Ann Ist Super Sanità 2004;40(1):57-63
Genetic analysis of the diversity in Toxoplasma gondii
Marie-Laure DARDÉ Service de Parasitologie-Mycologie, Faculté de Médecine, Université de Limoges, Limoges, France
Summary. - More than 50 different genetic markers are now available for Toxoplasma gondii isolate typing. For most of them, allelic polymorphism is low (2 to 4 alleles). A few markers, more polymorphic, can be used to fingerprint isolates for epidemiological studies. Phylogenetic analysis detects 2 main groups in Toxoplasma population. Group 2 is genetically heterogeneous. From a practical point of view, 3 main types (types I, II and III) are usually considered. Multilocus genotyping is necessary to reveal atypical or recombinant genotypes, more frequent among isolates collected in remote areas or in wild game. Toxoplasma isolates from human toxoplasmosis are mainly from type II (whatever the severity of clinical disease), but type I and atypical genotypes are found only in severe cases of human toxoplasmosis. Key words: Toxoplasma gondii, genetic typing, genetic markers, epidemiology, human toxoplasmosis. Riassunto (Analisi della variabilità genetica in Toxoplasma gondii). - Per tipizzare gli isolati di Toxoplasma gondii sono oggi disponibili più di 50 diversi markers genici. Per la maggior parte di essi il polimorfismo è basso (2 o 4 alleli). Per caratterizzare isolati a scopo di studi epidemiologici possono essere usati pochi marker più polimorfici. L’analisi filogenetica identifica 2 gruppi principali nella popolazione di Toxoplasma. Il gruppo 2 è geneticamente eterogeneo. Praticamente, sono definiti tre tipi principali (tipo I, II e III). Per rivelare genotipi atipici o ricombinanti più frequenti tra isolati ottenuti da zone relitte o da ospiti selvatici, è necessario fare tipizzazione genica su più loci. Gli isolati di Toxoplasma da toxoplasmosi umane sono soprattutto di tipo II (qualunque sia la gravità della patologia) ma i genotipi di tipo I e quelli atipici sono identificati solo nei casi gravi di toxoplasmosi umana. Parole chiave: Toxoplasma gondii, tipizzazione genetica, markers genetici epidemiologia, toxoplasmosi umana.
Introduction Toxoplasma gondii is an obligate intracellular parasite infecting all warm-blooded animals with a worldwide distribution. It causes a large range of clinical manifestations in humans. Besides, there are marked biological differences between stocks concerning their pathogenicity to mice: most of the stocks are avirulent for mice producing asymptomatic chronic infections, while few highly virulent stocks for mice produce acute toxoplasmosis killing all mice with less than 10 tachyzoites. With such a biological and epidemiological diversity, one could expect a large genetic diversity, reinforced by meiotic recombination in this protozoan with a well-described sexual cycle. In fact, the vast majority of isolates studied until now belong to only 2 or 3 clonal lineages designated types I, II and III, which occur in both animals and humans [1, 2]. This paper aims to present the different genetic tools used to analyze genetic diversity of T. gondii and the possible implications of this diversity concerning with epidemiology, biology and pathogenic role in human of this protozoan.
Genetic tools Technical aspects Genetic typing methods of T. gondii strains have been extensively perfected in the recent years. From a technical point of view, many tools have been successively used for analyzing genetic diversity of T. gondii, as well as that of other micro-organisms. The value of multilocus enzyme electrophoresis as a tool for genetic analysis has largely been demonstrated, but its main handicap is the need of a large quantity of purified Toxoplasma tachyzoites, difficult to obtain in the case of slow dividing strains [3, 4]. It could last between 1 or 2 months of repeated passages in cortisone-treated mice to obtain a sufficient amount of tachyzoites for electrophoresis analysis (about 7.106 tachyzoites for each enzymatic system). The genome restriction fragment length polymorphism (RFLP) analysis performed on total genomic DNA is a more direct approach of analysis but his application is limited by the same problem of production and purification of Toxoplasma parasites.
Indirizzo per la corrispondenza (Address for correspondence): Marie-Laure Dardé, Service de Parasitologie-Mycologie - EA3174 - Faculté de Médecine, Université de Limoges, 87025 Limoges, France. E-mail: darde@unilim.fr.
