Identification of a sporozoite-specific antigen from Toxoplasma gondii

Abstract

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.

Figure 1

Protein extracted from A, bradyzoite (bz), and B and E, sporozoite (sz), and D, tachyzoite (tz) stages of VEG strain T. gondii spots resolved on 2D-DIGE gels, pH range 4–9 in the 1st dimension, and 4–12% SDS gradient gel in the 2nd dimension. Protein samples from C, bz and sz or F, tz and sz T. gondii stage labeled with Cy2 (green; tz, bz), or Cy5 (red, sz). Equal concentrations of proteins from each stage were mixed loaded onto a 2D-DIGE gel for analysis. Stage-specific proteins from the bz or tz stage resulted in a green spot, proteins specific to the sz stage resulted in a red spot, and shared proteins resulted in a yellow spot. Sporozoite-specific protein selected from gel for assay development indicated by arrow. Molecular weight bars on left, A and D, top, 150kD, center, 30kDa, bottom, 14kDa.

Figure 2

Western blot of T. gondii extracted sporozoite proteins resolved on 2D gel, pH 4–9 in the 1st dimension, and 4–12% SDS gradient gel in the 2nd dimension. Proteins were transferred to PVDF nylon blotting membrane and incubated in a pool of porcine sera (diluted 1:500) from 10 pigs with acute oocyst-induced T. gondii infection, followed by horseradish peroxidase conjugated-goat anti-pig IgG. Immunoreactive, sporozoite-specific protein identified in DIGE gel was spot matched and selected from identical 2D gel for assay development; indicated by arrow.

Figure 3

A, MS/MS results from analysis of 11 kDa protein spot excised from DIGE 2-D gel from which the partial amino acid sequence for the sporozoite protein was derived. B, Amino acid sequence of TgERP as translated from DNA sequence of selected immunoreactive clone. C, Coding sequence of TgERP. D, Individual results of 1-dimensional Western Blots using sera diluted 1:100 from 10 pigs (lanes 1–10) with oocyst- or 10 pigs (lanes 11–20) with tissue cyst-induced T. gondii infection, using11 kDa sporozoite-specific protein as antigen. Antibody persisted ~6–8 months in pigs. E, Western blot of T. gondii total sporozoite protein extract, Lane 1, human sera from Group 2, T. gondii outbreak investigated by the CDC (EIA IgM positive = 4.2), Lane 2, serum from pig experimentally infected with T. gondii oocysts; Lane 3 and 4, T. gondii tachyzoite total protein extract probed with: Lane 3, human sera from Group 2, T. gondii outbreak investigated by the CDC (EIA IgM negative); Lane 4, serum from pig experimentally infected with T. gondii tissue cysts. F, Western blot of VEG strain TgERP with individual sera collected 60 days PI from mice infected with A, oocysts, or B, bradyzoites, of the ME-49 strain of T. gondii (Fig. 3F).

Figure 4

Western blots were carried out using human sera and the Factor Xa cleaved-fusion protein purified over a DEAE sepharose column (purified TgERP insert). A, Lane 1, Factor Xa-cleaved, DEAE column purified pMal-c2 vector without the TgERP insert. Lanes 2–7, purified insert, probed with individual sera from Group 1, 6 people from laboratory outbreak resulting from oocyst ingestion. Sera collected 6 weeks post-infection. B, Left, purified insert, probed with pooled sera from 6 Group 1 individuals, 12 weeks post infection; Right; same, probed with pooled sera from 4 chronically infected persons from Group 2. C, purified insert, probed with pooled sera from 6 Group 1 individuals, 5 months post infection D, purified insert, Lanes 1–9, probed with sera of 9 people from stable outbreak (Group 2), Lanes 10–12, probed with sera of 3 IgM positive women from Group 3.