# Toxoplasma Rhoptries:  Unique Secretory   Organelles  and   Source of Promising Vaccine Proteins  for  Immunoprevention of Toxoplasmosis

Authors: Henryka Dlugonska
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite classified in the phylum Apicomplexa, which includes numerous notable human and animal pathogens (Plasmodium species,
Cryptosporidium species,  Neospora caninum, etc.). The invasive stages of apicomplexans are characterized by the presence of an apical complex composed of specialized cytoskeletal and secretory organelles, including rhoptries. Rhoptries, unique apical secretory organelles shared exclusively by all apicomplexan parasites, are known to be involved in an active parasite's penetration into the host cell associated with the biogenesis of specific intracellular compartment, parasitophorous vacuole in which the parasite multiplies intensively, avoiding intracellular killing. Due to the key biological role of rhoptries, rhoptry proteins have recently become vaccine candidates for the prevention of several parasitoses, toxoplasmosis among them. The article presents current data on T. gondii rhoptries biology and new approaches to the development of effective vaccines against toxoplasmosis using rhoptry antigens.


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## Body

## 1. Introduction

Toxoplasma gondii is arguably the most successful protozoan
parasite on earth.  It infects humans and many species of endothermic animals
with a great infection rate,  achieving nearly 100% in some local populations
[1]. The course, symptoms, and consequences of T. gondii infection depend strongly on the virulence and inoculum
size of the parasite and the genetic background and immune status of the
infected host. In general, most postnatal cases of toxoplasmosis are mild and
subclinical, with lifelong persistence of the parasite in the host.  However, in immunosuppressed beings,
both primary infection and reactivation of chronic infection could occur very
serious, causing even death. Primary infection acquired during pregnancy can
lead to an abortion and neonatal malformations. Unfortunately, despite many
efforts there are no satisfactory immunoprevention methods against
toxoplasmosis. The only accepted vaccine against toxoplasmosis (Toxovax, Ovilis
Toxovac) contains live tachyzoites of incomplete nonpersistent S48 strain and
is used to prevent congenital toxoplasmosis in sheep. The vaccine causes a
decrease in abortion cases frequency but not complete eradication of the
parasite [2].


## 2.Toxoplasma gondii: Obligate Intracellular Apicomplexan Parasite

Successful parasitism byT. gondii is based
on its ability to form within the host cell a replication permissive niche—parasitophorous vacuole (PV). This growing
niche is delimited from
the host cell cytoplasm by PV membrane (PVM), a unique and dynamic “organelle”
found only in infected cells which prevents T.
gondii degradation by the host cell endocytic machinery and enables
parasite’s intracellular propagation. Unique to the apicomplexan protozoa
including Toxoplasma gondii are also three types of electrodense
secretory organelles: micronemes, rhoptries, and dense granules, carrying their
characteristic proteins: MICs, RONs, ROPs, and GRAs, respectively; see Table 1. Host cell
invasion is mediated by the sequential secretion of the contents of all three
organelles, which are exocytosed at the apical region when the parasite invades
the host cell. The processes, critical in the establishment of a productive
infection, are highly regulated at possibly two levels: physical separation of
exocytosis sites in the parasite’s plasma membrane and different trigger
mechanisms [3, 4].  Micronemes are involved in the attachment and penetration of T. gondii, while rhoptries are required for creating a transient
enigmatic structure,  moving junction and
then the establishment of PV. Host cell entry of the parasite is completed in
15–20 seconds [5, 6]. Dense granules secrete proteins throughout most of the parasite stages. The
secretion process coincides with the formation of intravacuolar network and
continues during the intracellular residence of T. gondii [3, 4].Table 1
The participation of secretory organelles proteins in the invasion of the host cell by protozoanToxoplasma gondii; PV: parasitophorous vacuole, MICs: micronemes proteins, TgAMA1: T. gondii apical membrane antigen 1, RONs: rhoptry neck proteins, ROPs: rhoptry proteins, GRAs: dense granule proteins.
Invasion stageSecretory organelles/secretion processsecreted proteinsAdhesionMicronemes/transientTgAMA1,TgMICs etc.Biogenesis of moving junction, PV and
its association with host mitochondria and
endoplasmic reticulumRhoptries/transientRONs, ROPsFormation of the specific architecture and
function of PVDense granules/continuousGRAs


