSE, MillerJL, KappeSH (2012) Malaria parasite pre-erythrocytic infection: preparation meets opportunity. Cell Microbiol 14: 316–324.

3. SturmA, GraeweS, Franke-FayardB, RetzlaffS, BolteS, et al. (2009) Alteration of the parasite plasma membrane and the parasitophorous vacuole membrane during exo-erythrocytic development of malaria parasites. Protist 160: 51–63.

4. GraeweS, RankinKE, LehmannC, DeschermeierC, HechtL, et al. (2011) Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathog 7: e1002224.

5. SturmA, AminoR, van de SandC, RegenT, RetzlaffS, et al. (2006) Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science 313: 1287–1290.

6. BaerK, KlotzC, KappeSH, SchniederT, FrevertU (2007) Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature. PLoS Pathog 3: e171.

7. MazierD, ReniaL, SnounouG (2009) A pre-emptive strike against malaria's stealthy hepatic forms. Nat Rev Drug Discov 8: 854–864.

8. PrudencioM, RodriguezA, MotaMM (2006) The silent path to thousands of merozoites: the Plasmodium liver stage. Nat Rev Microbiol 4: 849–856.

9. SzarfmanA, WallikerD, McBrideJS, LyonJA, QuakyiIA, et al. (1988) Allelic forms of gp195, a major blood-stage antigen of Plasmodium falciparum, are expressed in liver stages. J Exp Med 167: 231–236.

10. SturmA, HeusslerV (2007) Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites. Med Microbiol Immunol 196: 127–133.

11. Schmidt-ChristensenA, SturmA, HorstmannS, HeusslerVT (2008) Expression and processing of Plasmodium berghei SERA3 during liver stages. Cell Microbiol 10: 1723–1734.

12. ChandramohanadasR, DavisPH, BeitingDP, HarbutMB, DarlingC, et al. (2009) Apicomplexan parasites co-opt host calpains to facilitate their escape from infected cells. Science 324: 794–797.

13. RennenbergA, LehmannC, HeitmannA, WittT, HansenG, et al. (2010) Exoerythrocytic Plasmodium parasites secrete a cysteine protease inhibitor involved in sporozoite invasion and capable of blocking cell death of host hepatocytes. PLoS Pathog 6: e1000825.

14. PutriantiED, Schmidt-ChristensenA, ArnoldI, HeusslerVT, MatuschewskiK, et al. (2010) The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite. Cell Microbiol 12: 725–739.

15. RueckerA, SheaM, HackettF, SuarezC, HirstEM, et al. (2012) Proteolytic activation of the essential parasitophorous vacuole cysteine protease SERA6 accompanies malaria parasite egress from its host erythrocyte. J Biol Chem 287: 37949–37963.

16. MillerSK, GoodRT, DrewDR, DelorenziM, SandersPR, et al. (2002) A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle. J Biol Chem 277: 47524–47532.

17. AokiS, LiJ, ItagakiS, OkechBA, EgwangTG, et al. (2002) Serine repeat antigen (SERA5) is predominantly expressed among the SERA multigene family of Plasmodium falciparum, and the acquired antibody titers correlate with serum inhibition of the parasite growth. J Biol Chem 277: 47533–47540.

18. AlyAS, MatuschewskiK (2005) A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts. J Exp Med 202: 225–230.

19. DelplaceP, BhatiaA, CagnardM, CamusD, ColombetG, et al. (1988) Protein p126: a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites. Biol Cell 64: 215–221.

20. YeohS, O'DonnellRA, KoussisK, DluzewskiAR, AnsellKH, et al. (2007) Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131: 1072–1083.

21. BlackmanMJ (2008) Malarial proteases and host cell egress: an ‘emerging’ cascade. Cell Microbiol 10: 1925–1934.

22. CollinsCR, HackettF, StrathM, PenzoM, Withers-MartinezC, et al. (2013) Malaria Parasite cGMP-dependent Protein Kinase Regulates Blood Stage Merozoite Secretory Organelle Discharge and Egress. PLoS Pathog 9: e1003344.

23. Arastu-KapurS, PonderEL, FonovicUP, YeohS, YuanF, et al. (2008) Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum. Nat Chem Biol 4: 203–213.

24. KoussisK, Withers-MartinezC, YeohS, ChildM, HackettF, et al. (2009) A multifunctional serine protease primes the malaria parasite for red blood cell invasion. Embo J 28: 725–735.

25. Silmon de MonerriNC, FlynnHR, CamposMG, HackettF, KoussisK, et al. (2011) Global identification of multiple substrates for Plasmodium falciparum SUB1, an essential malarial processing protease. Infect Immun 79: 1086–1097.

26. BoyleMJ, RichardsJS, GilsonPR, ChaiW, BeesonJG (2010) Interactions with heparin-like molecules during erythrocyte invasion by Plasmodium falciparum merozoites. Blood 115: 4559–4568.

27. GoelVK, LiX, ChenH, LiuSC, ChishtiAH, et al. (2003) Band 3 is a host receptor binding merozoite surface protein 1 during the Plasmodium falciparum invasion of erythrocytes. Proc Natl Acad Sci U S A 100: 5164–5169.

28. LiX, ChenH, OoTH, DalyTM, BergmanLW, et al. (2004) A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3. J Biol Chem 279: 5765–5771.

