EGS Strains Research Articles

Inflammatory response and parasite regulation in acute toxoplasmosis: the role of P2X7 receptor in controlling virulent atypical genotype strain of Toxoplasma gondii.

Toxoplasmosis is a globally significant disease that poses a severe threat to immunocompromised individuals, especially in Brazil, where a high prevalence of virulent and atypical strains of Toxoplasma gondii is observed. In 1998, the EGS strain, exhibiting a unique infection phenotype, was isolated in Brazil, adding to the complexity of strain diversity. The P2X7 receptor is critical in inflammation and controlling intracellular microorganisms such as T. gondii. However, its genetic variability can result in receptor dysfunction, potentially worsening susceptibility. This study investigates the role of the P2X7 receptor during acute infection induced by the EGS atypical strain, offering insight into the mechanisms of T. gondii infection in this context. We infected the female C57BL/6 (WT) or P2X7 knockout (P2X7-/-) by gavage. The EGS infection causes intestinal inflammation. The P2X7-/- mice presented higher parasite load in the intestine, spleen, and liver. The absence of the P2X7 receptor disrupts inflammatory cell balance by reducing NLRP3, IL-1β, and Foxp3 expression while increasing IFN-γ expression and production in the intestine. In the liver, P2X7-/- animals demonstrate diminished inflammatory infiltrate within the portal and lobular regions concurrent with an enlargement of the spleen. In conclusion, the infection of mice with the EGS strain elicited immune alterations, leading to acute inflammation and cytokine dysregulation, while the P2X7 receptor conferred protection against parasitic proliferation across multiple organs.

Animal Models for Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects mammals and birds throughout the world. This protocol describes murine models of acute T. gondii infection, toxoplasmic encephalitis and toxoplasma retinochoroiditis. T. gondii infection in severe combined immunodeficient (SCID) mice, deficient in T and B cells, has allowed for the study of T cell-independent mechanisms of defense against intracellular organisms, as described here. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii induce protection against challenge with virulent strains of the parasite. They have allowed studies of immunization and adoptive-transfer experiments. A protocol is provided for infection with these mutant strains. The EGS strain of T. gondii has the unique feature of spontaneously forming tissue cysts in cell culture. Dual fluorescent reporter stains of this strain have allowed the study of tachyzoite to bradyzoite transitions in vitro and in vivo. A protocol for in vitro and in vivo growth of this strain and tissue cyst isolation is provided. Genetic manipulation of T. gondii and mice has led to the development of parasites that express fluorescent proteins as well as mice with fluorescently labeled leukocytes. This together with the use of T. gondii that express model antigens and transgenic mice that express the appropriate T cell receptor have facilitated the in vivo study of parasite host-interaction. In addition, parasites that express bioluminescent markers have made it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite that includes these newer methodologies. In addition, support protocols address the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of acute T. gondii infection in mice Basic Protocol 2: Model of toxoplasmic encephalitis and toxoplasma retinochoroiditis in chronically infected mice Basic Protocol 3: Assessment of T. gondii invasion into neural tissue Basic Protocol 4: T. gondii infection in scid/scid (SCID) mice Basic Protocol 5: Infection with the uracil auxotroph strain CPS1-1 or the temperature-sensitive TS-4 strain of T. gondii Basic Protocol 6: In vivo and in vitro maintenance of the EGS strain of T. gondii Support Protocol 1: Assessment of progression of infection and immune response to T. gondii Support Protocol 2: Maintenance of a bank of T. gondii cysts of the ME49 strain Support Protocol 3: Maintenance of T. gondii tachyzoites using human foreskin fibroblasts Support Protocol 4: Maintenance of T. gondii tachyzoites in mice Support Protocol 5: Preparation of T. gondii lysate antigens Support Protocol 6: Isolation of T. gondii tissue cysts from brain.

Abstract 15779: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons as Models for Toxoplasma Gondii Infection

