EXPERIMENTAL

PARASITOLOGY

72, 109-121 (1991)

MINIREVIEW Toxoplasma

gondii-New Advances Molecular Biology

RIMA McLEoD,*?S *Michael

DOUGLAS MACK,*~

in Cellular

and

AND CHARLES BROWN*,$

Reese Hospital and Medical Center, fThe University of Illinois, #The University §The Illinois Institute of Technology, Chicago, Illinois, U.S.A.

MCLEOD,R., MACK, D., AND BROWN, C. 1991. Toxoplasma gondii-New and molecularbiology. Experimental Parasitology 72, 109-121.

of Chicago,

and

advances in

cellular

Although our understanding of the cellular and molecular biology of Toxoplasma gondii is still limited, major advances have been made in these areas during the past decade. Some of these advances have intrinsic importance as they further characterize the structure, composition, physiology, metabolism, and function of this fascinating, obligate intracellular protozoan. They also provide a critical foundation for improved diagnosis as well as development of antimicrobial agents and protective preparations. ULTRASTRUCTURE AND INTERRELATIONSHIPOF TACHYZOITES, BRADYZOITES, AND OOCYSTS Earlier studies of the ultrastructure of T. gondii have been beautifully summarized by Chobotar and Schotyseck (1982). The major new advances have been made in ultrastructural localization of certain proteins in the obligate intracellular tachyzoite form of T. gondii which characterizes acute infection (Fig. 1). Studies of isolated organelle composition and function as well as characterization of T. gondii’s intracellular trafficking remain to be done. It has been known for a number of years that bradyzoites (the form that encysts approximately 8-10 days after acquisition in wivo and characterizes the chronic, latent phase of infection) differ from tachyzoites

in that they are more resistant to pepsin, have a slower generation time, and have cytoplasmic vacuoles which probably are carbohydrate stores (amylopectin) (Beyer er al. 1977) that may provide energy during latency. Recent data indicate that this is the only stage that has the ability to initiate the enteroepithelial cycle and transform into oocysts in the feline intestine (Freyre et al. 1989), and that bradyzoites have stagespecific antigens (Kasper and Ware 1989). Tachyzoites and bradyzoites also share common antigens (Kasper and Ware 1989). T. gondii antigen has been reported to be present in the cyst wall and Sims er al. (1988) recently also described the contribution of neuronal cytoskeletal components to the wall of tissue cysts in brain. Interferon-y (which depletes host intracellular tryptophan, Pfefferkorn et al. 1986) was reported to promote bradyzoite formation within astrocytes in vitro (Jones et al. 1986). Little else is known about factors which lead to formation of bradyzoites or oocysts, or genes which regulate transition or selection among these forms. Our knowledge of bradyzoite metabolism, cyst wall composition, accessibility of encysted bradyzoites to agents which could interfere with their metabolism or promote immune attack, and interaction of encysted bradyzoites and the host is meager. The formation of gametes and oocysts, their use for genetic analysis, and the mi109 0014-4894/91 $3.00 Copyright ,B 1991 by Academic Press. Inc. All rights of reproductmn m any form reserved.

110

MINIREVIEW: Toxoplasma gondii ANTERIOR MICROTUBULES

MICROTUBULE CYfOSKELETON OUTER MEMBRANE INNER MEW&WE

MITOCHONDRION

FIG. 1. Diagrammatic representation of T. gondii tachyzoite. Proteins and glycoproteins are indicated by a square outline which encloses them and by italic type. Their locations are indicated by arrowheads. *There are 22 microtubules that compose T. gondii’s cytoskeleton; these actually extend posteriorly but are indicated schematically in this figure only at the anterior end. **Parasitophorous vacuole. tLocations of ATPase activity, not necessarily the cloned 63-kDa protein.

croscopic appearance of the flagellated mi- pered study of their cellular and molecular crogamete (male), macrogamete (female), biology. and intermediate stages in gametogenesis and sporogony in the feline intestine have INVASION-ATTACHMENT, SECRETION, been reviewed comprehensively recently MOTILITY, AND FORMATION OF THE (Pfefferkorn 1990). The genetic potential to PARASITOPHOROUSVACUOLE become either a micro- or a macrogamete is There have been a number of new adpresent in a single sporozoite (reviewed in Pfefferkorn 1990). Antigens specific for vances in our understanding of mechanisms oocysts and antigens shared among oocysts underlying T. gondii’s entry into cells. These studies have demonstrated that invaand other stages have been identified (Kasper and Ware 1985). The present in- sion is a complex process, which occurs in ability to culture oocysts in vitro has ham- approximately 15 set (Werk 1985). Earlier

MINIREVIEW:

Toxoplasma

studies demonstrated that the host cell surface influences invasion and included the observations that invasion is enhanced when host cells are in the S phase of their cell cycle or by prior treatment of the host cells with enzymes such as B-glucuronidase. New evidence that T. gondii participates actively in invasion includes either inhibition or enhancement of invasion by alteration of extracellular ion concentration and thus motility of tachyzoites (Endo and Yagita 1990), data concerning effects of T. gondii extruded products which enhance penetration (Werk 1985; Schwartzman 1986), and very recently reported studies which indicated that during invasion, at entry, T. gondii tachyzoites create a vacuolar membrane depleted of certain cellular membrane markers and intramembranous particles that lacks (or has obscured) signals needed for fusion with other intracellular compartments (Joiner et al. 1990). In these elegant studies, native Chinese hamster ovary cells and these cells transfected with genes that encode Fc receptors were used. As others had demonstrated earlier for macrophages, it was shown that T. gondii is able to survive and replicate intracellularly by preventing fusion of its parasitophorous vacuole with host lysosomes, as well as other intracellular compartments, and preventing acidification of the vacuole. The tachyzoite’s ability to prevent fusion and acidification is dependent on whether it enters cells via Fc receptors (creating a fusion competent vacuole) or invades (producing a fusion incompetent vacuole) (Joiner ef al. 1990). Recently, it has also been demonstrated that laminin and laminin-binding proteins of 14-16 and 60 kDa are present on the tachyzoite surface and the laminin binding proteins appear to be involved in attachment of tachyzoites to the host cell (Joiner et al. 1989). If other host cell surface components also are used as receptors for attachment, they must be ubiquitous as almost all cells can be in-