29. CarvalhoTG, ThibergeS, SakamotoH, MenardR (2004) Conditional mutagenesis using site-specific recombination in Plasmodium berghei. Proc Natl Acad Sci U S A 101: 14931–14936.

30. CombeA, GiovanniniD, CarvalhoTG, SpathS, BoissonB, et al. (2009) Clonal conditional mutagenesis in malaria parasites. Cell Host Microbe 5: 386–396.

31. FalaeA, CombeA, AmaladossA, CarvalhoT, MenardR, et al. (2010) Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development. J Biol Chem 285: 3282–3288.

32. PfanderC, AnarB, SchwachF, OttoTD, BrochetM, et al. (2011) A scalable pipeline for highly effective genetic modification of a malaria parasite. Nat Methods 8: 1078–1082.

33. TarunAS, PengX, DumpitRF, OgataY, Silva-RiveraH, et al. (2008) A combined transcriptome and proteome survey of malaria parasite liver stages. Proc Natl Acad Sci U S A 105: 305–310.

34. OehringSC, WoodcroftBJ, MoesS, WetzelJ, DietzO, et al. (2012) Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum. Genome Biol 13: R108.

35. TreeckM, SandersJL, EliasJE, BoothroydJC (2011) The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries. Cell Host Microbe 10: 410–419.

36. FlorensL, WashburnMP, RaineJD, AnthonyRM, GraingerM, et al. (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419: 520–526.

37. Withers-MartinezC, SaldanhaJW, ElyB, HackettF, O'ConnorT, et al. (2002) Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells. Characterization, comparison with the parasite protease, and homology modeling. J Biol Chem 277: 29698–29709.

38. Withers-MartinezC, SuarezC, FulleS, KherS, PenzoM, et al. (2012) Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target. Int J Parasitol 42: 597–612.

39. BouillonA, GigantiD, BenedetC, GorgetteO, PetresS, et al. (2013) 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

40. JanseCJ, Franke-FayardB, MairGR, RamesarJ, ThielC, et al. (2006) High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol 145: 60–70.

41. LacroixC, GiovanniniD, CombeA, BargieriDY, SpathS, et al. (2011) FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nat Protoc 6: 1412–1428.

42. WangJ, SarovM, RientjesJ, FuJ, HollakH, et al. (2006) An improved recombineering approach by adding RecA to lambda Red recombination. Mol Biotechnol 32: 43–53.

43. GiovanniniD, SpathS, LacroixC, PerazziA, BargieriD, et al. (2011) Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa. Cell Host Microbe 10: 591–602.

44. SinnisP (1998) An immunoradiometric assay for the quantification of Plasmodium sporozoite invasion of HepG2 cells. J Immunol Methods 221: 17–23.

45. PinoP, SebastianS, KimEA, BushE, BrochetM, et al. (2012) A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host Microbe 12: 824–834.

46. SkarnesWC, RosenB, WestAP, KoutsourakisM, BushellW, et al. (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474: 337–342.

47. EckerA, LewisRE, EklandEH, JayabalasinghamB, FidockDA (2012) Tricks in Plasmodium's molecular repertoire–escaping 3′UTR excision-based conditional silencing of the chloroquine resistance transporter gene. Int J Parasitol 42: 969–974.

48. CollinsCR, DasS, WongEH, AndenmattenN, StallmachR, et al. (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol 88: 687–701.

49. MatuschewskiK, RossJ, BrownSM, KaiserK, NussenzweigV, KappeSHI (2002) Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J Biol Chem 277: 41948–41953.

50. GeraldN, MahajanB, KumarS (2011) Mitosis in the human malaria parasite Plasmodium falciparum. Eukaryot Cell 10: 474–482.

51. SeidahNG, PratA (2012) The biology and therapeutic targeting of the proprotein convertases. Nat Rev Drug Discov 11: 367–383.

52. IshinoT, BoissonB, OritoY, LacroixC, BischoffE, et al. (2009) LISP1 is important for the egress of Plasmodium berghei parasites from liver cells. Cell Microbiol 11: 1329–1339.

53. JanseCJ, Franke-FayardB, WatersAP (2006) Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat Protoc 1: 614–623.

54. HolderAA, FreemanRR (1981) Immunization against blood-stage rodent malaria using purified parasite antigens. Nature 294: 361–364.

55. KaraUA, StenzelDJ, IngramLT, BushellGR, LopezJA, et al. (1988) Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane. Infect Immun 56: 903–909.

56. SimmonsD, WoollettG, Bergin-CartwrightM, KayD, ScaifeJ (1987) A malaria protein exported into a new compartment within the host erythrocyte. EMBO J 6: 485–491.

57. JeanL, HackettF, MartinSR, BlackmanMJ (2003) Functional characterisation of the propeptide of Plasmodium falciparum subtilisin-like protease-1. J Biol Chem 278: 28572–28579.

58. GodiskaR, MeadD, DhoddaV, WuC, HochsteinR, et al. (2010) Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli. Nucleic Acids Res 38: e88.

59. PfanderC, AnarB, BrochetM, RaynerJC, BillkerO (2013) Recombination-mediated genetic engineering of Plasmodium berghei DNA. Methods Mol Biol 923: 127–138.

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2013 Číslo 12
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Eozinofilní granulomatóza s polyangiitidou
nový kurz
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa