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Toxoplasma secretory granules: one population or more? Corinne Mercier and Marie-France Cesbron-Delauw Laboratoire Adaptation et Pathoge´nie des Microorganismes (LAPM), CNRS UMR 5163 – Universite´ Joseph Fourier, Grenoble, France

In Toxoplasma gondii, dense granules are known as the storage secretory organelles of the so-called GRA proteins (for dense granule proteins), which are destined to the parasitophorous vacuole (PV) and the PV-derived cyst wall. Recently, newly annotated GRA proteins targeted to the host cell nucleus have enlarged this view. Here we provide an update on the latest developments on the Toxoplasma secreted proteins, which to date have been mainly studied at both the tachyzoite and bradyzoite stages, and we point out that recent discoveries could open the issue of a possible, yet uncharacterized, distinct secretory pathway in Toxoplasma. The T. gondii life cycle T. gondii is an apicomplexan parasite that can virtually infect any kind of warm-blooded animal, including human beings. The parasite is acquired orally, after ingestion of raw or undercooked meat containing cysts, or after ingestion of fresh water or vegetables spoiled with oocysts. In its intermediate hosts, Toxoplasma multiplies in an asexual manner. Once liberated from the resistant cyst or oocyst wall in the duodenum, the bradyzoites or the sporozoites, respectively, differentiate into tachyzoites that disseminate into the entire organism. Within immune-privileged organs (i.e., muscle, heart, brain, retina, and testicles), tachyzoites that had been dividing by endodyogeny within intracellular PVs differentiate into bradyzoites while the PVs transform into intracellular cysts that may remain dormant for years as long as the immune pressure remains stable, ensuring transmission of the parasite by carnivorism (for reviews, see [1,2]). In the small intestine of young felids, Toxoplasma behaves as a typical coccidian parasite, expanding by asexual endopolygeny to form merozoites. Eventually these merozoites differentiate into gametes. Fertilization in felid-definitive hosts leads to the production of millions of oocysts that are released into the environment with cat feces. The cells contained in the oocysts must then undergo a last step of division to generate infectious sporozoites. These mature oocysts, which are resistant to all normal laboratory disinfectants, are likely responsible for efficient Corresponding authors: Mercier, C. ([email protected], [email protected]); Cesbron-Delauw, M.-F. ([email protected]). Keywords: dense granules; GRA proteins; protozoa; Toxoplasma gondii. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.12.002

spreading of the parasite to all non-carnivorous animals (for reviews, see [1,2]). The host–parasite interactions that govern the sexual cycle of Toxoplasma in felids are likely highly specific and due to the refractoriness of the coccidian stages to cell culture, cell biology studies on this fascinating cycle are still limited. The recent determination of global gene expression of merozoites and oocysts harvested from cat intestines revealed dramatic differences from that of the tachyzoite and bradyzoite stages. In particular, most genes encoding secreted proteins, including those secreted by the dense granules, were shown to be downregulated [3–5]. This review will thus focus mainly on the tachyzoite and bradyzoite stages, which are amenable to cell culture and reverse genetics. Dense granule subpopulations or novel secretory organelles in Toxoplasma? The success of T. gondii as an intracellular parasite in intermediate hosts and in tissue culture relies on sequential secretion from Apicomplexa-specialized secretory organelles and mobilization of particular elements of its cytoskeleton to invade host cells and form a PV, inside which it multiplies [6]. Three types of morphologically distinct secretory organelles have been described in T. gondii. Among these, the Apicomplexa-conserved micronemes and rhoptries, which are localized at the apical part of the invasive stages, are involved in parasite attachment to the host cell and the early steps of PV formation, respectively [7]. In T. gondii, a third type of dense core secretory organelles, named the dense granules, has been characterized. The secretion of their contents, the GRA proteins, was shown to occur consecutively to the exocytosis from micronemes and rhoptries, at the end of the invasion process, once the PV has formed [8]. The postsecretory localization of the GRA proteins in both the PV and the cyst wall, combined with results obtained from the phenotypic analysis of parasites knocked-out for the expression of several GRA genes, have led to the understanding that GRA proteins are important for the maturation of the PV into a metabolically active compartment and to its subsequent transformation into a cyst [6,9]. Recent studies have challenged this view and have reported the specific targeting of some so-called dense granule proteins in the host cell nucleus, where they reprogram host gene expression [10]. Other ‘GRA’-like proteins have been recently annotated in the ToxoDB database (version 12.0 released September 10, 2014: http://toxodb.org/toxo/). Here, we Trends in Parasitology xx (2015) 1–12

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provide an update on the increasing number of newly characterized GRA proteins at both the tachyzoite and bradyzoite stages by summarizing first the discovery of the GRA proteins and reviewing the common properties of validated GRA proteins. We then discuss the possibility that some of these proteins actually do not originate from the conventional dense granules but from another type of Toxoplasma secretory organelle that is not yet characterized, leading us to propose new features to convey distinctions between validated GRA proteins and less well characterized proteins. Defining the dense granules Since the 1960s, as soon as transmission electron microscopy (EM) offered sufficient resolution to observe the internal organelles of Toxoplasma, dense granules have been so called because of their morphology: they were observed as one single population of microspheres of approximately 200 nm in diameter, enclosed in a unit membrane, and evenly dispersed on each side of the parasite nucleus. Most importantly, their dark appearance under the electron beam (Figure 1A) suggested that they could correspond to a protein storage compartment. Dense granules, which vary in number depending on the parasite stage, are most prevalent at the tachyzoite stage in Toxoplasma [11]. Discovery and nomenclature of the GRA proteins The development of monoclonal antibodies specific to Toxoplasma proteins secreted in vitro after the use of an artificial secretion inducer (heat-inactivated serum) led to the cloning of a unique gene encoding a protein that was localized by immuno-EM within the dense granules of (A)

The main features of the GRA proteins Today, the full proteome of dense granules still remains unknown, and awaits the purification of these organelles, which has not yet been reported for Toxoplasma or for any other Apicomplexa. Nevertheless, during the last two decades, 16 GRA genes (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA9, GRA12, GRA14, GRA19, GRA20, GRA21, GRA23, GRA25, here referred to as the ‘canonical’ GRA genes) and their encoded proteins have been characterized (Figure 2) [15,16]. Despite the fact that these proteins failed to share homology between themselves or with proteins of known functions, they were defined as the family of GRA proteins based purely on their unique colocalization within the dense granules: their localization was determined by immunofluorescence after using 0.1% of triton X-100, a non-ionic polyoxyethylene