gondii

111

vaded. It has been suggested that cholesterol (used as a receptor by rickettsia) might also be used for attachment by T. gondii (Pfefferkorn 1990) but this hypothesis has not been tested. As in other apicomplexa, the anterior portion of the tachyzoite is differentiated with organelles (Fig. 1) which assist entry and enters the host cell first (Aikawa et al. 1977; reviewed in Chobotar and Schotyseek 1982 and in Pfefferkom 1990). Isolation of apical components may be useful for identification of T. gondii apical components essential for entry. For other apicomplexa, it has been possible to isolate the surface membrane and apical components (Dubremetz and Dissous 1980) and isolation of T. gondii’s pellicle, microtubules, and conoid has been reported (Tyron 1979). The anterior end contains actin (Endo et al. 1988), myosin, the conoid, other unique microtubules, anterior polar rings, and a duct from the rhoptries to the tachyzoite surface (Fig. 1). No other specific attachment factors or unique surface epitopes responsible for oriented attachment and invasion have been identified. There are higher concentrations of laminin binding proteins (Joiner et al. 1989)and Fc binding sites (Budzko et al. 1989) at one end of the tachyzoite but it has not been established yet at which end. After the anterior end attaches, there is protrusion of the conoid and rhoptries extrude their contents, including a protein of approximately 60 kDa (Sadak et al. 1988; Schwartzman 1986). Rhoptry components appear to enhance penetration. Dense granule components are also extruded during invasion (Charif et al. 1990). It has been hypothesized that lipids from rhoptries or dense granules might be incorporated into and modify the vacuolar membrane as suggested for Plasmodium falciparum (Joiner et al. 1990). Motility is also important in invasion and is dependent on a pH gradient determined by extracellular ions. Motility occurs when

112

MINIREVIEW:

Toxoplasma gondii

the internal pH is greater than the external pH and likely involves actin (Endo et al. 1988) myosin (Schwartzman and Pfefferkom 1983), and microtubules (Schwartzman et al. 1985; Werk 1985). The host cell membrane and T. gondii’s outer membrane appear to form a moving, tight junction probably utilizing surface membrane components anchored to the cytoskeletal microtubules of T. gondii with actin and myosin providing the force. There is support for this hypothesis in the observations that there are connections between the subpellicular microtubules and the inner membrane complex, and between the subpellicular microtubules and the outer membrane (Cintra and De Souza 1985). During this process, the host cell membrane does not rupture but encircles the tachyzoite completely. As described above, the portion of the host cell membrane which contributes to the parasitophorous vacuole appears to be modified during this process. For example, this membrane loses its Mg+ATPase and 5’-nucleotidase but not its Na+K’ATPase or anionic sites (De Carvalho and De Souza 1989, 1990). It is possible that these modifications that are associated with fusion incompetence could also lead to an inability to acidify the parasitophorous vacuole if for example H+ATPase were not delivered to the vacuole (Joiner et al. 1990). Inability to acidify the vacuole could also be related to active exclusion or inactivation of plasma membrane proton pumps or other ion channels during formation of the vacuolar membrane (Joiner et al. 1990). A secretory network of tubular structures forms within the parasitophorous vacuole which contains T. gondii proteins, including the most abundant surface protein of 30 kDa and the dense granule-secreted proteins of 23 and 28.5 kDa. This network is continuous with the vacuolar cell membrane and the inner membrane of the parasitophorous vacuole is the former exterior face of the host cell membrane. T. gondii begins to synthesize DNA and

multiply by a process of binary fission called endodyogeny in which two daughter cells form from each tachyzoite. In the process, the nuclear membrane remains intact and chromosomes do not condense at metaphase (reviewed in Pfefferkom 1990). It is intriguing that after invasion T. gondii’s mitochondrial membrane potential is reduced (Tanabe and Murakami 1984; Kimata ef al. 1987) and host cell mitochondria collect around the parasitophorous vacuole. Interestingly, T. gondii has an extremely abundant, potent, and unique ATPase postulated to be important in purine salvage which is activated in an anionic environment (Asai et al. 1987;Johnson et al. 1989). As discussed above, at entry, when T. gondii invades, it prevents acidification of the parasitophorous vacuole as well as fusion of phagosomes and lysosomes (Sibley er al. 1985, Joiner et al. 1990). Recently parasitophorous vacuoles have been isolated, although not yet purified to homogeneity (K. A. Joiner, personal communication), which should greatly facilitate understanding how T. gondii interacts with the host cell. Exit of T. gondii tachyzoites from the parasitophorous vacuole can be elicited by the carboxylic calcium ionophore, monensin (Endo et al. 1982), and the exiting tachyzoites are highly motile. The processes causing egress from an infected cell have not been characterized. GENOME-COMPOSITION, TRANSCRIPTION, AND TRANSLATION

This subject has recently been reviewed in detail (J. C. Boothroyd and L. H. Kasper, in preparation, and A. Johnson, in preparation). Briefly, T. gondii’s nucleus is haploid except during sexual division in the feline intestine and prior to binary fission in the tachyzoite. The exact number of chromosomes in the haploid nucleus has not been determined but 8 have been identified using pulsed field gel electrophoresis and the total number is estimated to be 12 or