## 3. Rhoptries and Rhoptries Proteins

Unlike most secretory and lysosomal granules in
mammalian cells, which are of a semispheroidal shape, the mature rhoptries are club shaped with a bulbous
base and an extended duct (neck), connected to the extreme apical pole of the
parasite. The rhoptries in the number of 8–12 per cell
occupy 10–30% of the total
cell volume. They are the only known acidified organelles inT. gondii;
pH of immature rhoptries is 3.5–5.5 and of mature
rhoptries is 5.0–7.0 [7]. Most
probably each mature rhoptry originates from a separate immature rhoptry [8].Current findings suggest that rhoptries are
most analogous to secretory lysosomal granules because they receive material
from both biosynthetic and endocytic cell pathways. In the exit site of
endoplasmic reticulum, the rhoptry proteins (in a form of proproteins) are
loaded into coated vesicles and then travel to the Golgi apparatus where they
are sorted to an immature rhoptry using the specific sorting signals [9]. Both
tyrosine-based and dileucine sorting motifs were detected within cytoplasmic
tails of the predominant ROP2 family proteins [10]. Proproteins undergo
proteolytic cleavage of an N-terminal prodomain, which occurs in post-Golgi
compartment, probably in immature rhoptries [11]. In contrast to all known
prominent ROP2 family members, a new described ROP5 protein is not processed
during trafficking but is synthesized in the mature form [12]. Some rhoptry
cargo is delivered by endosomal pathway via
multivesicular body and immature rhoptry [9]. Immature rhoptries evolve into
mature rhoptries characterized by specific morphology and
subcompartmentalization of their contents: ROP proteins are located in the bulb
body whereas RONs in the neck of the rhoptry [13].A proteomic analysis revealed over 30 rhoptry
proteins, all localized in the contents of the rhoptries, although many of the
identified proteins have putative transmembrane domains [13]. As shown for
ROP2, a prototype protein of ROP2 protein family, apart from an N-terminal
signal sequence, the molecule is characterized by the high abundance of charged
amino acids and proline residues, N- terminal arginine-rich stretches and
hydrophobic sequence in C-terminus considered as transmembrane domain [14, 15].
The ROP2 protein was shown to be inserted in PVM during the invasion of a host
cell and its N-terminal domain is exposed to host cytosol [14] mediating the
association of parasitophorous vacuole with host cell mitochondria [14, 16].
Targeted depletion of ROP2 resulted in multiple effects, such as: (i) impairments
of rhoptry biogenesis and cytokinesis, (ii) reduction in the association of
host cell mitochondria with PVM of the parasite, and a reduced sterol uptake
from the host cell, and (iii) reduced capacity to invade and replicate in vitro
in human fibroblasts and attenuation of virulence in mice [17]. Besides,
anti-ROP2 antibodies caused a decrease in the invasion ability of T. gondii RH tachyzoites in vitro [18]. The observed effects of
ROP2 depletion or inactivation and simultaneous synthesis of several related
proteins (ROP2 family) suggest that they serve crucial biological functions. All
members of the ROP2 family contain a protein kinase-like domain but only some of them are
catalytically active [15]. The exocytosis of rhoptry contents coincides with
the formation of PV but the biological trigger of rhoptry secretion has not
been identified. Mobilization of intracellular Ca2+ by ionophores
triggers the discharge of micronemes but not rhoptries and dense granules [19].
In contrast to other secretory organelles, rhoptry proteins and lipids are
exocytosed through the duct, whereas delimiting membrane is retained in the
cell and the rhoptry is transiently empty. Secreted rhoptry proteins are
subsequently discharged into nascent parasitophorous vacuole and localized in
its membrane [14, 20].As mentioned earlier, certain rhoptry proteins
are preferentially localized in the neck of the rhoptry. During invasion, RON4
relocalizes at the moving junction and participates in the formation of this short living and very
enigmatic structure between the host cell and the parasite, sliding over the
parasite as it invades [21]. RON4, RON2, and RON5 (Ts4705) form protein trio
that associates with adhesion microneme protein TgAMA1 (T. gondii apical membrane protein 1) forming an unusual complex
derived from two distinct secretory organelles: rhoptries and micronemes [22].
Moving junction functions as a molecular sieve for the quick selective sorting
of parasite surface components (like RONs-TgAMA1 complex mentioned above) as
well as surface proteins of the host cell. Because of the exclusion of most
host cell membrane proteins, developing parasitophorous vacuole is not
recognized by the host cell fusogenic machinery enabling the vigorous parasite
proliferation in this safe intracellular compartment [5, 23].