(B)

C

M

both the tachyzoite and the bradyzoite stages of the coccidian parasite T. gondii [12,13]. Until that time, the Toxoplasma proteins had been referred to as their apparent molecular weight on SDS-PAGE, for example P30 being the 30-kDa major surface protein of the tachyzoite stage. However, with the growing number of characterized proteins, Sibley and collaborators proposed in 1991 the use of a common nomenclature for Toxoplasma genes and proteins [14]. This nomenclature was inspired by that of yeast and allowed for the dubbing of any characterized protein based on its enzymatic activity or its subcellular localization. Accordingly, the first characterized GRA protein that was localized in the dense granules of both tachyzoites and bradyzoites, for which no function could be proposed, was referred to as GRA1 [13].

IMC PM HC N GRA15, 16, 24

DG

Vacuolar space GRA1, GRA16, 24

R

N

1 µm

ER

PVM GRA3, 5, 7, 8, 14, 19, 20, 21, 23 GRA10, 15, 22, 24, 35

MNN GRA2, 4, 6, 9, 12

5 µm TRENDS in Parasitology

Figure 1. (A) The main intracellular organelles of Toxoplasma gondii as they appear on a longitudinal section of a tachyzoite observed by transmission electron microscopy (EM). Abbreviations: C, conoid; DG, dense granule; ER: endoplasmic reticulum; IMC, inner membrane complex; M, microneme; N, nucleus; PM, plasma membrane; R, rhoptry. (B) A schematic representation of the final localizations of the dense granule (GRA) proteins in infected cells. Human foreskin fibroblasts (HFFs) infected overnight with RH tachyzoites expressing green fluorescent protein [63] and fixed with 4% paraformaldehyde for 20 minutes. Host cell and parasite nuclei were labelled with Hoechst reagent No. 33342. Once secreted from the dense granules, GRA1, GRA16, and GRA24 can be detected within the vacuolar space; GRA2, 4, 6, 9, and 12 associate preferentially with the intravacuolar membranous nanotubular network (MNN); GRA3, 5, 7, 8, 14, 19, 20, 21, 23 and GRA10, 15, 22, 24, 25 associate with the parasitophorous vacuole membrane (PVM). GRA15, 16, and 24 are also targeted to the host cell nucleus (HC N).

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GRA1 (190 aa, 20.22 kDa) GRA2 (185 aa, 19.8 kDa) GRA3 (220 aa, 23.87 kDa) GRA4 (345 aa, 36.33 kDa) GRA5 (120 aa, 12.83 kDa) GRA6 (224 aa, 23.30 kDa) GRA7 (236 aa, 25.91 kDa) GRA8 (269 aa, 28.62 kDa) GRA9 (318 aa, 35.33 kDa) GRA10 (894 aa, 94.22 kDa) GRA11 (752 aa, 80.09 kDa) GRA11 bis (471 aa, 49.34 kDa) GRA12 (436 aa, 47.92 kDa) GRA12 bis (419 aa, 46.63 kDa) GRA14 (408 aa, 44.71 kDa) GRA15 (550 aa, 57.89 kDa) GRA16 (505 aa, 54.77 kDa) GRA20 (413 aa, 45.19 kDa) GRA21 (501 aa, 55.45 kDa) GRA22 (629 aa, 68.63 kDa) GRA23 (21 9 aa, 24.15 kDa) GRA24 (542 aa, 57.33 kDa) GRA25 (315 aa, 34.73 kDa) TRENDS in Parasitology

Figure 2. Schematic representation of the ME49 proteins identified as dense granule (GRA) proteins in ToxoDB version 12.0. aa, amino acids; signal peptide [http:// www.cbs.dtu.dk/services/SignalP/ and http://www.cbs.dtu.dk/services/SecretomeP/ available on the ExPASy server (http://www.expasy.org/proteomics)]; PEXEL-like motif (RxLxE/Q/D: processing and PVM targeting signal [19]); hydrophobic alpha-helix (http://embnet.vital-it.ch/software/TMPRED_form.html available on the ExPASy server (http://www.expasy.org/proteomics); amphipathic alpha-helix (http://heliquest.ipmc.cnrs.fr); Ca2+-binding EF hand [13]; nuclear localization signal (http:// elm.eu.og available on the ExPASy server (http://www.expasy.org/proteomics). Please note that in GRA16 one of the PEXEL-like motifs (position 63–67) covers one of the nuclear localization signals (position 62–79).

surfactant that permeabilized completely infected cells previously fixed with 4% formaldehyde, and also permeabilized fixed extracellular parasites [17]. This concentration of triton X-100 was shown to be sufficient to permeabilize even the most internal membranes of intracellular parasites. The tendency of the Toxoplasma scientific community is now to use 0.2% triton X-100 (Table 1), which allows a clearer signal of surface proteins in intracellular parasites but is unnecessary for intravacuolar GRA proteins. The choice of the fixative agent is as important as the permeabilization agent to preserve cellular morphology. Commercial formalin, which is formaldehyde stabilized by 10% methanol, should be avoided because the added alcohol causes dehydration of the cells while hardening their membranes [18] and may cause mis-localization of intravacuolar proteins (Mercier et al., unpublished data). Paraformaldehyde (4% final concentration), constituted of 8–100 units of polyoxymethylene, should be used as it naturally depolymerizes into formaldehyde in soft base solutions such as phosphate-buffered saline (PBS) and fixes cellular membranes while preserving them [18].