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Besides, it requires the use of 32P-labelled probes and RFLP patterns are difficult to interpret. Its main application is in basic Toxoplasma research for fingerprinting typing of isolates and laboratory stocks with repetitive BS or TGR probes [5-7]. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was carried out by Guo et al. [8]: four arbitrary primers were found to generate DNA fragments that discriminate mouse-virulent and mouse-non virulent genotype T. gondii strains. But with the increasing knowledge of Toxoplasma genome, PCR typing methods with sequence-specific primers are now most commonly used: sequencing, PCRRFLP, and analysis of the length polymorphism of microsatellite sequences. The choice between these three techniques depends on different criteria, among which cost, sequencer availability and technical support. DNA sequencing can be directly used as a typing method to detect nucleotide polymorphisms at various base pair level or deletions [9-15] or as a method to detect polymorphic endonucleases restriction sites in order to develop a PCR-RFLP method. According to the technique, a same marker could reveal more or less polymorphism: for instance, a high polymorphism was detected with GRA6 sequence analysis (9 allelic sequences among 30 strains), but the GRA6 PCR-RFLP method which was developed could simply differentiate three different groups among these same strains [13]. PCR-RFLP method on single-copy genes is the most commonly used method for typing T. gondii isolates [1, 16-22]. Microsatellites markers represent another class of genetic markers. They are short tandem repeats of two to six nucleotides. Markers generated from these repeats are known to be highly polymorphic because of length variation of these repeats, consequently they exhibit multiple alleles, which makes them very informative for genetic studies. Microsatellite polymorphism can be evaluated by PCR, which requires only a small amount of DNA, and allele sizing can be achieved with automatic sequencing by fluorescent primers and good reliability of results is assured. Microsatellites are considered the most informative technical tools for the study of DNA polymorphism, because of the considerable allelic polymorphism showed. The genetic origin of these genetic variability is due to a mechanism different from those of other genetic markers previously described. Hypervariability of microsatellites is explained by the accumulation of length mutations by intra-allelic polymerase slippage on microsatellite sequence during replication. The usefulness of microsatellite markers for Toxoplasma isolate typing was demonstrated by Costa et al. [23], Blackston et al. [24] and Ajzenberg et al. [2].
The sensitivity of all these PCR-based method ideally allows to directly analyze the parasite genotype from primary clinical sample [23, 25]. Actually, the low number of parasites present in some samples and the frequent presence of PCR inhibitors often led to negative results even with nested PCR [19, 26, 27]. Optimized PCR assays are needed for accurate genotyping on clinical samples. Phenotypic and genotypic markers More than 50 markers are now described for Toxoplasma isolate typing (Table 1). The finding of these markers was greatly facilitated by the development of the Toxoplasma genome project [28, 29]: more than 73 000 Toxoplasma genes and expressed sequence tags (EST) are now available in GenBank and can be analyzed through Toxoplasma Genome Web pages. The allelic diversity detected for most of these markers is low (2 to 4 alleles). This is the case for isoenzyme markers: the 6 polymorphic enzymatic systems (aspartate aminotransferase, glutathion reductase, amylase, glucose phosphate isomerase, acid phosphatase, propionyl esterase) exhibit only 2 to 3 isoforms in a population of 83 Toxoplasma isolates [2, 4]. This is also the case for the allelic diversity detected by PCR-RFLP or even sequencing of single copy genes: genes coding for major Toxoplasma antigens (surface antigens, dense granule or rhoptry antigens, cyst matrix antigen) [1, 11-14], genes coding for other Toxoplasma products (α-tubulin, β-tubulin, actin, dihydrofolate reductase-thymidilate synthase, nucleoside triphosphatase, DNA polymerase alpha) [15, 18, 20] or genes with unknown function (850, L328, 62B, 226, C19, B1) [1, 30]. In the study by Grigg et al. [14], all the 18 studied strains have one or the other of only 2 sequences for a large number of genes coding for surface and organite antigens, without intratypic variation. A slightly higher polymorphism (3 to 4 alleles) is detected for some microsatellites located in the intron of known genes (TUB2 coding for β-tubulin, TgM-A coding for myosin-A) or in an expressed sequence tag (EST W35487) [2]. Some multiple-copy loci, with unknown function, could detect a high polymorphism among Toxoplasma isolates. After a digestion of total genomic DNA, the BS probe, labeled with 32P, can provide a strainspecific fingerprint [6, 16, 31]. Similarly, TGR sequences [5], a family of repeated DNA sequences analyzed by PCR-RFLP revealed 10 different patterns in a population of 22 Toxoplasma isolates [7] and sequencing of the amplified products showed that each isolate had its own unique TGR sequence [32]. The REP elements are segments of repetitive DNA that transpose with relatively high frequency in the Toxoplasma genome and that are flanked by 2 distinct