## 4. Virulence ofT. gondii and New Described Rhoptry Antigens as Major Virulence Components

Despite the very high genetic homology
achieving ~98%,T. gondii strains
express dramatic differences in many phenotype aspects, including virulence.
The majority of T. gondii isolates from Europe and North America
belong to three clonal lines (types) significantly differing in their
virulence, which has been well characterized in the mouse model. Type I
isolates have an LD100 of a single organism and lead to rapid death of the infected
mice (6–10 days), whereas
types II and III (called nonvirulent or avirulent) typically have LD100 of ≥103 organisms; mice which survive acute phase of toxoplasmosis remain chronically
infected and highly seropositive [24].Dramatic
differences in acute virulence between three major types ofT. gondii enabled the mapping of
virulence genes by an analysis of F1 progeny from genetic crosses between three
toxoplasma types, using an experimental mouse model to test virulence. The
method applied to types I×III recombinants revealed first two closely
adjacent virulence loci on parasite chromosome VIIa and then single gene
encoding ROP18 protein as a major virulence component. Type III and type I
alleles of ROP18 differed significantly in nucleotide sequence and expression
level. The contribution of ROP18 to virulence was confirmed by the transfection
of the virulent ROP18 allele into nonpathogenic type III parasite strain. The
obtained transformants showed an increased growth rate in vitro and an enhanced mortality by 4 to 5 logs in CD1 mice,
as compared to wild nontransformed strain [25]. Saeij et al. [26] mapped
virulence in F1 progeny derived from crosses between type II and type III
strains. Five virulence loci were identified, and two of them coded rhoptry
proteins: ROP18 and ROP16, both hypervariable protein kinases that are
responsible for the high virulence of certain strains of the parasite. Contrary
to ROP2 and several other members of this family that lost detectable kinase
activity [15] and retained “molecular fossils” [27], newly described rhoptry
proteins ROP16 and ROP18 are true kinases, the substrates of which are unknown.
Both kinases presumably disrupt some signaling processes and alter
intracellular environment in a way that favors parasite growth. The work of El
Hajj et al. [28] brought many interesting data on ROP18. The protein showed a
very strong tropism to PVM and during the invasion it was translocated from
rhoptry to PVM. The mature recombinant ROP18 was able to phosphorylate two as
yet unknown parasite’s (but not host’s) proteins, a major one of 70 kDa and a
minor one of 68 kDa. The overexpression of ROP18 protein promoted dramatically
parasite’s proliferation, whereas the mutation (D394A) in ROP18 molecule
resulted in the loss of both the enzyme activity and accelerated growth. In
contrast to ROP18, the second recently described rhoptry protein, ROP16 is
translocated to the host cell nucleus like phosphatase 2C [29], subverts
STAT3/6 signaling, and, in consequence, IL-12 production in infected host
cells. Allele shared by types I and III of the parasite induce prolonged
phosphorylation of STAT3 (STAT3-PO4) which results in a much lower
level of IL-12 secretion, as compared to type II allele [30]. The observed
type-specific effects show the key role of ROP kinases in T. gondii virulence and could explain different disease outcomes
seen with type I, II, and III strains. The role of ROPs in virulence suggests that they could be
promising vaccine candidates against toxoplasmosis.In contrast to ROP18 and
ROP16, another novel rhoptry protein, bradyzoite rhoptry protein 1 (BRP1), is
expressed in bradyzoites but absent from tachyzoites. Bradyzoites are a
low-replicating, inactive developmental stage, sequestered in tissue cysts,
which are a typical marker of chronic toxoplasmosis. The biological function of
BRP1 is not defined yet. This protein does not play an essential role in the
development of the bradyzoite stage, brain cyst formation, and oral infection
of new hosts [31].