MINIREVIEW:

Toxoplasma

less (L. D. Sibley, in J. C. Boothroyd and L. H. Kasper, in preparation). They range in size from 2 to >lO Mbp (L. D. Sibley, in J. C. Boothroyd and L. H. Kasper, in preparation). There is less than 20% variation in chromosome size among the isolates of T. gondii studied (L. D. Sibley, in J. C. Boothroyd and L. H. Kasper, in preparation). T. gondii’s haploid, nuclear DNA contains approximately 8 x 10’ bp (Cornelissen er al. 1984) and has a GC content of approximately 55%. There are no methylated bases (Johnson et al. 1987), which are thought to be important in gene regulation. T. gondii’s mitochondrial DNA is circular, 36 kb, and has a IO-kb inverted repeat (Borst et al. 1984). Recent studies of T. gondii DNA have revealed few unique features which permit its characterization using conventional molecular biologic techniques. Specific information about T. gondii genes that have been partially or completely sequenced and the proteins they encode is given in Table I. General principles concerning the structure and transcription of T. gondii genes and the translation of its mRNA derived from studies of these genes are as follows: There is no information about RNA polymerases of T. gondii. The regions surrounding, particularly those upstream of, transcription start sites, for the genes which encode ~30, (Yand P-tubulin, and ~23, have been identified (Burg et al. 1988, Cesbron-Delauw et al. 1989; Nagel and Boothroyd 1988). However, there are no consensus sequences among the tubulin or p30 T. gondii genes or homology with upstream regions of higher eukaryotes such as TATA and CCAAT boxes, which might serve as promotors, activators, or enhancers of transcription, identified in the vicinity of these start sites (Boothroyd et al. 1987; Burg et al. 1988). In the p23 gene a CAAT box was identified (Cesbron-Delauw et al. 1989). Interestingly, in the p30 gene there is a series of five, 27-bp repeats, starting 60 bp upstream of the start site, but their role in expression

gondii

113

of the p30 gene is not yet defined (Burg et al. 1988). Four of the genes identified have introns (Table I), placing T. gondii higher on the evolutionary tree than the kinetoplastidia that have DNA which does not have introns. No transplicing has been found in either tubulin or p30 genes (Nagel and Boothroyd 1988; Burg et al. 1988). T. gondii can use the same splicing signals as the human HeLa cell line (J. C. Boothroyd and L. H. Kasper, in preparation). Among the genes studied, only the Bl gene is tandemly reiterated (Burg et al. 1989). In contrast, the fact that the tubulin gene exists as a single copy while its product, tubulin, is utilized in many diverse components of the tachyzoite suggests that T. gondii may have interesting mechanisms for post-translational modification of proteins. The p30 gene encodes signal peptides (Burg et al. 1988). The translation start sites of the genes encoding ~30, ~23, and tubulin conform approximately to Kozak’s criteria for initiation codons that in the - 3 position relative to the Met [ATG] codon there is a purine and in the +4 position there is a guanine (Burg et al. 1988; Cesbron-Delauw et al. 1989; Nagel and Boothroyd 1988). There is bias in codon usage to codons ending in C or G in preference to those ending in T and A for the abundantly expressed genes encoding p30 and NTPase (Burg et al. 1988; Johnson et al. 1989), but not for certain less abundantly expressed genes of T. gondii (J. C. Boothroyd and L. H. Kasper, in preparation). The locations of the proteins within T. gondii tachyzoites encoded by some of the genes that have been completely or partially sequenced (Table I) are shown in Fig. 1 and discussed below. The product of the Bl gene is unknown but this tandemly repeated gene may be useful for PCR detection of T. gondii in body fluids and tissues (Burg et al. 1989). The p30 gene has also been used with PCR (Saava et al. 1990). Sequences encoding the small subunit rRNA have been used to establish T. gondii

lntrons

Yes

Yes

COPY no.

I

I

p.54, a-tubulin

~54, S-tubulin

I.1 kb

1500 nt 1400 nt

Yes

Yes Yes

Not certain

No

NO

I

I

I

gp28

gp36

~23

a Not stated.

Yes

Yes

Yes

Yes

Yes Unknown

Yes

Yes

Post translational modification

24

34.7

S8

130

NS 25

54

54

Fusion protein size Wa)

-^=.----_“--_.^..-._^, .“.,.

I600 nt

NO

NO

I

gp22

1.6 kb 2800 nt

Yes NO

Yes

No

Gene B 1, unknown ~63, NTPase

SO

1362 nt

1347 nt

No

NO

mRNA size

l-5

product

Total gene sequenced

_”

NO

Yes

NO

Yes

NS NS

NS

NS”

GPI anchor

,”

Yes

Yes

Yes

NS

NS NS

NS

NS

ESA

TABLE I Cloned Genes and the Proteins They Encode

“,

Dense granules: beneath plasmalemma; golgi; vacuolar network Surface; vacuolar network Dense granules, vacuolar network

Unknown Submembrane; mitochondria Surface

Subpellicular: microtubules

Location

“.

Calcium binding

Unknown

Unknown

Unknown (Purine salvaeet Unknown

Motility (invasion)

Function (postulated)

,,

Cesbron-Delauw er al. (1989)

Burg cr al. (1988)

,,I

Boothroyd er al. ( 1987) Schwartzman et al. (1985) Nagel and Boothroyd (1988) Burg et al. (1989) Johnson er al. ( 1989) Boothroyd CI al. ( 1987) prince cl nl. ( 1989)

.”