Moreover, all the ‘canonical’ GRA proteins described to date were detected within the PV by epifluorescence after fixation of infected cells and further permeabilization with 0.002% of saponin, a non-ionic surfactant [17]. This low percentage of saponin was shown to be sufficient to create pores (by forming complexes with membrane cholesterol molecules) within both the host cell membrane and the PV membrane, without affecting the parasite plasma membrane. This allowed a selective detection of the proteins that had been secreted into the PV without detecting those that remained within the dense granules [17]. The latter would indeed lead to a very strong fluorescence signal, which could mask that originating from the PV. However, it is worth mentioning that any new batch of saponin (which is a mixture of molecules extracted from a plant) needs to be tested and its final concentration adjusted to achieve this selective permeabilization. Interestingly, the ‘canonical’ GRA proteins were shown to present several other common features, as described hereafter. 3

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Table 1. GRA proteins (co)labeling(s) in the tachyzoite dense granulesa GRA protein

GRA1

ToxoDB v.12.0 (10.09.2014) (ME49) TGME49_270250

Number of amino acids (ME49) 190

GRA2

TGME49_227620

185

GRA3

TGME49_227280

220

GRA4

TGME49_310780

345

GRA5

TGME49_286450

120

GRA6

TGME49_275440

224

GRA7

TGME49_203310

236

GRA8

TGME49_254720

269

GRA9

TGME49_251540

318

GRA10

TGME49_268900

894

GRA11 GRA1 bis GRA12

TGME49_212410 TGME49_237800 TGME49_275850

752 471 436

GRA12 bis GRA13 GRA14

TGME49_288650 Not found TGME49_239740

419

GRA15

TGME49_275470

550

GRA16

TGME49_208830

505

GRA17 GRA18 GRA19

Not found Not found Not found in v.12.0 YGME49_087740 in a previous version of ToxoDB was reported as being present only in type II strains TGME49_200010

GRA20

4

408

413

Detection in tachyzoite dense granules and co-labellings

Refs

Immunogold detection of GRA1 (mouse specific anti-serum) in RH extracellular tachyzoites: labeling of the dense granules Immunogold detection of GRA2 (mAb) in RH extracellular tachyzoites: labeling of the dense granules Immunofluorescent labeling of GRA3 (rabbit specific serum) in the dense granules of HFF cells infected with RH, fixed with paraformaldehyde-periodate, permeabilized with 0.1% triton X-100 Immunogold detection of GRA4 (rabbit specific serum) and GRA2 (mAb) in RH extracellular tachyzoites: labeling of the dense granules Immunogold detection of GRA5 (mAb) in RH extracellular tachyzoites: labeling of the dense granules Immunogold detection of GRA6 (mouse specific serum) in RH extracellular parasites: labeling of the dense granules Immunogold detection of GRA6 (mAb) and GRA2 (rabbit specific serum) in RH extracellular tachyzoites: labeling of the dense granules Immunogold detection of GRA7 (mAb) in RH extracellular tachyzoites: labeling of the dense granules Co-labeling of GRA8 (mAb) and GRA6 (mAb) in the dense granules of RH extracellular tachyzoites fixed with 2% formaldehyde, permeabilized with 0.1% triton X-100; Immunogold detection of GRA8 (mAb) in the dense granules of RH extracellular tachyzoites IF co-labelings of GRA9 (rabbit specific serum) with GRA1 (mAb), GRA2 (mAb) and GRA5 (mAb) in the dense granules of extracellular BK (type I) tachyzoites fixed in 3% formaldehyde and permeabilized in 0.1% triton X-100; Immunogold detection of GRA9 by rabbit serum anti-GRA9 in HFF infected with RH tachyzoites: labeling of the dense granules IF labeling of GRA10 (mAb) in methanol-fixed/permeabilized extracellular RH tachyzoites: no co-labeling with canonical dense granule proteins Co-labeling of GRA10 (rat-specific serum) with GRA1 (specific-rabbit serum) in the dense granules of extracellular RH tachyzoites fixed in 3% formaldehyde and permeabilized in 0.2% triton X-100:partial overlap Not reported Not reported IF co-labeling of GRA12 (rat-specific serum) with GRA3 (mAb) in the dense granules of extracellular RH parasites permeabilized with cold acetone; Immunogold detection of GRA12 by rat serum anti-GRA12 in HFF infected with RH: labeling of the dense granules Not reported

[12] [12] [64]

[23] [12] [65] [23] [66] [67]

[68]

[69]

[70]

[71]

Immunogold detection of GRA14-HA (mAb anti-HA) of RH tachyzoites infecting HHFs: labeling of the dense granules IF co-localization of GRA15II-HA (mAb anti-HA) with GRA7 (rabbitspecific serum) at the PVM and in the PV of HFF cells fixed with 3% formaldehyde and permeabilized with 100% ethanol OR 0.2% triton X-100 (no details on the permeabilization protocol), the localization of GRA15 or GRA15II HA in extracellular parasites was not reported IF co-localization of GRA16-HA (rabbit anti-HA or rat anti-HA) with GRA1 (mAb anti-GRA1) or with GRA7 (mAb anti-GRA7) in the PV space in infected HFF cells fixed with 3% formaldehyde and permeabilized with 0.2% triton X-100. Partial perinuclear co-localization ofGRA16-HA (rabbit anti-HA or rat anti-HA) with GRA1 (mAbanti-GRA1) in extracellular parasites: unusual perinuclear localization of GRA1

[72]

Co-labeling of GRA19-HA (specific mouse serum anti-HA tag) with GRA7 (rabbit specific serum) in extracellular DHXGPRT (PRU) tachyzoites fixed/permeabilized with 3% formaldehyde

[19]

co-labeling of GRA20-HA (specific mouse serum anti-HA tag) with GRA7 (rabbit specific serum) in extracellular DHXGPRT (PRU) tachyzoites fixed/permeabilized with 3% formaldehyde

[19]

[40]

[41]

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Table 1 (Continued ) GRA protein

