Selective Condensation Drives Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating


Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.


Vyšlo v časopise: Selective Condensation Drives Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating. PLoS Pathog 6(4): e32767. doi:10.1371/journal.ppat.1000835
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000835

Souhrn

Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.


Zdroje

1. AdamRD

2001 Biology of Giardia lamblia. Clin Microbiol Rev 14 447 475

2. GillinFD

BoucherSE

RossiSS

ReinerDS

1989 Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro. Exp Parasitol 69 164 174

3. LujanHD

MowattMR

ByrdLG

NashTE

1996 Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia. Proc Natl Acad Sci U S A 93 7628 7633

4. DacksJB

FieldMC

2007 Evolution of the eukaryotic membrane-trafficking system: origin, tempo and mode. J Cell Sci 120 2977 2985

5. MartiM

RegosA

LiY

SchranerEM

WildP

2003 An ancestral secretory apparatus in the protozoan parasite Giardia intestinalis. J Biol Chem 278 24837 24848

6. MorrisonHG

McArthurAG

GillinFD

AleySB

AdamRD

2007 Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317 1921 1926

7. LloydD

HarrisJC

2002 Giardia: highly evolved parasite or early branching eukaryote? Trends Microbiol 10 122 127

8. HehlAB

MartiM

2004 Secretory protein trafficking in Giardia intestinalis. Mol Microbiol 53 19 28

9. MartiM

LiY

SchranerEM

WildP

KohlerP

2003 The secretory apparatus of an ancient eukaryote: protein sorting to separate export pathways occurs before formation of transient Golgi-like compartments. Mol Biol Cell 14 1433 1447

10. LujanHD

MarottaA

MowattMR

SciakyN

Lippincott-SchwartzJ

1995 Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. J Biol Chem 270 4612 4618

11. JarrollEL

ManningP

LindmarkDG

CogginsJR

ErlandsenSL

1989 Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine. Mol Biochem Parasitol 32 121 131

12. GerwigGJ

van KuikJA

LeeflangBR

KamerlingJP

VliegenthartJF

2002 The Giardia intestinalis filamentous cyst wall contains a novel beta(1–3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology 12 499 505

13. DasS

GillinFD

1996 Giardia lamblia: increased UDP-N-acetyl-D-glucosamine and N-acetyl-D-galactosamine transferase activities during encystation. Exp Parasitol 83 19 29

14. Van KeulenH

SteimlePA

BulikDA

BorowiakRK

JarrollEL

1998 Cloning of two putative Giardia lamblia glucosamine 6-phosphate isomerase genes only one of which is transcriptionally activated during encystment. J Eukaryot Microbiol 45 637 642

15. MacechkoPT

SteimlePA

LindmarkDG

ErlandsenSL

JarrollEL

1992 Galactosamine-synthesizing enzymes are induced when Giardia encyst. Mol Biochem Parasitol 56 301 309

16. BulikDA

van OphemP

ManningJM

ShenZ

NewburgDS

2000 UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated. J Biol Chem 275 14722 14728

17. HehlAB

MartiM

KohlerP

2000 Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Mol Biol Cell 11 1789 1800

18. StefanicS

MorfL

KulangaraC

RegosA

SondaS

2009 Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote. J Cell Sci 122 2846 2856

19. LujanHD

MowattMR

ConradJT

BowersB

NashTE

1995 Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem 270 29307 29313

20. DuBoisKN

AbodeelyM

SakanariJ

CraikCS

LeeM

2008 Identification of the major cysteine protease of Giardia and its role in encystation. J Biol Chem 283 18024 18031

21. TouzMC

NoresMJ

SlavinI

CarmonaC

ConradJT

2002 The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia. J Biol Chem 277 8474 8481

22. DavidsBJ

MehtaK

FesusL

McCafferyJM

GillinFD

2004 Dependence of Giardia lamblia encystation on novel transglutaminase activity. Mol Biochem Parasitol 136 173 180

23. MartiM

HehlAB

2003 Encystation-specific vesicles in Giardia: a primordial Golgi or just another secretory compartment? Trends Parasitol 19 440 446

24. StefanicS

PalmD

SvardSG

HehlAB

2006 Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia. J Biol Chem 281 7595 7604

25. GottigN

EliasEV

QuirogaR

NoresMJ

SolariAJ

2006 Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia. J Biol Chem 281 18156 18166