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Table 1. - Genotypic and phenotypic markers used in genetic diversity studies of Toxoplasma gondii
Phenotypic or genetic markers with low polymorphism (2 to 4 alleles) Isoenzymes aspartate aminotransferase, amylase, propionyl esterase, glucose-phosphate isomerase, glutathion reductase, acid phospatase Genes coding for major antigens surface antigens: SAG1, SAG2, SAG3, SAG4, MAG1, BSR4, SRS1,SRS2, SRS3 organite antigens: GRA1, GRA2, GRA3, GRA4, GRA6, ROP1 nucleoside triphosphatase: NTP Other genes with known function actin (ACT1), α-tubulin (TUB1), β-tubulin (TUB2), B10, dihydrofolate réductase (FOL1) ADN Polymerase a (POL1) Genes with unknown function 850, L328, 62B, 226, C19, B1 Microsatellites β-tubulin (TUB2), myosin-A (TgM-A), EST W35487 References
[2, 4] [10, 14, 16, 17] [11, 13, 14, 16] [18, 40] [15] [20] [16, 30] [2, 23]
Genetic markers with high polymorphism Sequences repeated in the genome BS TGR REP (mobile genetic elements) Microsatellites ESTs (N60608, N82375, N83021, N61191, AA519150) Loci M6, M33, M48, M95, M102, M163 [16] [5, 7, 32] [27] [2] [24]
repeats, found in both direct and inverted formation [27]. This peculiar structure allows them to be amplified by a single primer PCR. Banding patterns observed after PCR of REP elements are identical for all mouse-virulent isolates but all mouse-avirulent isolates had different banding patterns. The fingerprinting of isolates allowed by this kind of markers can facilitate epidemiological studies on human and animal outbreaks. Another approach for the individual characterization of strains is a multilocus typing with several moderately polymorphic markers. For instance, the discriminatory power of an association of 8 microsatellite markers is 0.997 in a population of 83 Toxoplasma stocks, as calculated by Simpson’s index (maximum = 1.0 indicating that with the typing method used, all stocks have a different genotype) [2]. The high discriminatory power of the 8 microsatellite markers appears to be very useful in epidemiological studies of T. gondii, but also for strain genotype control in laboratory or for detection of mixed infections. For this last indication, microsatellite typing is superior to multicopy loci which cannot distinguish mixed infection in samples [33]. Since T. gondii is haploid, only one peak is expected for a given locus corresponding to one allele. More than one peak will be detected if mixed infections with different alleles are present in the sample [2].
Toxoplasma sample Another tool of genetic analysis, too often neglected, is the Toxoplasma sample under survey. Many studies were performed on a small sampling of stocks which forbids any valuable conclusions about the population structure of T. gondii. Population genetic analysis could only be performed on a large population of stocks. This population should be representative of Toxoplasma isolates circulating in nature. From a geographical point of view, a restrictive origin (for instance isolates originating from a limited area) can be interesting to explore the possibilities of recombination events, avoiding the bias due to geographical distance [34]. On the contrary, different geographic origins, with isolates originating from different parts of the world, are necessary to appreciate the real genetic diversity of T. gondii. The current conclusions about population structure of Toxoplasma were drawn from data obtained with isolates originating from Europe (mostly France) and North America [1-3]. Few isolates originating from remote areas of South America were analyzed and interestingly, they showed different multilocus genotypes suggesting that other lineages could circulate in other parts of the world [35, 36]. Another aspect of sampling that should be considered is the origin of the host from which Toxoplasma was isolated. Most of the studies were