ToxoDB v.12.0 (10.09.2014) (ME49) TGME49_241610 Found only in ME49

Number of amino acids (ME49) 501

GRA22

TGME49_215220

629

GRA23

TGME49_297880

219

GRA24

TGME49_230180

542

GRA25

TGME49_290700

625

GRA21

Detection in tachyzoite dense granules and co-labellings

Refs

co-labeling of GRA21-HA (specific mouse serum anti-HA tag) with GRA7 (rabbit specific serum) in extracellular DHXGPRT (PRU) tachyzoites fixed/permeabilized with 3% formaldehyde Co-labeling of GRA22 (mouse-specific serum) with GRA1 (rabbit specific serum) in HFF cells infected with RH tachyzoites, fixed with 4% formaldehyde and permeabilized with 0.2% tritonX-100: potential localization in the dense granules that should have been confirmed by the study of extracellular parasites Co-labeling of GRA23 (rabbit-specific serum) with GRA4 (mouse specific serum) and of GRA23 (mouse-specific serum) with GRA7 (rabbit specific serum) in the dense granules of RH extracellular tachyzoites fixed with 4% paraformaldehyde and permeabilized with 0.5% triton X-100; Immunogold detection of GRA23 (rabbit specific serum) of RH tachyzoites infecting Vero cells: labeling of dense granules Co-labeling of GRA24-HA-FLAG with anti-HA (rat mAb) and GRA7 (mAb) or GRA1 (mAb) in extracellular RH tachyzoites fixed in 3% formaldehyde and permeabilized with 0.1% triton X-100: no overlay of the labelings IF co-labeling of GRA25 (mouse-specific serum to GRA25 from Me49) with GRA7 (rabbit-specific serum) in the dense granules of extracellular parasites fixed in 4% formaldehyde and permeabilized in 0.2% triton X-100 IF co-labeling of GRA25 (mouse-specific serum to GRA25 from Me49) and GRA7 (rabbit-specific serum) in the PV of HFF cells infected with RH, fixed in 4% formaldehyde and permeabilized in 0.2% triton X-100

[19]

[52]

[20]

[42]

[22]

a

Abbreviations: RH, initials of the infant who died of congenital toxoplasmosis and from whom this type I strain was isolated [25]; mAb, monoclonal antibody; HFF, human foreskin fibroblasts; IF, immunofluorescence; BK, type I virulent strain [73]; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; DHXGPRT (PRU), Prugniaud strain (type II) deleted of its hypoxanthine-xanthine-guanine phosphoribosyl transferase gene.

With the exception of GRA20, ‘canonical’ GRA proteins are predicted to contain either a classical or non-classical N-terminal hydrophobic signal peptide (Table 2) that correlates to their secretory profile: they are indeed secreted into the PV as soon as 3 minutes post-invasion, with a peak of secretion occurring 10–30 minutes post-invasion [8]. They are detected in the PV as long as the parasites multiply, and up to the step of parasite egress, indicating that their secretion into the PV likely goes on at a basal level as long as the PV develops. The ‘canonical’ GRA monomeric forms are of relatively low calculated molecular weight, being mostly between 20 and 50 kDa (Figure 2 and Table 2). Except for GRA1, which was described as a soluble protein, GRA proteins were all shown to adopt an intriguing dual behavior, being partly soluble and partly membraneassociated within the PV. More precisely, they localize and interact either directly or indirectly with: (i) the PV membrane (GRA3, GRA5, GRA7, GRA8, GRA14, GRA19, GRA20, GRA21, GRA23) and its long and thin extensions that plunge into the host cell cytoplasm [6,19,20]; (ii) the vacuolar HOST structures (host sequestering tubulostructures) (GRA7) [21]; or (iii) the intravacuolar membranous nanotubular network (MNN) that connects the parasites within the PV and links them to the vacuolar membrane (GRA2, GRA4, GRA6, GRA9, GRA12) (Figure 1B) [6]. In agreement with this direct interaction with PV membranes, most of these GRA proteins were predicted to contain either at least one hydrophobic a helix (GRA3, GRA5, GRA6, GRA7, GRA8, GRA12, GRA14, GRA25) that could help them span their respective membrane, or at least one

amphipathic a helix (GRA2, GRA9) [6,22]. Of interest, GRA4 [23] and GRA23 [20] were shown to be peripherally associated with their respective membranes, likely via protein–protein interactions, despite the prediction of a typical hydrophobic a helix within the GRA4 amino acid sequence [16]. Since they also lack a potential transmembrane domain, it is probable that GRA19, GRA20, and GRA21 [19] are peripheral membrane proteins that interact with the PV membrane via protein–protein interactions. The partition of GRA19 in the triton X-114 aqueous phase of PV proteins [19] could be in favor of protein–protein interactions relying on ionic rather than on hydrophobic interactions, which would have been disrupted by triton X-114. The high molecular complexes containing several of these GRA proteins that were revealed both within the dense granules and the PV [23,24] also favor such peripheral association to membranes via protein–protein interactions. Many ‘canonical’ GRA proteins were shown to be nonessential in the virulent RH strain (type I; note that ‘RH’ were the initials of the child who died in 1939 in Cincinnati, Ohio of toxoplasmic encephalitis and from whom the strain was isolated [25]): deletion of the GRA2, GRA3, GRA5, GRA6, GRA7, or GRA14 gene did not hamper parasite growth in vitro [6]. Nevertheless, decreased virulence phenotypes were observed in mice infected with either Dgra2 or Dgra6 knockout (KO) mutants [6,26]. Moreover, correlating with important functions in the PV maturation, phenotypic analyses of these KO parasites showed that both GRA2 and GRA6 trigger the formation of the membranous tubules that constitute the MNN [27–29]. Furthermore, GRA7 was shown to encircle the HOSTs, these 5

Localization in cells infected by type I or type II tachyzoites

RNA expression during the cell cycle at the tachyzoite stage e

Classical

Not predicted

PV

High, constant

GRA2

TGME49_227620 185 (19.8; 10)

Classical

Not predicted

PV

GRA3 GRA4 GRA5

TGME49_227280 220 (23.87; 10.22) TGME49_310780 345 (36.33; 6.8) TGME49_286450 120 (12.83; 5.26)

Classical Classical Classical

160–178 276–293 75–93

PV PV PV

GRA6

TGME49_275440 224 (23.30; 5.19)

Non-classical

153–171

PV

GRA7

TGME49_203310 236 (25.91; 5.25)

Classical

181–202

PV

GRA8

TGME49_254720 269 (28.62; 10.56)

Classical

225–243

PV

GRA9

TGME49_251540 318 (35.33; 5.28)

Classical

Not predicted

PV

GRA10

TGME49_268900 894 (94.22; 6.51)