## 5. Immunoprophylaxis of Toxoplasmosis

As mentioned earlier, the only vaccine against
toxoplasmosis has been licensed for use in sheep in Europe and New Zealand [2]. This conventional vaccine contains live attenuated tachyzoites of the
nonpersistent T. gondii S48 strain
and it cannot be considered for an application in humans because of the risk of
reverting to tissue cyst formation. From both humanitarian and economic points
of views, an effective anti-T. gondii vaccine(s) for humans and animals is still strongly desired. Taking into
account the main sources and transmission routes of T. gondii, host groups of a particularly high infection risk and
consequences of congenital infection, as well as postnatal infections in
immunocompromised individuals, targets for vaccination strategy should include (i)
preventing tissue cyst formation in consumption animals (to avoid parasite
transmission to humans and animals) and in people (to protect against
reactivation of infection in immunosuppression state of persistently infected
individuals), (ii) reducing oocyst shedding in cats (to limit environmental
contamination and infection risk for all intermediate hosts), and (iii)
preventing development of parasitemia in pregnant women and farm livestock (to
avoid transplacental transmission to the fetus and congenital toxoplasmosis)
[32].Vaccine candidates should induce protective
cellular TH1 and humoral responses as well, both at the level of intestinal
mucosa (local) and whole organism (systemic). Many studies have shown that
protective immunity toT. gondii is
characterized by the development of cell-mediated immunity dominated by the
production of IFN-γ
by T lymphocytes (a TH1 type response). Whereas immune CD8+ lymphocytes are
cytotoxic for T. gondii-infected host
cells, CD4+ lymphocytes exert their protective activity primarily by the
production of many proinflammatory cytokines (IFN-γ,
TNF-α,
IL-12) and providing growth factor, IL-2. Specific antibodies limit
multiplication of T. gondii by
killing of extracellular tachyzoites, either by activating complement, or
opsonizing the parasites for phagocytosis and next killing by macrophages [33].
The major challenge in novel vaccine construction is a selection of relevant T.
gondii antigens and then their presentation to host immune system in an
appropriate manner to induce strong and long-lasting protective immunity.
Because of the complex life cycle of the parasite, the expression of numerous
common and stage-dependent antigenic epitopes and host-dependent immune responses,
the vaccination with multiantigen preparation comprising antigens from
different life cycle stages is likely to be more efficacious than with a single
antigen. Previous and current vaccine approaches focus not only on classic
formulations (live attenuated or mutant T.
gondii strains) [2, 34, 35] but more often on the defined subcellular
components of the parasite such as biosynthetic (recombinant) antigens [36] and
naked T. gondii DNA [37] or RNA [38].
Nonpathogenic recombinant microorganisms with the expression of
immunoprotective antigens of T. gondii (live
vector vaccines) [39] and synthetic
peptides have also been tested as interesting antitoxoplasmic vaccine
alternatives [40]. These new generation subunit vaccines require an addition of
potent adjuvants enhancing the intensity of the immune response and inducing
the desired TH1 profile. Several new compounds, for instance unmetylated CpG
nucleotides, present satisfactory adjuvant TH1 activity [41].