MINIREVIEW: Toxoplasma

as ancient, near Sarcocystis, and quite separate from malaria in a phylogenetic tree (Johnson and Baverstock 1989). The major practical advance resulting from the characterization, sequencing, and cloning of T. gondii DNA has been the development of PCR probes to detect antigen in patient specimens such as amniotic fluid. Also, sequence data are being used in attempts to develop improved, standardized diagnostic reagents and are being tested for vaccine development. RNA T. gondii ribosomal RNA has the typical large and small molecule. Its messenger RNA has a 3’-polyadenylated tail which has been translated in heterologous systems producing T. gondii proteins (Prince ei al. 1985). PROTEINS: ULTRASTRUCTURAL LOCATIONS, POSSIBLE FUNCTIONS, AND UTILITY AS DIAGNOSTIC REAGENTS OR VACCINE COMPONENTS T. gondii contains more than 1000 proteins (Pfefferkorn 1990). Recent studies have begun to provide insight into the structure, function, use as diagnostic reagents, or role in producing protective immune responses of some of these proteins. The major surface proteins of T. gondii are shown in Fig. I and some are listed in Table I. Different methods of preparation and analyses likely have resulted in ascribing slightly different molecular weights to the same proteins. The 43-, 35, 30-, and 22-kDa surface proteins are anchored by a glycosylphosphatidylinositol anchor (Tomavo et al. 1989) which is antigenically similar for all of them as well as for the VSGs of trypanosomes. Their removal under physiologic conditions with PI-specific phospholipase should permit analysis of their reexpression at the cell surface as well as invasive properties of surface antigen free tachyzoites (Tomavo et al. 1989). Mu-

gondii

115

tant T. gondii tachyzoites (Kasper et al. 1982) that do not express these proteins also should be useful in determining their functions. The most abundant of these surface proteins, gp30, has been studied most extensively (Santoro et al. 1986, Burg et al. 1988; reviewed in J. C. Boothroyd and L. H. Kasper, in preparation): Its apparent molecular weight is 30-35 kDa under reducing conditions and 27-28 kDa under nonreducing conditions. It is conserved among most strains of T. gondii tested. It is detected only in tachyzoites (not bradyzoites or sporozoites) and constitutes approximately 5% of the total tachyzoite protein. It is homogeneously distributed on the tachyzoite surface, within the tachyzoite, and in the tubular network of the parasitophorous vacuole. It is hydrophobic with a slightly acidic isoelectric point. Two-dimensional gel electrophoresis resolves several closely related forms of this protein. The primary translation product has a carboxy-terminal hydrophobic tail, which predicts posttranslational cleavage and modification with a glycolipid anchor. The p30 mRNA transcript is approximately 1500 nucleotides in length and polyadenylated. Antibodies to p30 have been identified in sera from acutely and chronically infected adults and congenitally infected individuals and in human secretory IgA (D. Mack and R. McLeod, in preparation). Passive protection studies using monoclonal antibodies to p30 paradoxically have reported either partial protection or increased mortality. Administration of p30 with adjuvants to mice has resulted in substantial protection (L. H. Kasper, personal communication). Interestingly, this protein can stimulate IgG, IgM, IgE, IgA, and secretory IgA responses as well as lymphocytes that produce interferon--y and cytotoxic lymphocytes (Khan et al. 1988). These observations suggests that p30 can sensitize both THl and TH2 lymphocytes. It has also been useful in the development of a sensi-

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Toxoplasma gondii

tive diagnostic test for congenital toxoplasmosis (Decoster ef al. 1988b). Excreted-secreted antigens (ESA) have been found to confer enhanced survival in mouse and rat models (Duquesne et al. 1990; Sharma et al. 1984) and thus have been intensively studied. One of these, gp28.5, has been identified by a monoclonal antibody (Sharma et al. 1984) that also cross-reacts with a 58-kDa protein. ESA gp28.5 is present in dense granules, beneath the outer membrane of T. gondii tachyzoites, in the Golgi apparatus where it is likely packaged for secretion, and in the parasitophorous intravacuolar network (Charif et al. 1990; Sibley and Sharma 1987). It is also present in bradyzoites and in the cyst wall (Charif et al. 1990). Its function in the parasite is unknown. It elicits an antibody response in sera from all chronically infected individuals tested (Decoster et al. 1988a) and certain of its hydrophilic peptides have conferred minimal protection in a mouse model (Araujo et al. 1990). Another ESA that has been studied, p23 (27 native), has been cloned (CesbronDelauw et al. 1989)and has calcium binding properties. It is located in the dense granules of tachyzoites and bradyzoites and in the parasitophorous vacuoles of the host cell (Cesbron-Delauw ef al. 1989). Its further characterization should help to define function(s) of dense granules. Regulation of calcium, its interaction with calcium binding proteins, and roles of calcium and calcium binding proteins in T. gondii’s intracellular processes are likely to be productive areas of future research. Wang (1982) presented a comprehensive summary of earlier knowledge of coccidial biochemistry and metabolism stressing features unique to coccidia. This also provided bases for strategies of rational drug design. He described empirically discovered coccidiostatic and coccidiocidal agents which have provided insight into coccidial metabolism and biochemistry. Recent elegant