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performed on isolates originating from humans or from domestic animals (sheep and pigs) and detected a vast majority of type II strains [1, 4, 22]. Here again, the few isolates originating from other animals, specially from wild game, revealed atypical multilocus genotypes or unusual alleles for some genes [1, 14, 15, 37]. The double influence of geographical and host origins could be suggested by the results of Brazilian chicken isolate typing: these isolates exhibited an unusual genotype (predominantly SAG2 type I genotype) and it could be questioned if this is due to a selection of this genotype by chicken immune system or to the geographical origin of the isolates [38]. Clonal population structure and multilocus genotype Few studies considered a large population of stocks with several independent genetic markers: 6 enzymatic systems on 86 stocks [3, 4, and unpublished data), PCR-RFLP of 6 independent single-copy loci on 106 stocks [1], and 8 microsatellite markers on 83 stocks [2]. Two other studies were performed on a more limited sample, but with the use of several loci: sequencing of 7 independent single-copy genes on 16 stocks [15] and sequencing of 15 unlinked loci on 18 stocks [14]. The analysis of these different data led to propose that T. gondii exhibits a basically clonal population structure, like many other parasitic protozoa species [1, 2, 16, 34]. Phylogenetic studies demonstrated that T. gondii is subdivided into two major clonal groups [2, 15] although Howe and Sibley [1] postulated the existence of 3 lineages, designated types I, II and III, instead of 2. In fact, group 2 is genetically heterogeneous and type III is only a subgroup of group 2 [2]. Grigg et al. [14] from the observation that many loci exhibit only 2 alleles, hypothesized that T. gondii population possessed 2 distinct ancestries and that the 3 observed lineages are only the result of successful recombination between these 2 ancestries. The main criteria for a clonal population structure in T. gondii are: a) the isolation of identical multilocus genotypes over large geographic areas and at interval of several years; b) the small number of different multilocus genotypes and the overrepresentation of the observed genotypes by comparison with panmixic expectations, providing evidence of a high linkage disequilibrium; and c) the correlation between independent set of markers. This high linkage disequilibrium was observed even if linkage disequilibrium tests were performed on geographical subdivisions of the Toxoplasma sample to avoid the bias due to geographical distance [2]. This clonal structure led to a typing strategy of Toxoplasma isolates with only 1 or 2 loci, mainly SAG2 and/or SAG1 locus, capable to differentiate the 3
main genotypes. This typing strategy was adopted in many studies [19, 21, 22, 26, 37-39]. Actually, the clonal population structure does not rule out the possibility of recombinants between the 3 main types even if these are less frequent than expected in a panmixic population [34]. Recombinant genotypes or genotypes with atypical alleles represent only 5 to 10% in most collections of isolates [1, 4], but they are more frequent among isolates sampled from exotic host species or geographically remote areas or from patients with unusual clinical presentation [4, 14, 25, 35, 36, 37]. A genetic typing based on 1 or even 2 loci led to the misidentification of recombinant strains (shuffled combinations of classical alleles) and of atypical strains characterized by unusual alleles at other loci. For instance, among 14 stocks exhibiting a type I with SAG2 PCR-RFLP, 4 belonged to type II, according to 6 isoenzymatic markers and 8 microsatellite loci [2]. Among 12 isolates from patients with ocular toxoplasmosis, RFLP analysis at one locus would have misidentified 5 recombinant strains and even analysis at 3 loci mistakenly identified a recombinant isolate as type I [25]. This demonstrates that multilocus analysis should be preferred for typing Toxoplasma. For typing other micro-organisms, a minimum of 10 loci, randomly selected, is generally required. Multilocus typing is necessary to appreciate the real genetic diversity of Toxoplasma population, to find genetic factors that could influence virulence, to understand an eventual mechanism of genotype selection according to host species or to try to find relationships between human disease and genotype.
Toxoplasma genotype and biological characteristics The relationships between Toxoplasma main genotype and some biological characteristics are now well established (summarized in Table 2). The main biological characteristic concerns behavior in mice: infection with a type I strain led to widespread parasite dissemination and rapid death of mice; in contrast mice survived to infection with a type II strain and tachyzoite dissemination is much less extensive. In vitro interconversion from tachyzoite to bradyzoite is easier in type II strains [40]. Strain genotype also influences host immune response: hyperinduction of pro-inflammatory cytokines (mainly IFN-gamma) was found during a type I infection in mice whereas a controlled production of these same cytokines had a protective effect after infection with a type II strain [41]. This effect of strain characteristics on host immune response can be itself determinant in parasite virulence. It should be underlined that strain virulence is not the same according to the host: type I strains, highly virulent in mice, are not pathogenic in rats [42].
