Corinne Mercier

Stable DNA transformation of Toxoplasma gondii using phleomycin selection

Toxoplasma gondii (Tg) is an obligate intracellular protozoan parasite that is an important opportunistic pathogen in humans. To provide additional tools for molecular genetic analysis, we have developed a set of vectors for DNA transformation in Tg based on selection with the antibiotic phleomycin (Ph). These new vectors rely on the flanking sequences from the parasite genes GRA1, GRA2 or SAG1 to drive expression of the Tn5 ble gene encoding resistance to the DNA intercalating drug Ph (PhR). Treatment of extracellular parasites was used to select stable PhR transformants by plaque formation on host cell monolayers. Transfection of linear or circular forms of the pGRA1/ble, pGRA2/ble or pSAG1/ble vectors by electroporation resulted in stable transformation with an efficiency of approx. 10(-4)/micrograms DNA. Stable transformants contained 1-5 copies of ble that were integrated at non-homologous sites in the parasite nuclear genome. Ble provides a new dominant selectable marker for safe, efficient and rapid isolation of stable DNA transformants in Tg.

Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.

ABSTRACT Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii

Following secretion into the parasitophorous vacuole, dense granule proteins, referred to as GRA proteins, are targeted to different locations including a complex of tubular membranes that are connected with the vacuolar membrane. To further define the formation of this intravacuolar network, we have investigated the secretion, trafficking and membrane association of GRA4 and GRA6 within the parasitophorous vacuole. In extracellular parasites, GRA4 and GRA6 were found exclusively in dense secretory granules where they were packaged primarily as soluble proteins. Following release into the vacuole, GRA6 was rapidly translocated to the posterior end of the parasite where, like previously reported for GRA2, it bound to a cluster of multi-lamellar vesicles that give rise to the network. In contrast, GRA4 was distributed throughout the lumen of the vacuole and only later became associated with the mature network that is found dispersed throughout the vacuole. Cell fractionation and treatment with denaturing agents established that the association of GRA4 with the network membranes was mediated by strong protein-protein interactions. In contrast, GRA6 was predominantly influenced by hydrophobic interactions, and a phosphorylated form of this protein present within the vacuole showed increased association with the network membranes. Cross-linking studies established that GRA4 and GRA6 specifically interact with GRA2 to form a multimeric complex that is stably associated with the intravacuolar network. Formation of this protein complex, which is based on both protein-protein and hydrophobic interactions, may participate in nutrient or protein transport within the vacuole.

Enzyme-Linked Immunosorbent Assays Using the Recombinant Dense Granule Antigens GRA6 and GRA1 of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies

ABSTRACT The potential of the dense granule antigens GRA1 and GRA6 ofToxoplasma gondii to be used as diagnosis reagents in a recombinant form was evaluated. Both proteins were expressed inEscherichia coli as glutathione-S-transferase (GST) fusions. The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide. Separate expression of the two N- and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot. One hundred T. gondii-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein. Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96%. The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers. In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%. Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigen-based test for serodiagnosis of toxoplasmosis.

Role of Secretory Dense Granule Organelles in the Pathogenesis of Toxoplasmosis

Multiplication of Toxoplasma gondii only occurs intracellularly inside a specialized compartment called the parasitophorous vacuole (PV). Regulated secretory processes are key to the success of the intracellular parasitism of Toxoplasma as the parasite extensively modifies the newly formed vacuole using secreted proteins (Beckers et al. 1994; Charif et al. 1990; Sibley and Krahenbuhl 1988). The main structural modification of the PV consists of elaboration of a network of tubular membranes that are continuous with the vacuolar membrane (Sibley and Krahenbuhl 1988; Sibley et al. 1986, 1995).

Limited value of assays using detection of immunoglobulin G antibodies to the two recombinant dense granule antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women.

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e., Neospora, Plasmodium, Babesia, and Cryptosporidium).

Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8.

The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agents worldwide. Besides its sexual reproduction in cats, T. gondii can also infect a wide spectrum of other warm-blooded animals. These include animals used for human consumption such as pigs or chickens. Nevertheless, the role of turkeys for the epidemiology of T. gondii infections has not been studied thoroughly. We have established a kinetic ELISA (KELA) for the detection of T. gondii-specific IgG antibodies in turkey serum samples. The test is based on the recombinant dense granule antigens GRA7 and GRA8. These proteins were used as an antigen mixture at a concentration of 0.13 μg per well. The overall sensitivity of the assay was between 92.6% and 100% and the specificity ranged from 78.1% to 100%, depending on the method used to calculate these parameters. Using this KELA we examined 1913 turkey serum samples from 14 turkey farms from different areas of Germany. From these sera, 387 produced a signal in the KELA, corresponding to a true seroprevalence of up to 20.2%. The seropositivity rate in individual fattening cycles at individual farms ranged from 0.0% to 77.1%, whereas the rates were highly variable within the individual farms and individual fattening cycles. Consequently, conditions of animal husbandry could not be associated with particular seroprevalence rates. Although seropositivity cannot be linked directly to infectious tissue cysts in the muscle tissue of commercially produced turkey meat, we state that there is a potential risk of being infected by consuming turkey meat products that were not heat treated.

Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen

Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.