work with mutant host cells and mutant parasites (reviewed in Pfefferkorn 1990; Schwartzman and Pfefferkom 1982) has defined some of the critical enzymes of T. gondii, which provides some insight into why T. gondii is an obligate intracellular parasite and may provide a basis for development or use of novel antimicrobial agents. One of the most important observations is that T. gondii is incapable of purine synthesis and depends on the host cell for preformed purines (reviewed in Pfefferkom 1990; Schwartzman and Pfefferkom 1982). ATP is largely degraded to adenosine before it can be used by T. gondii (Pfefferkorn 1990). T. gondii hypoxanthine-guanine phosphoribosyltransferase acting alone can provide adequate purine salvage (Pfefferkorn 1990). A number of other purine salvage enzymes have also been identified in T. gondii (Krug et al. 1989). Using the observation that uracil phosphoribosyltransferase is present in T. gondii, an in vitro effect of an analogue of the uracil substrate was recently described (Pfefferkorn et al. 1989). In addition, it has been demonstrated that a variety of host cell constituents or processes are not essential for T. gondii’s intracellular survival. These include a host cell nucleus and host cell DNA synthesis, oxidative phosphorylation by ATP, mRNA synthesis, protein synthesis, thymidine, or a pyrimidine nucleotide pool (reviewed in Pfefferkorn 1990). Since T. gondii cannot use preformed folates as mammalian cells can, the dihydrofolate reductase (DHFR) of T. gondii has been a major target for antimicrobial agents directed against T. gondii (Kovacs et al. 1990; Derouin and Chastang 1989). Therefore, recently it has been characterized more completely (Kovacs et al. 1990). T. gondii DHFR has been found to be a bifunctional enzyme with both DHFR and thymidylate synthetase activity as described for other protozoa (Kovacs et al. 1990). Another potential target, dihy-

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Toxoplasma gondii

dropteroate synthase, which is also responsible for de aovo synthesis of folates, has also recently been characterized (Allegra et at. 1990). Nucleoside triphosphate hydrolase (NTPase) is localized in the cytosol and is about 8% of the total tachyzoite protein (Asai et al. 1987; Johnson et al. 1989). ATPase activity was present most abundantly between the layers of the tachyzoite membrane, and also in mitochondria, microsomes, Golgi complex, cytoplasmic vacuoles, and rhoptries. NTPase has broad substrate specificity for both ribo- and deoxyribonucleoside triphosphates. Dithiol compounds are essential activators and magnesium is the most effective activating metal ion. One unique feature of this enzyme among protozoa is that in the presence of dithiothreiotol, its hydrolytic activity is markedly enhanced (500-fold) in contrast to the substantially lesser effect of dithiothreitol on activity of NTPase in other protozoa. It has four subunits of 63 kDa and is detected in peripheral blood during acute infection in a mouse model (Asai et al. 1987). A rhoptry protein of 43 or 55-60 kDa with post-translational modification has been identified (Shwartzman 1986; Sadak et al. 1988) and its relationship to previously identified penetration enhancing factor is being studied. Phospholipase activity also recently has been identified. It is likely that some of these extruded rhoptry components alter the host cell membrane and facilitate invasion (Pfefferkorn 1990). Recently, Darde et al. (1988) used isoelectric focusing in polyacrylamide gels to study T. gondii strain differences in enzyme content. Fourteen enzymes had the same patterns for all strains tested, and four enzymes (aspartate amino transferase, glutathione redutase, amylase, and glucose phosphate isomerase) had three patterns which appeared to have an approximate relationship between pathogenicity and

oocyst production. Such enzyme patterns (as well as RFLPs) could be useful as markers in genetic linkage studies. Strain differences in antigens have also been described. As discussed above, stage-specific epitopes have been described (Kasper and Ware 1989) and may prove to be useful for serodiagnosis. Epitopes common to tachyzoites and bradyzoites (D’Arcy et al., in press) may prove to be useful for vaccine development. CARBOHYDRATES

AND LIPIDS

There is little new information about T. carbohydrates. Antibody to a 6kDa carbohydrate antigen appears to be a marker for acute infection (Erlick et al. 1983) but has not been characterized further. Maurus et al. (1980) found 3 x lo4 sites for concanavalin A binding (a low number) but no binding of wheat germ agglutinin on tachyzoites and no sialic acid. The GPI anchor for p30 is a diglycerol linked via phosphoinositol to sugars which include mannose (J. C. Boothroyd and L. H. Kasper, in preparation). The cyst wall binds lectins. Little is known about T. gondii’s lipid composition or that of the parasitophorous vacuole. T. gondii has a low cholesterol/ phospholipid ratio, and was reported to have many unsaturated fatty acid chains, relatively large amounts of phosphatidylcholine, and low amounts of sphingomyelin (Gallois et al. 1988). It is intriguing that one type of cell (plant protoplasts) that T. gondii cannot invade has a low ratio of cholesterol to lipids (Werk 1985).

gondii’s

THE FUTURE

The studies described above provide a foundation for further characterization of the cellular and molecular biology of T. gondii. Some areas likely to yield fundamental and useful information include preparation of genomic linkage maps, determination of signals that regulate T. gondii’s

118

MINIREVIEW: Toxoplasma gondii

genes, study of relationships of isolates, and characterizations of virulence factors. Transformation/transfection systems would be useful in determining functions of T. gondii’s proteins and it will be of interest to determine which antigens will be useful for diagnosis and protection. Establishment of how T. gondii attaches to and invades host cells, particularly characterization of the role of its lipids, as well as identification of unique metabolic pathways or T. gondii components, may provide new strategies to induce protection and for antimicrobial therapy. Information about bradyzoite metabolic pathways accessible to antimicrobial agents could be especially important in therapy of immunocompromised individuals with toxoplasmosis and patients with congenital toxoplasmosis, as encysted organisms play a major role in the pathogenesis of these diseases. It would be of great interest to determine why T. gondii is an obligate intracellular parasite and what is needed to grow it axenically as well as factors that influence selection of tachyzoites versus bradyzoites and/or genes and gene products that trigger switches from tachyzoite to bradyzoite and from bradyzoite to sporozoite. It would also be useful to learn what is unique about the feline intestine that permits oocyst formation and to produce oocysts in vitro, especially for characterization of T. gondii genetics. Studies of these aspects of T. gondii cellular and molecular biology are likely to be of considerable interest and medical importance. ACKNOWLEDGMENTS This work was supported by Grant AI 19645from the National Institutes of Health. We appreciate Ms. D. Patton’s assistance with the preparation of the manuscript and Drs. Ben Stark and Lloyd Kasper’s thoughtful reviews of and suggestions concerning parts of the manuscript.