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Table 2. - Summary of some biological and epidemiological characteristics of the main Toxoplasma genotypes
Type I Rarely isolated (10% of strain collections in Europe and USA, mainly from human origin) Highly virulent for mice: death of all mice inoculated with less than 10 tachyzoites In vitro: high rate of multiplication, reduced interconversion tachyzoite-bradyzoite Type II The most commonly isolated (human, sheep, pigs) (80% of strain collections in Europe and USA) Non-virulent for mice: chronic infection with persistence of tissue cysts In vitro: slow rate of multiplication, easier interconversion tachyzoite-bradyzoite and formation of cysts Type III, recombinant genotypes genotypes with atypical alleles and unusual
typing performed on all the strains consecutively isolated in French laboratories revealed that they nearly all belonged to type II [23, and Dardé et al. unpublished data). Type II isolates are found in the different aspects of congenital disease: lethal infection, severe neuroocular involvement, isolated chorioretinitis or latent toxoplasmosis. The main factor for the severity of congenital infection remains the stage of pregnancy at the time of contamination. So, type II strains, non virulent in mice, can be very pathogenic in human fetus, but they are also the only ones found in benign or latent congenital toxoplasmoses. The few type I or atypical strains isolated from congenital toxoplasmosis are usually observed in severe cases of congenital toxoplasmosis. Immunodeficient patients Typing of isolates originating from immunodeficient patients was reported by Howe et al. [19] and Honoré et al. [20]. In these 2 studies, typing was based on only 1 locus (SAG2 PCR-RFLP) and some atypical genotypes could have been misidentified. Type II strains are equally predominant (75%) in cerebral and in extra-cerebral toxoplasmoses, in AIDS patients and in other immunocompromised patients (lymphoma, organ transplant). In mice, type II strains produce high numbers of cysts and are more prone to reactivate in experimentally immunocompromised mice. If they have the same behavior in humans, it could be hypothesized that this would explain their predominance in toxoplasmic encephalitis. However, about 15% of isolates observed in these patients belong to type I, suggesting that type I strains could also give rise to cysts in human tissues. Immunocompetent patients T. gondii is rarely isolated in immunocompetent symptomatic patients. The few strains originating from toxoplasmic lymphadenopathies belong to type II [4]. In ocular toxoplasmosis, genetic typing, performed directly from toxoplasmic DNA extracted from vitreous fluid by Grigg et al. [25], revealed an unusual abundance of type I (3/12) or recombinant genotypes (5/12). The only type II isolates were observed in chororetinitis due to cyst reactivation in immunocompromised patients. These data suggested that severe or acquired ocular toxoplasmosis are more likely to be due to type I or recombinant genotypes. The role of atypical strains is also apparent in the rare cases of severe toxoplasmosis observed in immunocompetent patients with multiorgan failures [35, 36, 45]. These cases described after wild game consumption in French Guiana suggested that human immune system is less adapted to atypical Toxoplasma genotypes than to Toxoplasma genotypes usually circulating in domestic environment.
Rare among Toxoplasma isolates originating from Europe and USA More frequent among isolates originating from wild animals, from remote areas and from unusual human disease Usually more virulent for mice than type II isolates
Toxoplasma genotype and human disease Isolates from human toxoplasmosis originated predominantly from cases of congenital toxoplasmosis or from immunodeficient patients [2, 4, 19, 39], less frequently from symptomatic acquired toxoplasmosis in immunocompetent patient [25, 35, 36]. But to interpret the influence of Toxoplasma genotype in different clinical aspects of human toxoplasmosis, we will need strain typing for the vast majority of human infection which are asymptomatic infections. With the present typing methods, this is unfeasible and we have to await the development of non-invasive methods. The large number of polymorphic genes coding for major Toxoplasma antigens allow a kind of serotyping and a preliminary study found differences in immune responses according to the infecting strain [43, 44]. Type II isolates are largely predominant (about 80%) in human symptomatic toxoplasmosis. One could think that type II strains will also predominate in asymptomatic infection as this genotype is also the most frequently isolated in livestock destined for human consumption, at least in Europe and USA. Congenital toxoplasmosis Congenital toxoplasmosis is the main source of Toxoplasma isolation in humans (amniotic fluid, placenta, cord blood, tissues of aborted fetuses). Isolate
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Conclusions Physiopathological aspects of the behavior of the different Toxoplasma genotypes in humans are totally ignored. All our hypothesis (widespread parasite dissemination in organs of type I strains, cyst formation and reactivation with type II strains) are derived from mouse experimental toxoplasmosis and studies are needed to better understand the influence of strain genotype on human toxoplasmosis.
Submitted on invitation. Accepted on 10 February 2003.
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