Not predicted Not predicted

PV

GRA11

TGME49_212410 752 (80.09; 3.97)

Classical

GRA11 bis TGME49_237800 471 (49.34; 4.42)

Classical

TGME49_288650 436 (47.92; 9.30)

Classical

114–135, 194-212, Not determined 299–315 112–133, 188–208, Not determined 282–300 Not predicted PV

Non-classical

203–226, 237–254

Classical

290–308

GRA12

GRA12 bis TGME49_275850 419 (46.69; 9.77) Not identified GRA13 TGME49_239740 408 (44.71; 8.81) GRA14

Neospora Neospora Neospora Neospora Not identified Neospora Neospora Neospora Neospora, Eimeria

Very low, constant

Low,  constant

Low, constant

Low,  constant

Not identified

High, maximum at the SZ and TK stages Low, constant

Neospora, Eimeria

Not determined

High, maximum in M and C phases Low, constant

PV

High, constant Medium, maximum in G1 and S phases Not reported

High, maximum at the SZ and then, the TK stages Low, maximum at the TK stage

Neospora

Not reported

Neospora

Medium, maximum at the TK and BZ stages

Neospora

Low, maximum at the late BZ and the TK stages High to medium, maximum at the SZ, then TK and late BZ stages

Neospora, Eimeria

TGME49_275470 550 (57.89; 9.16)

Not predicted 54–72

PV, HCN

GRA16 GRA17 GRA18 GRA19 GRA20

TGME49_208830 505 (54.77; 9.92) Not identified Not identified Not identified TGME49_200010 413 (45.19; 7.76)

Classical

PV, HCN

Not predicted Not predicted

PV

GRA21

TGME49_241610 501 (55.45; 9.77)

Classical

PV

GRA22

TGME49_215220 629 (68.63; 5.22)

Not predicted 39–62

Not predicted

High, maximum at the TK stage, then SZ and BZ stages High,  constant High, maximum at the TK, then the BZ stages High, constant High in TK and BZ stages High, constant High in TK and BZ stages High, constant High, maximum at the TK, then the BZ stages High,  constant High, maximum at the TK, then SZ stages High, maximum High, maximum at the SZ and at the M and C phases TK stages, then late BZ High, constant High, maximum at the TK and SZ stages High, maximum in the C Medium, maximum at the BZ stage phase Medium,  constant Medium, maximum at the SZ stage

Homologies in Apicomplexa g

Neospora, Eimeria, Cryptosporidium, Babesia, Theileria, Plasmodium Not identified

GRA15

Not predicted

Transcriptomic analysis during the parasite life cycle f

PV

High, maximum in M phase of the first cycle Medium, constant

Neospora, Eimeria Neospora

Neospora, Eimeria

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Predicted transmembrane hydrophobic alpha helix d

ID in ToxoDB version 12.0 (10.09.2014)

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

Predicted signal peptide c

GRA1

Predicted number of amino acids (calculated molecular weight in kDa; calculated isoelectric point) b TGME49_270250 190 (20.22; 3.86)

GRA protein

Review

6

Table 2. Characteristics of the dense granule proteins: ‘canonical’ GRAs (light blue background) and ‘GRAs-like’ (mid-blue background) (http://toxodb.org/toxo/)a

TREPAR-1339; No. of Pages 12

http://web.expasy.org/blast/ and ToxoDB v. 12.0.

‘‘Expression profiling of oocyst, tachyzoite, and bradyzoite development in strain M4’’ from H. Fritz, K.R. Buchholz, P. Conrad, and J.C. Boothroyd. ToxoDB v. 12.0. [5].

g

f

http://embnet.vital-it.ch/software/TMPRED_form.html available on the ExPASy server (http://www.expasy.org/proteomics).

‘‘Cell cycle expression profiles’’ from M.S. Behnke, J.C. Wooton, M. Lehmann, J.B. Radke, O., Lucas, J. Nawas, L.D. Sibley, and M.W. White. ToxoDB v. 12.0 [3].

e

http://web.expasy.org/compute_pi/ available on the ExPASy server (http://www.expasy.org/proteomics).

d

c

Abbreviations: BZ, bradyzoite; HCN, host cell nucleus; PV, parasitophorous vacuole; SZ, sporozoite; TK, tachyzoite.

b

a

PV Classical TGME49_290700 315 (34.73; 9.51) GRA25

123–140

PV, HCN Not predicted Classical TGME49_230180 542 (57.33; 10.26) GRA24

http://www.cbs.dtu.dk/services/SignalP/ and http://www.cbs.dtu.dk/services/SecretomeP/ available on the ExPASy server (http://www.expasy.org/proteomics).

Neospora, Eimeria, Plasmodium Medium, maximum at the SZ and TK stages

Medium, maximum at the TK and Neospora, Sarcocystis, BZ stages Eimeria Medium, maximum at the SZ stage Not identified Classical TGME49_297880 219 (24.15; 9.56)

Not predicted

PV

High, maximum in M phase of the first cycle High, maximum in G1 phase Medium to high, maximum in M and C phases High, maximum in M and C phases GRA23

Predicted number of amino acids (calculated molecular weight in kDa; calculated isoelectric point) b ID in ToxoDB version 12.0 (10.09.2014)

Table 2 (Continued )