## 6. Rhoptry Antigens as a Potential Vaccine Against Toxoplasmosis

Most of the current vaccine
candidates forT. gondii and other apicomplexa
are either surface or secreted antigens that appear to be essential for the
invasion process, rhoptry proteins among them. Until now several rhoptry
proteins of T. gondii have been tested
as vaccines. Garcia et al. [42] used
crude native rhoptry antigens incorporated in the immunostimulating
complexes (ISCOMs) to protect pigs against an infection with oocysts of T.
gondii VEG strain (of type III). Two-fold subcutaneous immunization
procedure led to a partial protection against cysts formation in muscles and
brain during chronic infection (as confirmed by bioassay in mice) but it was
not protective against acute infection, probably because of the lack of
intestinal immunity after subcutaneous immunization. Taking into account
epidemiological role of cats as an important source of T. gondii dissemination by shedding millions of oocysts in feces, the authors tested the
same antigen material in domestic cats [43]. The cats were immunized
intranasally three times (0, 21, 42 day) with crude rhoptry proteins adjuvanted
with Quil-A and then challenged with tissue cysts of the T. gondii VEG
strain. This vaccination procedure resulted in a partial protection only—two from three
immunized cats did not shed oocysts.Till now vaccine trials with individual ROPs ofT. gondii have mostly focused on the
ROP2 antigen, prominent member of ROP2 protein family. In majority, the
experiments were performed with DNA vaccines which are believed to elicit a
predominant TH1 response. Vercammen [44] showed that the immunization with ROP2
gene protected mice against lethal challenge with a highly virulent RH strain,
but the observed protection was mouse strain dependent—inbred C3H mice
were protected, whereas C57BL/6 and BALB/c were not.Similar results were obtained by Leyva et al. [45] in mice of three
inbred strains (CBA/J, C57BL/6, and BALB/c), vaccinated with plasmid encoding
antigen ROP2. The mice did not resist a challenge with RH strain but the
protection was achieved by vaccination with live tachyzoites of the T. gondii nonpersistent thermosensitive
ts-4 strain. In the mice immunized with ROP2-plasmid, a mixed TH1/TH2 immune
profile instead of a desirable TH1 profile and a significant variability in the
response between individual mice was observed. Using similar vaccine material,
that is, ROP2-plasmid, Wei et al. [46] reported both a high immune response and
significant immunoprotection against a lethal dose of T. gondii RH
strain in BALB/c mice.Recently, live nonpathogenic microbes (viruses
or bacteria) were used as vectors of selected ROP genes. Recombinant vaccinia
virus carrying ROP2 gene induced in mice a specific humoral response very similar,
in intensity and profile, to the thermosensitive ts-4 strain ofT. gondii.
Because the ts-4 strain as vaccine was able to fully protect mice against the
challenge with the highly virulent RH strain, the authors conclude by analogy
that ROP2-vaccinia virus could be equally effective in toxoplasmosis
immunoprotection [47]. Mishima et al. [18] obtained recombinant feline
herpesvirus 1 (FHV1) encoding ROP2 and evaluated its ability to induce antitoxoplasmic
activity and protective immune responses. Antisera of ROP2-FHV1-immunized cats
reduced significantly the invasion capacity of T. gondii RH tachyzoites in
vitro. The number of parasites in the brains of immunized cats lowered, however
oocyst shedding intensity was not reduced. Recently, Wang et al. [48] tested
the efficacy of recombinant Mycobacterium bovis BCG expressing ROP2 gene
as a vaccine, using mouse experimental toxoplasmosis model. BCG, widely applied
in immunoprophylaxis of tuberculosis, is a particularly attractive vector for
delivering heterologous antigens because mycobacteria elicit TH1-mediated
immune response without an additional adjuvant [49]. Vaccine ROP2-BCG induced
strong specific humoral and cellular (IL-2 and IFN-γ
in supernatants of splenocytes cell cultures) immunity. A significant delay in
mortality after the infection with the highly virulent RH strain was also
observed [48].The use of recombinant ROP2 antigen, instead of
ROP2-plasmid, revealed again in mouse experimental model that the protective
activity of ROP2 protein is mouse strain-dependent; ROP2 vaccination resulted in the partial
protection (decrease in tissue cyst burden) only in C3H mice but not C57BL/6
mice [50]. The efficacy of ROP2 protein vaccine significantly increased by
including new immunostimulatory components, Leishmania
infantum heat shock protein 83 (Hsp83) fused with the ROP2 [51].
Immunization with this fusion protein elicited a predominant TH1 response in
all mouse strains used (BALB/c, C57Bl/6, and C3H), whereas ROP2 alone or in
mixture with Hsp83 induced a mixed TH1/TH2 profile.Multiple endogenous and exogenous parameters
can influence the type and intensity of vaccine-induced immune response. The
results obtained on inbred mouse strains proved that the protection induced by
ROP2 and other vaccine antigens ofT.
gondii is strongly dependent on the host genetic background [43, 50, 52].
In relation to the vaccine itself, the composition seems a very important
factor in the context of both selected ROP antigens and adjuvants used. In the
light of our current knowledge on rhoptry antigens, ROP18 protein, described
recently as a major component of pathogenicity of T. gondii, seems a very promising candidate
for a vaccine, as a single antigen or, more possibly, accompanied by other T.
gondii antigens because many previous studies showed greater effectiveness
of combined than single-antigen vaccines. However, the usefulness of ROP18 as a
vaccine antigen for toxoplasmosis needs to be experimentally confirmed,
especially in relation to the polymorphism of this protein. Whether protection
across T. gondii species could be
achieved with that polymorphic antigen remains an open question.The advantages of combined vaccines as compared
to single-antigen vaccines were recently emphasized by Zhang et al. [53] who
constructed a multiantigenic DNA vaccine consisting of plasmid encoding both
ROP2 and  surface antigen 1 (SAG1)
antigens and inoculated it to BALB/c mice with or without plasmid encoding
murine IL-12. Multiantigenic (cocktail) vaccine elicited stronger antibody and
cellular responses than single-gene vaccines and, additionally, coimmunization
with IL-12 gene significantly enhanced the intensity of immune responses. The
most immunostimulating vaccine, pSAG1+ROP2+pIL-12, delayed about three times
the survival time of mice after a lethal challenge with RH tachyzoites. As
found by Guo et al. [54] another genetic adjuvant, plasmid carrying IFN-γ,
revealed also a potent stimulatory activity, enhancing the cellular immune
response induced in mice by ROP1-plasmid.In summary, all the above presented
experimental vaccination strategies with ROP antigens still remain suboptimal
but the vaccination results appear promising and therefore future research on
ROP vaccine seems rational.


