Selection and Identification of Dense Granule Antigen GRA3 by Toxoplasma gondii Whole Genome Phage Display

Abstract

Toxoplasma gondii is a ubiquitous, unicellular, eukaryotic parasite with a complex intracellular life cycle capable of invading and chronically infecting a wide variety of vertebrate host species, including man. Although normally opportunistic in healthy adults, it is a lethal pathogen in immunocompromised humans, particularly in AIDS patients. We present the application of a genomic phage display as a tool for the direct identification of antigens with potential value in diagnosis and/or as subunit vaccine components. Using a polycosmid cloning strategy, we constructed a large phagemid display library (>10 9 independent clones) of mixed short genomic restriction fragments (< 500 bp) of T. gondii genomic DNA (80 Mbp genome size) fused to gene III of the filamentous phage M13. Biopanning of the library with monoclonal Toxoplasma antibodies resulted in the isolation and identification of an epitope of GRA3, an antigen located in the dense granules of T. gondii tachyzoites. The reactivity of the phage displaying the GRA3 epitope with the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. These results demonstrate the accessibility of midsized eukaryotic genomes to display technology and the feasibility to screen these whole genome display libraries with antibodies for isolating novel antigenic determinants.

Serotyping ofToxoplasma gondiiinfections in Humans Using Synthetic Peptides

“…Nucleotide sequences from archetypal type I (RH), II (Me49 and Prugniaud), and III (VEG and CEP) strains were obtained from GenBank, from Toxoplasma expressed sequence tags, or by direct sequencing for the following 14 Toxoplasma immunogens: dense granule proteins GRA1 [20], GRA3 [21], GRA4 [22], GRA6 [23], and GRA7 [24,25]; NTPase I and III [26,27]; surface antigens SAG1, SAG2, SAG3, SAG4, BSR4, and SRS2 [13,16,28]; and the rhoptry protein ROP1. Polymorphic regions were identified after alignment of all synthesized alleles and allele-specific peptides.…”

Cellular Location of Starch Synthesis and Evolutionary Origin of Starch Genes

“…The starch‐storing apicomplexan Toxoplasma gondii , whose apicoplast is apparently derived from a red alga by endosymbiosis, also has a gene coding for a UGPase but not for an AGPase, and the extracted enzyme activity shows faster reaction with UDP‐glucose than with ADP‐glucose (Coppin et al 2005). The nuclear genome of T. gondii is not obviously reduced (Robben et al 2002), unlike the two cyanidiophyte red algae.…”

“…However, these two sequenced cyanidiophytes have small genomes arrived at by reduction, and no sequence data are available for the florideophyte macroalga Gracilara gracilis from which Sesma and Iglesias (1998) characterized an AGPase. The starch-storing apicomplexan Toxoplasma gondii, whose apicoplast is apparently derived from a red alga by endosymbiosis, also has a gene coding for a UGPase but not for an AGPase, and the extracted enzyme activity shows faster reaction with UDP-glucose than with ADP-glucose (Coppin et al 2005).The nuclear genome of T. gondii is not obviously reduced (Robben et al 2002), unlike the two cyanidiophyte red algae.…”

Genome-wide characterisation of the binding repertoire of small molecule drugs

“…Even if only a single phage particle is selected for affinity to a small molecule, it is possible to identify the gene product responsible because the virus can be grown in quantities sufficient for sequencing the viral DNA which contains an insert coding for the gene product displayed on the surface. A similar approach using genomic libraries is also possible, whereby whole genomes are chemically fragmented and cloned into the phage genome [75-79]. A principal shortcoming of this method is that many gene products do not fold properly on a phage surface, particularly large proteins and membrane proteins.…”