26. SunCH

McCafferyJM

ReinerDS

GillinFD

2003 Mining the Giardia lamblia genome for new cyst wall proteins. J Biol Chem 278 21701 21708

27. ShawMK

RoosDS

TilneyLG

1998 Acidic compartments and rhoptry formation in Toxoplasma gondii. Parasitology 117 (Pt 5) 435 443

28. CowanAT

BowmanGR

EdwardsKF

EmersonJJ

TurkewitzAP

2005 Genetic, genomic, and functional analysis of the granule lattice proteins in Tetrahymena secretory granules. Mol Biol Cell 16 4046 4060

29. ArvanP

CastleD

1998 Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem J 332 (Pt 3) 593 610

30. SobotaJA

FerraroF

BackN

EipperBA

MainsRE

2006 Not all secretory granules are created equal: Partitioning of soluble content proteins. Mol Biol Cell 17 5038 5052

31. GaechterV

SchranerE

WildP

HehlAB

2008 The Single Dynamin Family Protein in the Primitive Protozoan Giardia lamblia Is Essential for Stage Conversion and Endocytic Transport. Traffic 9 57 71

32. MironovAA

MironovAAJr

BeznoussenkoGV

TruccoA

LupettiP

2003 ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains. Dev Cell 5 583 594

33. ReinerDS

McCafferyM

GillinFD

1990 Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia. Eur J Cell Biol 53 142 153

34. McCafferyJM

FaubertGM

GillinFD

1994 Giardia lamblia: traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching. Exp Parasitol 79 236 249

35. McCafferyJM

GillinFD

1994 Giardia lamblia: ultrastructural basis of protein transport during growth and encystation. Exp Parasitol 79 220 235

36. LujanHD

MowattMR

ConradJT

BowersB

NashTE

1995 Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem 270 29307 29313

37. DavidsBJ

ReinerDS

BirkelandSR

PreheimSP

CiprianoMJ

2006 A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein. PLoS ONE 1 e44 doi:10.1371/journal.pone.0000044

38. Van DellenKL

ChatterjeeA

RatnerDM

MagnelliPE

CipolloJF

2006 Unique posttranslational modifications of chitin-binding lectins of Entamoeba invadens cyst walls. Eukaryot Cell 5 836 848

39. FrisardiM

GhoshSK

FieldJ

Van DellenK

RogersR

2000 The most abundant glycoprotein of amebic cyst walls (Jacob) is a lectin with five Cys-rich, chitin-binding domains. Infect Immun 68 4217 4224

40. ChatterjeeA

GhoshSK

JangK

BullittE

MooreL

2009 Evidence for a “wattle and daub” model of the cyst wall of entamoeba. PLoS Pathog 5 e1000498 doi:10.1371/journal.ppat.1000498

41. de la VegaH

SpechtCA

SeminoCE

RobbinsPW

EichingerD

1997 Cloning and expression of chitinases of Entamoebae. Mol Biochem Parasitol 85 139 147

42. Villagomez-CastroJC

Lopez-RomeroE

1996 Identification and partial characterization of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin. Antonie Van Leeuwenhoek 70 41 48

43. BelliSI

FergusonDJ

KatribM

SlapetovaI

MaiK

2009 Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina. Int J Parasitol 39 1063 1070

44. FergusonDJ

BelliSI

SmithNC

WallachMG

2003 The development of the macrogamete and oocyst wall in Eimeria maxima: immuno-light and electron microscopy. Int J Parasitol 33 1329 1340

45. BernanderR

PalmJE

SvardSG

2001 Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol 3 55 62

46. KimT

Tao-ChengJH

EidenLE

LohYP

2001 Chromogranin A, an “on/off” switch controlling dense-core secretory granule biogenesis. Cell 106 499 509

47. KimT

Gondre-LewisMC

ArnaoutovaI

LohYP

2006 Dense-core secretory granule biogenesis. Physiology (Bethesda) 21 124 133

48. KlumpermanJ

KuliawatR

GriffithJM

GeuzeHJ

ArvanP

1998 Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles. J Cell Biol 141 359 371

49. MidlejV

BenchimolM

2009 Giardia lamblia behavior during encystment: how morphological changes in shape occur. Parasitol Int 58 72 80

50. PalmD

WeilandM

McArthurAG

Winiecka-KrusnellJ

CiprianoMJ

2005 Developmental changes in the adhesive disk during Giardia differentiation. Mol Biochem Parasitol 141 199 207

51. ManningP

ErlandsenSL

JarrollEL

1992 Carbohydrate and amino acid analyses of Giardia muris cysts. J Protozool 39 290 296

52. ReinerDS

McCafferyJM

GillinFD

2001 Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway. Cell Microbiol 3 459 472

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2010 Číslo 4
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