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GRA protein

Predicted signal peptide c

Predicted transmembrane hydrophobic alpha helix d

Localization in cells infected by type I or type II tachyzoites

RNA expression during the cell cycle at the tachyzoite stage e

Transcriptomic analysis during the parasite life cycle f

Homologies in Apicomplexa g

Review

structures being defined as intravacuolar digitations of the PV membrane sustained by a single shortened host cell microtubule and involved in acquisition of host cell endosomes into the PV [21]. Most of the ‘canonical’ GRA proteins were also detected in the cyst wall that derives from the PV. This correlates with the maintained expression of the genes encoding these GRA proteins at the bradyzoite stage (ToxoDB version 12.0). Consistently, the dense granules, as described morphologically, are restricted to a subset of apicomplexan parasites: those forming tissue cysts, namely the Toxoplasma, Neospora, Sarcocystis, Hammondia, Besnoitia, and Frankelia genera. Toxoplasma cysts are permeable and round structures up to 150 mm in diameter. Cysts are limited by a unit membrane and can contain several hundred to several thousand bradyzoites characterized by slow metabolism and asynchronous and slow division. The cyst wall, which can reach up to 240 nm in thickness, is composed of two layers, an external compact layer and an inner looser layer, while the cyst matrix contains a network of tubular membranes that link the bradyzoites together (in a similar manner to the MNN, which links the tachyzoites within the PV), filamentous material, and two populations of vesicles [6,30]. Interestingly, even if the distribution of all the ‘canonical’ GRA proteins in the cyst wall has not been investigated yet, the localization of GRA1, 2, 3, 5, 6, and 7 in the various layers of the cyst wall [30,31] suggests that the GRA proteins would constitute critical components for the biogenesis of this structure. This is supported experimentally by the dramatic reduction of cyst burden (91–99%) observed in the brain of mice infected by cystogenic type II Prugniaud parasites knocked-out for their GRA4 and/or their GRA6 gene(s) [32]. Recent reports [26,33] established that ‘canonical’ GRA proteins, in addition to contributing to PV formation, could be involved in controlling host cell effectors. Polymorphic GRA6 was indeed shown to activate the host transcription factor nuclear factor of activated T cells 4 (NFAT4), possibly via the direct interaction of PV membrane-associated GRA6 with calcium signal-modulating cyclophilin ligand (CAMLG), an integral membrane protein of the host endoplasmic reticulum [26]. The translocation (and activation) of NFAT4 into the host cell nucleus of infected mouse embryonic fibroblasts (MEFs) stably expressing m-cherry-tagged NFAT4 was shown to depend upon GRA6. Translocation was indeed significantly reduced in MEFs infected by GRA6-deficient parasites but restored in MEFs infected with complemented parasites. However, although direct interaction between FLAG-tagged GRA6 and human influenza hemagglutinin (HA)-tagged CAMLG, both transiently expressed in human epithelial kidney T cells, was demonstrated by co-immunoprecipitation [26], direct interaction between both proteins was not established in infected cells. Furthermore, GRA7, which becomes phosphorylated within the host cell, was shown to be part of a protein complex that includes rhoptry kinase ROP18 and the pseudokinases ROP8 and ROP2 on the cytosolic face of the PV membrane. In vitro, GRA7 binds to the active dimer of immunity-related GTPase a6 (IRGa6), leading to its polymerization, its rapid turn-over and disassembly, thus potentially complementing the functions of ROP18 and ROP5 to thwart the mouse 7

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Review macrophages IRG defense system and protect the parasite from destruction [33]. In summary, from the several functions defined for the ‘canonical’ GRA proteins at the tachyzoite stage and from their persistent expression in the cyst wall, it appears that these proteins would play major structural functions within the PV and later in the cyst wall. Moreover, their particular location within the PV at the interface between the host cell and the parasite allows them to be involved in various interactions with the host cell [26,33–39]. GRA proteins versus host nucleus-targeted GRA proteins To date, three secreted proteins, which were named GRA15 [40], GRA16 [41], and GRA24 [42] (Figure 2), have been described as being stored within the dense granules, secreted into the PV and eventually targeted to the host cell nucleus (Figure 1B), where they modulate the host cell response. The presence in GRA15 and GRA16 of a HT (host targeting signal)/PEXEL-like (Plasmodium Export Element) motif (described in Plasmodium as being a signal motif allowing the export of proteins across the PV membrane into the red blood cell cytoplasm [43,44]), sustains the export of GRA15 and GRA16 into the host cell and may indicate that the HT/PEXEL-like motif found in many Toxoplasma secreted proteins [19] (Figure 2) is also functional in this parasite. Interestingly, in Plasmodium, for export into the erythrocyte cytoplasm, many proteins use a PV membrane translocon constituted of five proteins secreted from the dense granules within the first ten minutes consecutive to erythrocyte invasion (for a review, see [45]). The existence of a translocon associated to the Toxoplasma PV membrane has been suggested [10] and, such as in Plasmodium, GRA proteins targeted to the PV membrane might be components of this translocon. The studies conducted to dissect the interactions established between GRA15, GRA16, GRA24, and the host nuclear proteins were extensive and proved three key points. Firstly, GRA15-type II induces nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 nuclear translocation and NF-kB-mediated host cell transcription, in a tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibitor of kappa B kinase (IKK)dependent manner [40], thus activating the transcription of genes involved in the proinflammatory response [interleukin-12 (IL12) p40/p70] in infected macrophages [46]. Secondly, GRA16 binds to the Herpes virus-associated ubiquitin specific protease (HAUSP) deubiquitinase and to the protein phosphatase 2 (PP2A) holoenzyme (PR55/B phosphatase), two host enzymes that regulate the P53 tumor suppressor pathway and the cell cycle [41]. As such, GRA16 could control host cell arrest in the G2/M phase associated with Toxoplasma early infection. Finally, GRA24 induces the prolonged Thr180 autophosphorylation (and activation) and nuclear translocation of host P38a mitogen-activated protein (MAP) kinase, thus increasing the production of proinflammatory cytokines (IL12 p40) in macrophages infected with type II parasites [42]. These three proteins, two of which display a typical Nterminal signal peptide and nuclear localization sequences (GRA16 and GRA24) (Figure 2), have thus been shown to 8