plasma gondii. American

Journal

of Pathology

87,

285-296. ALLEGRA, C. J., BOARMAN, D., KOVACS, J. A., MORRISON, P., BEAVER, J., CHABNER, B. A., AND MASUR, H. 1990. Interaction of sulfonamide and sulfone compounds with Toxoplasma gondii dihydropteroate synthase. Journal of Clinical Investigation 85, 371-379. ARAUJO, F. G., PRINCE, J. B., JUDD, A. K., AND REMINGTON, J. S. 1990. Immunogenicity of synthetic peptides from P28 of Toxoplasma gondii. In

“Seventh International Congress of Parasitology, Abstracts, August 2624.” ASAI, T., KIM, T., KOBAYASHI, M., AND KOJIMA, S. 1987. Detection of nucleoside triphosphate hydrolase as a circulating antigen in sera of mice infected with Toxoplasma gondii. Infection and Immunity 55, 1332-1335. BEYER, T., SIM. V., AND HUTCHINSON, W. 1977. Cytochemistry of Toxoplasma gondii. VI Polysaccharides, lipids and phosphatase on cyst forms. Tsitogia 19, 979. BOOTHROYD, J. C., BURG, J. L.. NAGEL, S. D., PERELMAN, D., KASPER, L. H., WARE, P. L., PRINCE, J. B., SHARMA, S. D., AND REMINGTON, J. S. 1987.Antigen and tubulin genes of Toxoplasma Strategies of Parasitic gondii. In “Molecular

Invasion” (N. Agabian, H. Goodman, and N. Nogueira, Eds.1, pp. 237-250. A. R. Liss, New York. BORST, P., OVERDULVE, J. P., WEWERS, P. J., FASEFOWLER, F., AND VANDENBERG, M. 1984. DNA circles with cruciforms from Isospora Toxoplasma gondii. Biochimica Biophysics Acta 781, 100. BUDZKO, D. B., TYLER, L., AND ARMSTRONG, D. 1989. Fc receptors on the surface of Toxoplasma gondii trophozoites: A confounding factor in testing for anti-Toxoplasma antibodies by indirect immunofluorescence. Journal of Clinical Microbiology 27, 959-%l. BURG, J. L., PERELMAN, D., KASPER, L. H., WARE, P. L., AND BOOTHROYD, J. C. 1988. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. Journal of Immunology 141, 3584-3591. BURG, J. L., GROVER, C. M., POULETTY, P., AND BOOTHROYD, J. C. 1989. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii. by polymerase chain reaction. Journal of Clinical Microbiology 27, 1787-1792. CESBRON-DELAUW, M. F., GUY, B., TORPIER, G., PIERCE, R. J., LENZEN, G., CESBRON, J. Y.,

REFERENCES

CHARIF, H., LEPAGE, P., DARCY, F., LECOCQ, J. P., AND CAPRON, A. 1989. Molecular character-

AIKAWA, M., KOMATA, Y., ASAI, T., AND MrDORIKAWA, 0. 1977. Transmission and scanning

ization of a 23kilodalton major antigen secreted by

electron microscopy of host cell entry by Toxo-

Toxoplasma gondii. Proceedings of the National Academy of Sciences, U.S.A. 86, 7537-7541.

MINIREVIEW:

Toxoplasma

gondii

119

CHARIF, H., DARCY, F., TORPIER, G., CESBRONnude rats against Toxoplasma infection by excretedDELAUW, M. F., AND CAPRON, A. 1990. Toxosecreted antigen-specific helper T cells. Infection plasma go&ii: Characterization and localization of and Immunity 58, 2120-2126. ENDO, T., SMITH, K. K., AND PIEKARSKI, G. 1982. antigens secreted from tachyzoites. Experimental Parasitology 71, 114-124. Toxoplasma gondii: Calcium ionophore A23 187mediated exit of trophozoites from infected murine CHOBOTAR, B., AND SCHOTYSECK,E. 1982. Ultramacrophages. Experimental Parasitology 53, 17% structure. In “The Biology of the Coccidia” (P. L. 188. Long, Ed.), pp. 101-165. Univ. Park Press, BaltiENDO, T., YAGITA, K., YASUDA, T., AND NAKAmore. MURA,T. 1988.Detection and localization of actin in CINTRA, W. M., AND DE SOUZA, W. 1985. ImmunoToxoplasma gondii. Parasitology Research 75, 102cytochemical localization of cytoskeletal proteins 106. and electron microscopy of detergent extracted ENDO, T., AND YAGITA, K. 1990. Effect of extraceltachyzoites of Toxoplasma gondii. Journal of Submicroscopy Cytology 17, 503-508. lular ions on motility and cell entry in Toxoplasma gondii. Journal of Protozoology 37, 133-138. CORNELISSEN,A. W. C. A., OVERDULVE,J. P., AND VAN DER PLOEG,M. 1984.Determination of nuclear ERLICH, H. A., RODGERS,G., VAILLANCOURT, P., ARAUIO, F. G., AND REMINGTON,J. S. 1983. IdenDNA of five Eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella. tification of an antigen-specific immunoglobulin M E. acervulina, and Plasmodium berghei, with speantibody associated with acute Toxoplasma infeccial reference to gamontogenesis and meiosis in I. tion. Infection and Immunity 41, 683-690. ( T. ) gondii. Parasitology 88, 53 l-553. FREYRE.A., DUBEY, J. P., SMITH, D. D., AND FRENKEL, J. K. 1989.Oocyst-induced Toxoplasma gondii DARDE, M. L., BOUTEILLE, B., AND PESTREALEXANDRE. M. 1988. Isoenzymic characterization infections in cats. Journal of Parasitology 75, 750755. of seven strains of Toxoplasma gondii by isoelectrofocusing in polyacrylamide gels. American Journal GALLOIS, Y., FOUSSARD,F.. GIRAULT, A., HODBERG, of Tropical Medicine and Hygiene 39, 55 l-558. J.. TRICAUD, A., MAURAS, G., AND MOTTA, C. DE CARVALHO, L., AND DE SOUZA, W. 1989. Cyto1988. Membrane fluidity of Toxoplasma gondii: A chemical localization of plasma membrane enzyme fluorescence polarization study. Biology of the Cell markers during interiorization of tachyzoites of Tox62, 11-15. oplasma gondii by macrophages. Journal of ProtoJOHNSON, A. M.. ILLANA. S., DUBEY, J. P., AND zoology 36, 164170. DAME, J. B. 1987. Toxoplasma gondii and Hammondia hammodiic DNA comparison using cloned DE CARVALHO, L., AND DE SOUZA, W. 1990. Internalization of surface anionic sites and phagosomerRNA gene probes. Experimental Parasitology 63, 272-278. lysosome fusion during interaction of Toxoplasma gondii with macrophages. European Journal of CelJOHNSON, A. M., AND BAVERSTOCK, P. R. 1989. lular Biology 51, 21 I-219. Rapid ribosomal RNA sequencing and the phylogeDECOSTER,A., DARCY. F., AND CAPRON, A. 1988a. Today 5, netic analysis of protists. Parasitology Recognition of Toxoplasma gondii excreted and se102-105. creted antigens by human sera from acquired and JOHNSON,A. M.. ILLANA, S.. MCDONALD. P. J.. AND congenital toxoplasmosis: Identification of markers ASAI, T. 1989. Cloning, expression and nucleotide of acute and chronic infection. Clinical and Experisequence of the gene fragment encoding an antigenic mental Immunology 73, 375-382. portion of the nucleoside triphosphate hydrolase of DECOSTER,A., DARCY, F., CARON, A., AND CAPRON, Toxoplasma gondii. Gene 85, 215-220. A. 1988b. IgA antibodies against p30 as markers of JOINER, K. A., FURTADO. G., MELLMAN, I., KLEINcongenital and acute toxoplasmosis. Lancer 2, 1104MAN, H.. MIETTINEN, H., KASPER, L. H.. HALL, 1107. L., AND FUHRMAN, S. A. 1989.Cell attachment and DEROUIN, F.. AND CHASTANG, C. 1989. In vitro efinvasion by tachyzoites of Toxoplasma gondii. Jourfects of folate inhibitors on Toxoplasma gondii. Annal of Cellular Biochemistry 13E, 64. timicrobial Agents and Chemotherapy 33, 1753- JOINER.K. A., FUHRMAN, S. A., MIETTINEN, H. M., 1759. KASPER, L. H., AND MELLMAN, I. 1990. ToxoDUBREMETZ,.I. F., AND Drssous, C. 1980.Characterplasma gondii: Fusion competence of parasitophoistic proteins of micronemes and dense granules rous vacuoles in Fc receptor-transfected tibroblasts. from Sarcocystis tenella endozoites (protozoa, cocScience 249, 641446. cidia). Molecular and Biochemical Parasitology 1, JONES, T. C., BIENZ. K. A., AND ERB, P. 1986. In 279-289. vitro cultivation of Toxoplasma gondii cysts in asDUQUESNE, V., AURIAULT, C., DARCY, F., DEtrocytes in the presence of gamma interferon. lnfecCLAVEL, J. P., AND CAPRON.A. 1990. Protection of tion and Immunity 51, 147-156.

120

Toxoplasma gondii

MINIREVIEW:

lular Immunological and Molecular Aspects,” KASPER, L. H., CRABB, J. H., AND PFEFFERKORN, (D. J. Wyler, Ed.), pp. 26-50. Freeman, New York. E. R. 1982. Isolation and characterization of a monoclonal antibody-resistant antigenic mutant of PRINCE, J. B., KOVEN-QUINN, M. A., REMINGTON, Toxoplasma gondii. Journal of Immunology 129, J. S., AND SHARMA, S. D. 1985. Cell free synthesis 1694-1699. of ToxoplASmA gondii antigens. Molecular and Biochemical Parasitology 17, 163-170. KASPER,L. H., AND WARE, P. L. 1985. Recognition and characterization of stage specific oocyst sporo- PRINCE, J. B., ARAUJO, F. G., REMINGTON, J. S., BURG, J. L., BOOTHROYD, J. C., AND SHARMA, zoite antigens of Toxoplasma gondii by human anS. D. 1989.Cloning of cDNAs encoding a 28 kilodaltisera. Journal of Clinical Investigation 15, 157G ton antigen of Toxoplasma gondii. Molecular and 1577. Biochemical Parasitology 34, 3-14. KASPER,L. H., AND WARE, P. L. 1989. Identification SAAVA, D., MORRIS,J. C., JOHNSON,J. D., AND HOLof stage specific antigens of T. gondii. Infection and LIMAN, R. E. 1990. Polymerase chain reaction for Immunity 57, 668-672. detection of ToxoplASmA gondii. Journal of Medical KHAN, I. A., SMITH, K. A., AND KASPER, L. H. Microbiology 32, 25-3 1. 1988. Induction of antigen-specific parasiticidal cytotoxic T cell splenocytes by a major membrane pro- SADAK, A., TAGHY, Z., FORTIER, B., AND DuBREMETZ,J. F. 1988.Characterization of a family of tein (P30) of Toxoplasma gondii. Journal of Immurhoptry proteins of Toxoplasma gondii. Molecular nology 141, 3600-3605. and Biochemical Parasitology 203-211. KIMATA, I., TANABE, K., IZUMO, A., AND TAKADA, SANTORO,F., CHARIF, H., AND CAPRON, A. 1986. S. 1987. Host cell ATP level and invasion of ToxoThe immunodominant epitope of the major memplasma gondii. Transactions of the Royal Society of brane tachyzoite protein (~30) of Toxoplasma gonMedicine and Hygiene 81, 377-380. dii. Parasite Immunology 8, 631-639. KOVACS, J. A., ALLEGRA, C. J., AND MASUR, H. SCHWARTZMAN, J. D., AND PFEFFERKORN, E. R. 1990.Characterization of dihydrofolate reductase of 1982. Toxoplasma gondii: Purine synthesis and salPneumocystis carinii and Toxoplasma gondii. Exvage in mutant host cells and parasites. Experimenperimental

Parasitology

11, 60-68.

KRUG, E. C., MARR, I. J., AND BERENS,R. L. 1989. Purine metabolism in Toxoplasma gondii. Journal of Biological Chemistry 264, 10,601-10,607. MAURAS,G., DODEUR,M., LAGET, P., SENET,J. M., AND BOURILLON, R. 1980. Partial resolution of the sugar content of Toxoplasma gondii membrane. Biochemical and Biophysical tions 91, 906-912.

Research

Communica-

NAGEL, S. D., AND BOOTHROYD,J. C. 1988. The alpha and beta tubulins of Toxoplasma gondii are encoded by single copy genes containing multiple introns. Molecular and Biochemical Parasitology 29, 267-273. PFEFFERKORN,E. R. 1981. Molecular genetics of Toxoplasma: Toxoplasma gondii as a model parasite for genetic studies. In “Modem Genetic Concepts and Techniques in the Study of Parasites” (F. Michal, Ed.) pp. 195-212. Schwabe, Basel. PFEFFERKORN,E. R., ECKEL, M., AND REBHUN, S. 1986. Interferon-gamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan. Molecular and Biochemical Parasitology 20, 215-222. PFEFFERKORN,E. R., ECKEL, M. E., MCADAMS, E. 1989. Toxoplasma gondii: The biochemical basis of resistance to enimycin. Experimental Parasitology 69, 129-139. PFEFFERKORN,E. 1990. The cell biology of Toxoplasma gondii. In “Modern

Parasite Biology:

Cel-

tal Parasitology

53, 77-86.

SCHWARTZMAN, J. D., AND PFEFFERKORN, E. R. 1983. Immunofluorescent localization of myosin at the anterior pole of the coccidian Toxoplasma gondii. Journal

of Protozoology

30, 657-l.

SCHWARTZMAN,J. D., KRUG, E. C., BINDER, L. J., AND PAYNE, M. R. 1985. Detection of the microtubule cytoskeleton of the coccidian Toxoplasma gondii and the hemoflaggelate Leishmania donovani by monoclonal antibodies specific for Beta tubulin. Journal of Protozooiogy 32, 747-749. SCHWARTZMAN,J. D. 1986. Inhibition of penetrationenhancing factor by TOXOplASmA gondii by monoclonal antibodies specific for rhoptries. Infection and Immunity

51, 760-764.

SHARMA, S. D., ARAUJO, F. G., AND REMINGTON, J. S. 1984. Toxoplasma antigen isolated by affinity chromatography with monoclonal antibody protects mice against lethal infection with Toxoplasma gondii. Journal of Immunology 133, 2818-2820. SIBLEY, L. D., WEIDNER, E., AND KRAHENBUHL, J. L. 1985. Phagosome acidification blocked by intracelhdar TOXOp/ASmA gondii. Nature (London) 315,416-419. SIBLEY, L. D., AND SHARMA, S. D. 1987. Ultrastructural locahzation of an intracehular Toxoplasma protein that induces protection in mice. Infection and Immunity 55, 2137-2141. SIMS, T. A., HAY, J., AND TALBOT, I. C. 1988. Host parasite relationship in the brains of mice with con-

MINIREVIEW:

Toxoplasma

genital toxoplasmosis. Journal of Pathology 156, 255-261. TANABE, K., AND MURAKAMI, K. 1984. Reduction in the mitochondrial membrane potential of Toxoplasma gondii after invasion of host cells. Journal of Cell Science 70, 73-81. TOMAVO,

S..

SCHWARZ,

R. T.,

AND

DUBREMETZ,

J. F. 1989. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens. Molecular and Cellular Biology 9, 45764580. TYRON, I. C. 1979. Toxoplasma gondii: Ultrastructure

gondii

121

and antigenicity of purified tachyzoite pellicle. Experimental Parasitology 48, 198-205. WANG, C. C. 1982. Biochemistry and physiology of coccidia. In “Biology of the Coccidia” (P. L. Long, Ed.), pp. 167-228. University Park Press, Baltimore. WERK, R. 1985. How does Toxoplasma gondii enter host cells? Review of Infectious Diseases 7, 449457. Received 7 August 1990; accepted 13 September 1990