Selection of Polymorphic Peptides from GRA6 and GRA7 Sequences of Toxoplasma gondii Strains To Be Used in Serotyping

Abstract

The evaluation of Toxoplasma gondii isolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III). GRA6 and GRA7 are polymorphic genes that have been used for the genotyping of Toxoplasma. The coding regions of GRA6 and GRA7 from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for GRA6 and GRA7, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of Toxoplasma infections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the GRA7 locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the GRA6 and the GRA7 loci. Serum samples from humans infected with Toxoplasma strains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.The vast majority of Toxoplasma gondii isolates from human patients and domestic animals in Europe and North America belong to three archetypal clonal lineages, namely, types I, II, and III (1,11,30). However, nonarchetypal strains with atypical genotypes have recently been described in unusual hosts such as sea otters (10,42,43) and in tropical areas such as South America and Africa (2,32,39,46,54). Genotyping studies that distinguish different types of strains are important to gain knowledge of the biodiversity of the parasite in order to understand the molecular epidemiology of Toxoplasma and to highlight the correlation between the genotype of the parasite and the pathogenesis of human toxoplasmosis.The dense granules (GRA) are parasitic organelles involved in cell invasion and in the intracellular survival of the parasite. GRA proteins are expressed by the three stages of T. gondii: the tachyzoite, bradyzoite (38), and sporozoite (55) stages. GRA6 is a GRA antigen of 32 kDa described for the first time by Lecordier et al. (38). In extracellular parasites, GRA6 exists in dense secretor granules mostly as soluble proteins. Like the other GRA proteins, GRA6 is involved in host cell invasion. GRA6 is a glycine-rich protein and behaves like an integral membrane protein within the parasitophorous vacuole (36,41). GRA6 is considered a good marker of acute infection (27,28,52). However, the immune response to GRA6 is very heterogeneous (25).GRA7 is a GRA antigen of 29 kDa (26, 31). Like GRA6, it is involved in host cell invasion. This pro...

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“…Therefore, a larger sample size should be carried out in human patients to identify specific T. gondii strains using high resolution typing methods such as multiplex nested PCR-RFLP which can genotype some DNA samples extracted directly from infected tissues. Serotyping of T. gondii strains has proved to be a promising tool using serum samples to overcome the diagnostic challenge (Kong et al, 2003;Peyron et al, 2006;Morisset et al, 2008;Sousa et al, 2008;Sousa et al, 2009). However, due to the limitation of serotyping it is not possible to differentiate type II from non-type II strains particularly in South America where there is a high diversity of T. gondii strain types.…”

“…Genetic analyses have shown that the vast majority of all T. gondii-strains typed to date fall into one of three clonal lineages, type I, II, and III (Howe & Sibley, 1995), which differ in virulence but do not show clear host or geographic boundaries (Lindström et al, 2006). Studies from different parts of the world have produced similar findings in which the genotyping of the SAG2-locus revealed that the type II allele was the most disease-causing strain (reactivation of chronic infections) found in immunocompromised individuals (Dardé et al, 1992;Howe and Sibley, 1995;Howe et al, 1997;Fuentes et al, 2001;Lindström et al, 2006;Ajzenberg et al, 2009). This high prevalence of type II strains in human toxoplasmosis may simply reflect the source of the strains that led to human infections (Howe et al, 1997).…”

“…One study interestingly indicates that the type of infecting parasitic strain does not predominantly influence the pathogenesis of toxoplasmosis in immunocompromised patients and fully support the need for specific prophylaxis in patients infected by T. gondii, regardless of the strain genotype (Honoré et al, 2000). In addition, other host factors are more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis in immunocompromised hosts (Ajzenberg et al, 2009). A total of 50 studies have been reported from different parts of the world including Asia, Europe, North America, South America and Africa (Table 1).…”

High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

“…The Fmoc protecting group was removed by using a mild base treatment with 2% piperidine 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) in dimethyl sulfoxide-70% domethylformamide with in situ activation. The peptide sequence chosen for SAG-1 is based in KATO et al 19 ; for GRA-1 in BEGHETTO et al 3 ; and for GRA-7 in SOUSA et al 31 .…”

Peptide Microarray Analysis ofIn Silico-Predicted Epitopes for Serological Diagnosis of Toxoplasma gondii Infection in Humans

“…Several studies have shown that synthetic peptides can be used for the serological diagnosis of viral and bacterial as well as parasitic diseases (1,31,36,44,46,54). Large amounts of synthetic peptides can be produced with a high level of purity within a relative short period of time.…”