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be important effectors of Toxoplasma in controlling the host cell response following infection [10]. However, the storage of these three GRA proteins within dense granules is questionable. The localization of GRA16 [41] and GRA24 [42] was determined, in part, in infected cells using HA or HA-FLAG tagged proteins overexpressed in Toxoplasma under the strong GRA1 promoter. The infected cells were fixed with 3% formaldehyde, permeabilized with 0.2% or 0.1% triton X-100, labeled with anti-HA antibodies, and observed by immunofluorescence (Table 1). The localization of GRA15 [40] was presumably determined using similar treatments before immunofluorescence, but it is unknown due to the lack of technical details provided. GRA15 clearly colocalized with GRA7 within the PV and at the PV membrane. Localization at the PV membrane was in agreement with the labeling of the vesicles that are secreted from Toxoplasma into the host cell cytoplasm at the time of parasite invasion and which then align along a host cell microtubule to reach the PV and form a PV extension. Even if this colocalization of GRA15 and GRA7 within the PV and at the PV extensions could indeed evoke a step of storage within the dense granules, the colocalization of GRA15 and GRA7 in these organelles was not demonstrated in labelled intracellular parasites [40]. The localization of GRA16–HA within the PV was also unambiguous [41]. By contrast, the examination of extracellular parasites expressing GRA16–HA following fixation with 3% formaldehyde, permeabilization with 0.2% triton X-100, and labeling with anti-HA revealed a very fine dotted pattern located around the parasite nucleus. Intriguingly, the co-labeled GRA1, instead of appearing as the expected dotted pattern spread throughout the Toxoplasma cell and which is typical of ‘canonical’ dense granule proteins, appeared in this experiment as a rim of fluorescence around the nucleus, which could suggest stacking of the protein along the earliest compartments of the secretory pathway (since the endoplasmic reticulum and the nuclear envelope are contiguous in Toxoplasma), rather than storage within the dense granules. GRA24–HA–FLAG secreted in the PV 16 hours postinvasion could be detected within the PV only when the GRA24 coding sequence was overexpressed under the strong GRA1 promoter [42]. A low level of expression at the tachyzoite stage is not common for ‘canonical’ GRA proteins (Table 2). Moreover, the co-labeling of GRA24– HA–FLAG with GRA1 or GRA7 in extracellular parasites [42] clearly revealed two populations of vesicles, one labeled by ‘canonical’ anti-GRA antibodies and the other by anti-HA antibodies. It is therefore likely that GRA24–HA– FLAG originates from cytoplasmic vesicles distinct from dense granules. The presence of other kinds of secretory vesicles has actually already been reported in Toxoplasma: (i) the fusion protein CD46–HA–FLAG stably expressed in Toxoplasma was observed as trafficking to vesicles as big as dense granules but distinct from them within the parasite cytoplasm [47]; (ii) the patatin-like protein TgPL1 (TGME49_232600), which was found within the PV formed in macrophages 5 days after their activation, was localized in cytoplasmic vesicles different from dense granules and from acidocalcisomes [48]. Finally, without establishing any functional parallel, it can be stated that: (i) micronemes,

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Review although having been described since the 1960s as forming an homogenous population of vesicles, were recently shown by stimulated emission depletion (STED) to be organized into two distinct subsets or sub-compartments [49]; and (ii) several novel types of secretory organelles, such as exonemes [50] and mononemes [51], have been recently discovered in the closely-related Plasmodium species. In summary, while the trafficking of the three nucleustargeted GRA proteins to the PV on their way to the host cell nucleus cannot be questioned, their localization within the parasite dense granules or within any other kind of Toxoplasma cytoplasmic vesicles remains to be established using conventional or more elaborated microscopic techniques such as STED [49]. Revisiting the recently annotated ‘GRA’ proteins? Browsing the last release of ToxoDB (version 12.0) currently reveals 23 annotated GRA genes (Figure 2 and Table 2), with GRA19 (TGME49_245432) [19] missing from the list of annotated GRA proteins. Identifying groups of proteins among these predicted proteins is not an easy task, and localization experiments of the identified GRA11, GRA11 bis, and GRA12 bis still await to be performed. The timing of the mRNA expression peak of the annotated ‘GRA’ proteins during the parasite life cycle (Table 2) might prove to be an additional criterion to distinguish conventional GRA proteins from GRA-like proteins. Except for GRA21, which was reported to be faintly expressed, all the ‘canonical’ GRA genes appear to be expressed abundantly and, importantly, peak mRNA expression covers at least the tachyzoite and/or the bradyzoite stage(s) (Table 2), in agreement with their major role in the biogenesis of the PV and the cyst wall. By contrast, the putative ‘GRA’ proteins tend to be expressed moderately or at low levels, and their expression does not seem to peak during the promotion of cyst development. Instead, their expression peaks at the sporozoite stage (GRA10, GRA24) or remains low and constant throughout the various parasite stages (GRA11, GRA11 bis, and GRA12 bis). GRA22 has been shown to be involved in regulating tachyzoite egress from the PV [52]. However, its peak of expression at the sporozoite stage (Table 2) tends to place it among the non-‘canonical’ GRA proteins. Moreover, the observed granular pattern of GRA22 partially overlaps that of GRA1 in fixed infected cells permeabilized with 0.2% triton X-100 and observed by immunofluorescence [52]. The very limited GRA1 signal located between the parasites, within the PV, is unexpected as GRA1 is a very abundant soluble protein commonly used to label the whole vacuolar space when using 0.002% of saponin or even 0.1% triton X-100 as a permeabilization agent. Investigating whether or not GRA22 could be detected in the cyst wall could thus likely allow its definitive classification within the ‘canonical’ GRA proteins. A recent study [53] reported that host mitochondrial association (HMA) to the PV membrane depends on a polymorphic secreted parasite protein, dubbed mitochondrial association factor 1 (MAF1; TGGT1_220950 in ToxoDB version 12.0). MAF1 fits most of the criteria defined for ‘canonical’ GRA proteins: that is, it is of rather small molecular weight (446 amino acids); it displays an N-terminal

