Molecular weight analysis of soluble antigens from Toxoplasma gondii

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoites were fractionated into a water-soluble and a deoxycholate-soluble fraction. Polyclonal immune mouse serum was prepared by challenging chronically-infected mice with viable RH strain tachyzoites. The parasite fractions were labelled with 125I, and the radio-labelled antigens were precipitated by the immune mouse serum or a monoclonal anti-Toxoplasma antibody (FMC 20), that reacts only in the indirect hemagglutination antibody test. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates showed that the water-soluble fraction contained 10 antigenic polypeptides, and the deoxycholate-soluble fraction contained seven antigenic peptides. The FMC 20 reacted against a 98,000-dalton antigen that was present in the water-soluble fraction only.     

High-resolution typing of Toxoplasma gondii using microsatellite loci.

High-resolution typing of Toxoplasma gondii is essential to understand the effect of genetic differences among strains on the variation in disease manifestation and transmission patterns. Current typing methods discern 3 lineages with minimal within-lineage variation. Described here are 6 new variable loci. These loci, including a minisatellite and 5 microsatellites, were more polymorphic than allozymes, restriction fragment length polymorphisms, and sequence variation in introns. Most importantly, these loci revealed, for the first time, substantial within-lineage variation that was over 6-fold higher than that detected by other markers. Genotyping at these loci facilitates classification of isolates beyond the lineage level.     

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.     

Silencing of GRA10 protein expression inhibits Toxoplasma gondii intracellular growth and development

Toxoplasma gondii dense granule proteins (GRAs) are secreted abundantly in both the tachyzoite and bradyzoite stages of the parasite and are known to localize to various compartments of the parasitophorous vacuole (PV) that interfaces with the host cell milieu. Thus, GRAs may play significant roles in the biogenesis of the PV that is important for survival of intracellular T. gondii. GRA10 is a dense granule protein whose role in T. gondii has not yet been characterized. Therefore, in this study, we endeavored to determine the role of GRA10 in the growth and survival of intracellular T. gondii by using phosphorodiamidate morpholino oligomers (PPMOs) antisense knockdown approach to disrupt the translation of GRA10 mRNA in the parasites. We expressed and purified a truncated recombinant GRA10 protein to generate anti-GRA10 polyclonal antibodies that we used to characterize GRA10 in T. gondii. We found that GRA10 is a soluble, dense granule-associated protein that is secreted into the parasite cytosol and the parasitophorous vacuole milieu. Using in vitro cultures, we found that knockdown of GRA10 results in severe inhibition of T. gondii growth in human fibroblasts and in ovine monocytic cells. Together, our findings define GRA10 as a dense granule protein that plays a significant role in the growth and propagation of intracellular T. gondii in human fibroblasts and in ovine monocytic cells.     

Molecular evolution and multilocus sequence typing of 145 strains of SARS-CoV

In this study, we have identified 876 polymorphism sites in 145 complete or partial genomes of SARS-CoV available in the NCBI GenBank. One hundred and seventy-four of these sites existed in two or more SARS-CoV genome sequences. According to the sequence polymorphism, all SARS-CoVs can be divided into three groups: (I) group 1, animal-origin viruses (such as SARS-CoV SZ1, SZ3, SZ13 and SZ16); (II) group 2, all viruses with clinical origin during first epidemic; and (III) group 3, SARS-CoV GD03T0013. According to 10 special loci, group 2 again can be divided into genotypes C and T, which can be further divided into sub-genotypes C1-C4 and T1-T4. Positive Darwinian selections were identified between any pair of these three groups. Genotype C gives neutral selection. Genotype T, however, shows negative selection. By comparing the death rates of SARS patients in the different regions, it was found that the death rate caused by the viruses of the genotype C was lower than that of the genotype T. SARS-CoVs might originate from an unknown ancestor.     

Molecular phylogenetic analysis of Candida tropicalis isolates by multi-locus sequence typing

Multi-locus sequencing data for 242 different isolates of Candida tropicalis generated a dendrogram showing 235 strains assigned to a single large recently evolved group which contained several small clonal clusters. Haplotype analysis of a representative strain subset revealed a high level of recombination events in an otherwise clonal population. Pairs of isolates from single sources showed non-identity attributable to loss of heterozygosity in some genes in a manner similar to that established for C. albicans.     

Selection and identification of dense granule antigen GRA3 by Toxoplasma gondii whole genome phage display

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 (< or = 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.     

Molecular characterization of tgd057, a novel gene from Toxoplasma gondii

The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Preservation of laboratory strains of Toxoplasma gondii in vitro

A procedure to obtain viable stabilates of virulent laboratory strains of Toxoplasma gondii with a prolonged storage life is described. Viable endozoites recovered from the sediment of mouse exudate or tissue cultures (LEP, HeLa) are suspended in Eagle's MEM medium supplemented with 10% calf serum and 10% dimethylsulfoxide and sealed into glass ampoules of 1-2 ml in volume. The ampoules are placed in an apparatus for gradual cell freezing, frozen to --35 degrees C at a rate of --1 degree C/min, and stored in liquid nitrogen. Reinoculation experiments on mice given the suspension intraperitoneally confirmed that such Toxoplasma gondii strains retain viability for at least 4 years. This in vitro preservation technique is compared with the analogous T. gondii preservation procedures described in the literature.     