## 7. Concluding Remarks

Toxoplasmosis, widely dispersed in the
populations of humans and endothermic animals all over the world, is a serious
problem of human and veterinary medicine in the case of immunocompromised
individuals (fetuses, AIDS patients, etc.). The consequences of primary
infection in pregnant females and the possibility of reactivation of latent
infection in immunodepressed individuals cause that both immunoprophylactic and
immunotherapeutic vaccines are needed. Natural infection withToxoplasma leads to strong, lifelong nonsterile
protective immunity. The persistence of the memory T lymphocytes, stimulated by
the regular rupture of T. gondii tissue
cysts or by recurrent contact with infected food, prevents a reinfection in
most individuals [33]. The effective and safe vaccine should mimic the immune
response observed in natural infections and protect the hosts against T. gondii infection, however without the
potential risk of tissue cyst generation in intermediate hosts and development
of oocysts in cats, associated with the vaccination using live attenuated or
incomplete parasite strains. In this
context, for T.
gondii ROP antigens as key molecules in biogenesis of parasite
intracellular niche in the host cells and their association with host
organelles, major virulence factors showing satisfactory immunogenicity [55]
seem particularly valuable vaccine candidates for toxoplasmosis prophylactic
approaches but their vaccine potential needs experimental confirmation. It is
worth mentioning that rhoptry proteins are also vaccine candidates in other apicomplexa,
including  rhoptry-associated protein 1 (RAP1)
and RAP2 proteins of Plasmodium
falciparum [56]. Several rhoptry proteins are polymorphic, with T and B
epitopes that are conserved among strains but not across species and over
species, as found for instance for Plasmodium [57] and Eimeria [58]. The question, if it would be possible to construct
universal anti-T. gondii vaccine,
independent of the host and vaccination target, remains open. The results of
the studies on ROP-vaccine presented in this paper are promising and could be a
good starting point to develop an effective vaccine not only for the prevention
of toxoplasmosis but also other parasitoses caused by apicomplexan protozoa.
Recent advances in bioinformatics and experimental tools will make the
selection of vaccine candidates easier.



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