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signal peptide; it is highly expressed at both the tachyzoite and bradyzoite stages; once secreted into the PV, it associates with the PVM – this association being in agreement with the presence of a potential transmembrane domain in the amino acid sequence. Accordingly, HA-tagged MAF1 colocalized perfectly with GRA7 in the dense granules of parasites forced-lysed by syringing from PVs tightly fitting to unique parasites (remnants of the PV membrane still attached to the parasites were also likely labeled) [53]. Furthermore, it is important to mention that several other proteins with proven enzymatic function or with significant homology with proteins of known functions have also been localized in the dense granules and within the PV after secretion. These proteins include the nucleoside triphosphate isomerases NTPase I and NTPase II [54–56], the protease inhibitors TgPI and TgPI2 [57–59], and the cathepsins TgCPC1 and TgCPC2 [60]. TgPI was colocalized with GRA4 in the dense granules of extracellular parasites fixed with 3% formaldehyde and 0.027% glutaraldehyde before permeabilization with 0.1% triton X-100 [58]. TgCPC1 and TgCPC2 were also colocalized with GRA3 in the dense granules of intracellular parasites fixed and permeabilized with 0.2% triton X-100 [60]. Interestingly, while the expression of TgNTPase II and TgCPC2 peaks at the tachyzoite stage, the expression of TgCPC1 and TgPI2 peaks at the sporozoite stage, and TgPI expression remains almost constant (and low) during the three tested parasite stages [58]. Except for NTPases that have been labeled in cysts obtained from infected mice [31], to our knowledge, the localization of TgPIs and TgCPCs in cysts has not been investigated and it is thus not known whether these proteins contribute efficiently or not to the formation of the cyst structure. Concluding remarks and future perspectives The first conclusion that can be drawn from our literature review on the dense granule proteins is that standardization of the immunofluorescence protocols used to label the GRA proteins in both extracellular and intracellular parasites would greatly help comparisons of results obtained in different laboratories around the world. A consensus on using human foreskin fibroblasts (HFFs; ATCC CRL-2429) as host cells to study the localization of Toxoplasma proteins has been achieved over the years in the Toxoplasma scientific community as these cells are non-transformed cells that are easy to amplify in cell culture and, most importantly, they are flat cells, allowing high resolution of the observations performed by epifluorescence, confocal microscopy, or EM. The consensus should now go one step further and the most appropriate immunofluorescence protocols to label particular organelles should be defined. Regarding the localization of GRA proteins, although epitope tagging at the endogenous locus using the CRISPR (clustered regularly interspaced short palindromic repeats)/CAS9 (CRISPR-associated protein 9) technology recently developed for Toxoplasma [61,62] allows rapid localization of newly described proteins, co-labeling with specific antibodies to a ‘canonical’ GRA protein and further observation by immuno-EM performed on extracellular parasites is necessary to confirm the localization of a given 9

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Review Box 1. Localization of GRA proteins (i) Localization in the dense granules  Technique of choice: co-labeling of extracellular parasites with specific antibodies to a ‘canonical’ GRA protein and further observation by immuno-EM.  Alternative technique: indirect immunofluorescence on extracellular parasites fixed for 20 minutes in 4% paraformaldehyde (not formalin) diluted in PBS and permeabilized with 0.1% triton X-100; colocalization with specific antibodies to a ‘canonical’ GRA protein. (ii) Localization in the PV  Fixation of infected HFFs with 4% formaldehyde for 20 minutes.  Permeabilization with the appropriate dilution of saponin that allows selective permeabilization of both the host cell plasma membrane and the PV membrane (typically 0.002%).  Colocalization with specific antibodies to a ‘canonical’ GRA protein (since saponin pores are labile, saponin must be maintained during the whole labeling procedure).

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the host–parasite interface, some of them are likely involved in host cell manipulation, as recently demonstrated for GRA6 and GRA7 [26,33]. The other ‘GRA’ proteins, which may actually be released preferentially during the sporozoite stage or constantly during the parasite life cycle, are likely secreted into the PV before being directed, for some of them, into the host cell nucleus (Box 2). These proteins may contribute to other crucial functions, such as the reprogramming of the host cell. Refining the localization of these ‘GRA’ proteins in the parasites by immuno-EM or STED, analysis of their kinetics of secretion into the PV, and their localization in the cyst (if any) using specific antibodies now seems a necessity. The results of these experiments will help validate their GRA name or, on the contrary, will highlight a novel secretary pathway in Toxoplasma. Acknowledgments

protein within the dense granules. Indirect immunofluorescence performed on extracellular parasites fixed in 4% paraformaldehyde (not formalin) diluted in PBS and further permeabilized with 0.1% triton X-100 also allows clear results to be obtained (Box 1). To localize as precisely as possible any protein secreted in the PV, the use of infected HFFs that have been fixed with 4% formaldehyde and incubated with the appropriate dilution of saponin to permeabilize both the host cell and PV membranes remains the gold standard (Box 1). We have also addressed the possible existence of distinct secretory granules, the ‘canonical’ dense granules on one side (Box 2) and still uncharacterized cytoplasmic vesicles on the other side. The ‘canonical’ GRA proteins GRA1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 14, 20, 21, 23, and 25, most of which are expressed in abundance during the tachyzoite and bradyzoite stages, would contribute to the development of a functional PV and to the formation of the cyst wall that derives from this PV. Being located at Box 2. The criteria that could define most GRA proteins (i) ‘Canonical’ GRA proteins  Peak of mRNA expression at the tachyzoite and/or the bradyzoite stage(s)  Monomeric proteins of relatively low molecular weight (20–75 kDa)  Classical or non-classical N-terminal hydrophobic signal peptide  Expression at the tachyzoite stage: co-labeling with ‘canonical’ GRA proteins in the dense granules  Secretion into the tachyzoite PV: partly soluble and partly membrane-associated, often via a single hydrophobic a-helical domain  Persistence within the PV up to the parasite egress  Maintained expression at the bradyzoite stage: storage within the dense granules and detection at the cyst wall  Possible functions: (1) development of a functional PV; (2) formation of the cyst wall; (3) possible effects on host cell proteins (ii) GRA-like proteins  Storage within parasite secretory vesicles that resemble dense granules (dense granule subpopulation?)  Secretion into the tachyzoite PV  Possible targeting to the host cell nucleus  Possible functions include reprogramming of the host cell 10

We thank J.F. Dubremetz (CNRS UMR 5539 – Montpellier 2 University, France), C. Bisanz, S. Cristinelli, and D. Jublot (CNRS UMR 5163 – Joseph Fourier University, Grenoble 1, France) for the gift of the photos included in Figure 1 and L.D. Sibley (Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA) for proofreading and advice on the manuscript before submission. Work in our lab was supported by the Labex Parafrap (ANR-11-LABX0024 to MFCD), Fondation pour la Recherche Me´dicale (MFCD), and ANR grants (ANR-11-BSV3-010-02 to MFCD and ANR 11 EMMA 032-01 to CM).

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Toxoplasma secretory granules: one population or more?

In Toxoplasma gondii, dense granules are known as the storage secretory organelles of the so-called GRA proteins (for dense granule proteins), which a...
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