Diversity of Toxoplasma gondii strains shaped by commensal communities of small mammals

Commensal rodent species are key reservoirs for Toxoplasma gondii in the domestic environment. In rodents, different T. gondii strains show variable patterns of virulence according to host species. Toxoplasma gondii strains causing non-lethal chronic infections in local hosts will be more likely to persist in a given environment, but few studies have addressed the possible role of these interactions in shaping the T. gondii population structure. In addition, the absence of validated techniques for upstream detection of T. gondii chronic infection in wild rodents hinders exploration of this issue under natural conditions. In this study, we took advantage of an extensive survey of commensal small mammals in three coastal localities of Senegal, with a species assemblage constituted of both native African species and invasive species. We tested 828 individuals for T. gondii chronic infection using the modified agglutination test for antibody detection in serum samples and a quantitative PCR assay for detection of T. gondii DNA in brain samples. The infecting T. gondii strains were genotyped whenever possible by the analysis of 15 microsatellite markers. We found (i) a very poor concordance between molecular detection and serology in the invasive house mouse, (ii) significantly different levels of prevalence by species and (iii) the autochthonous T. gondii Africa 1 lineage strains, which are lethal for laboratory mice, only in the native African species of commensal small mammals. Overall, this study highlights the need to reconsider the use of MAT serology in natural populations of house mice and provides the first known data about T. gondii genetic diversity in invasive and native species of small mammals from Africa. In light of these results, we discuss the role of invasive and native species, with their variable adaptations to different T. gondii strains, in shaping the spatial structure of T. gondii genetic diversity in Africa.     

Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3o-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5o-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.     

Expression variance,biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7

During intracellular stay, Toxoplasma gondii secretes dense granule proteins (GRA) which remodel the parasitophorous vacuole and are considered functional in parasite-host interrelation. Comparative analysis of parasites from mouse-virulent strain BK and an in vitro attenuated variant revealed that the level of GRA7 expression correlates with T. gondii virulence: proteome analysis and quantitation by immunoblot demonstrated a massive decrease in GRA7 steady-state synthesis parallel to the loss of virulence. Properties of GRA7 that are pertinent to its membrane targeting and to GRA7-directed immune resistance were studied in detail. GRA7 is exclusively membrane-associated in both parasites and infected host cells as demonstrated by subcellular fractionations. Triton X-114 partitioning of isolated parasites substantiated that GRA7 is an integral membrane protein, the hydrophobic stretch from amino acid 181 to 202 providing a possible membrane anchor. A fraction enriched for membranous material from infected host cells contained additional forms of GRA7 with reduced mobility in gel electrophoresis, indicating that the protein is modified after exocytosis from the parasite. By flow cytometric analysis, GRA7 was detected on the surface of intact host cells. An intracellular origin of surface-associated GRA7 seems likely since GRA7 released from extracellular parasites failed to label the host cell surface. Consistent with a role at a parasite-host interface, GRA7 proved to be a target antigen of the intracerebral immune response as evidenced by the presence of GRA7-specific antibodies in mouse cerebrospinal fluid during chronic infection.     

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.     

Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes

Molecular characterization of Toxoplasma gondii isolates is central for understanding differences in disease transmission and manifestations. Only 3 subgroups (lineages) have been discerned with subtle within-lineage variation, permitting low-resolution classification of isolates. Because proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of housekeeping genes, which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of 5 housekeeping genes with 2 antigen-coding genes. Introns of housekeeping genes were slightly more polymorphic than coding and noncoding regions of antigen-coding genes and only the former showed intralineage variation. Intragenic linkage disequilibrium was complete, but intergenic linkage, although highly significant, was incomplete, suggesting that genes are partially uncoupled. Six of 7 substitutions found within the region coding for the tachyzoite surface antigen, SAG2, were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates on the basis of housekeeping and antigen-coding genes was consistent, but the phylogenetic relationships among the resulting groups was inconsistent. A cougar isolate typed as lineage II using a restriction fragment length polymorphism assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of housekeeping genes are preferred markers for phylogenetic study, and that multilocus genotyping is preferred for typing parasites, especially from feral or unstudied environments.     

Molecular typing of Yersinia frederiksenii strains by means of 16s rDNA and gyrB genes sequence analyses

The phenospecies Yersinia frederiksenii is known to comprise three genospecies, indistinguishable on the basis of phenotypic characteristics. In previous works, 13 strains, identified biochemically as Y. frederiksenii, were characterized using Multilocus enzyme electrophoresis and Ribotyping. In order to elucidate the phylogenetic position of these strains we performed their molecular typing by means of 16S rDNA and gyrB sequences analyses. Results demonstrated that gyrB is a better phylogenetic marker than 16S rDNA. The classification achieved by gyrB sequences analyses was in agreement with results obtained with more laborious methods. Moreover, a good phylogenetic identification could be reached also with partial gyrB sequences of only 350 bp.     

Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii

This work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR–RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge.     

Molecular evolution of typical enteropathogenic Escherichia coli: clonal analysis by multilocus sequence typing and virulence gene allelic profiling

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.