Introduction: Toxoplasma gondii infects all nucleated cells in warm-blooded vertebrates. Infection may be asymptomatic in healthy people but may result in the lifelong chronic persistence of cysts containing bradyzoites in specific tissues such as muscle and neurons. Hypothesis: Stem cell-derived neurons and cardiomyocytes will recapitulate the tissue-specific parasite-host interaction underlying chronic persistence in Toxoplasma infection. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (iCMs), cardiac fibroblasts (iCFs) and neurons (iNEs) were used as biologically relevant models to study the interactions of T. gondii with human hosts in vitro . iCM, iCF and iNE were infected with T. gondii TypeI/III EGS strain (dual fluorescent stage-specific reporter strain: mCherry driven by SAG1 promoter and sfGFP driven by the LDH2 promoter). Results: We observed higher rate of bradyzoite formation in iCM and iNE, compared to iCF from the same donor at 72 hours post-infection by fluorescence microscopy of live cells and flow cytometry. Furthermore, we have analyzed the changes in the gene expression of host cells during Toxoplasma infection using high-throughput RNA-seq. Compared to uninfected iCMs, iCMs infected for 24 hours showed 1090 upregulated and 753 downregulated differentially expressed genes (DEGs); iCMs infected for 72 hours showed 1956 upregulated and 1253 downregulated DEGs. Compared to uninfected iCFs, iCFs infected for 24 hours showed 1654 upregulated and 1971 downregulated DEGs; iCFs infected for 72 hours showed 2742 upregulated and 1916 downregulated DEGs. In comparison to uninfected iNEs, iNEs infected for 24 hours showed 190 upregulated and 321 downregulated DEGs; iNEs infected for 72 hours showed 244 upregulated and 91 downregulated DEGs. Enrichment analysis of gene sets regulated by infection will be presented. Conclusions: Our in vitro generated human iCMs, iCF and iNEs have provided us a valuable tool for better understanding the impact on the host cells during Toxoplasma infection. A detailed analysis of validation and intervention of the top dysregulated genes in all different cell types will inform the design of potential therapeutic targets.

In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii

Toxoplasma gondiiis an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline tonew derivatives improved the anti-T. gondiiactivity 3.4 fold. The most active derivative presented ED50in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.

Tissue dissemination and humoral response after experimental reinfection with atypical Toxoplasma gondii strains obtained from congenital human toxoplasmosis in Brazil

The paradigm that Toxoplasma gondii infection generates sterilizing protective immunity was broken by case studies in which reinfections were observed in immunocompetent pregnant women in the chronic phase of toxoplasmosis. Since then, several murine models have suggested that immunoprotection against a previous T. gondii infection may be violated after reinfection with strains of different genotypes. This study aimed to evaluate the dissemination of the parasite after reinfection with the virulent TgCTBr9 and EGS strains in BALB/c mice chronically infected with the avirulent TgCTBr5 strain. Three mice were euthanized at 2, 4, 8, 12, 24 and 48 h post challenge (p.c.) and at 7, 14 and 30 days p.c. Intestines, mesenteric lymph nodes, lungs and brains were collected for PCR-RFLP. Blood samples were collected to measure total IgG, IgG1 and IgG2a by ELISA. The reinfected animals survived and presented reduced morbidity after challenge with the virulent strains. Mice challenged with the TgCTBr9 strain showed a slight increase in anti-T. gondii IgG1. The spread of the TgCTBr5 strain was observed to occur earlier than the dissemination of the virulent TgCTBr9 or EGS strains. The TgCTBr9 strain was observed in the mesenteric lymph node at 7 days post challenge (d.p.c.); in the intestine and lungs at 14 d.p.c.; and in the brain at 30 d.p.c. EGS strain was demonstrated in the mesenteric lymph node and lung at 7 d.p.c and in the intestine and brain at a later time point. The immune response promoted by the primary infection with the avirulent strain (TgCTBr5) protected the animals from death after challenge with the virulent strains (TgCTBr9 or EGS).

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 μM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4days of infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.

New naphthoquinones and an alkaloid with in vitro activity against Toxoplasma gondii RH and EGS strains

The efficacy of three amino-terpenyl naphthoquinones and the alkaloid liriodenine were examined against tachyzoites and tissues cysts of the RH and EGS strains, respectively. Monolayers of 2C4 fibroblasts infected with tachyzoites of the RH strain were incubated with different concentrations of the compounds for 48 h. Specifically, 7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-6), 6-(4-methylpentyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-11), and 8 h-benzo[g]-1,3-benzodioxolo[6,5,4-de]quinolin-8-one,9Cl-1,2-methylene dioxiaporfina (liriodenine) inhibited intracellular replication of T. gondii. The IC(50) values obtained for compounds QUI-5 and QUI-6 were 69.35 and 172.81 μM (i.e., 21.4 and 53.4 μg/mL), respectively. The naphthoquinone QUI-11 and liriodenine significantly inhibited intracellular replication of T. gondii. The IC(50) values obtained with these experiments were 0.32 and 0.07 μM (i.e., 0.1 and 0.02 μg/mL), respectively. Compounds QUI-5, QUI-6, QUI-11 and liriodenine demonstrated lower toxicity for 2C4 fibroblasts compared to atovaquone. In addition, cysts isolated from the brains of mice chronically infected with the EGS strain were exposed to the compounds. Infectivity of the cysts after incubation with the compounds was assessed by infection of mice. The data obtained showed that in vitro incubation with QUI-6, QUI-11 and liriodenine inhibited the infectivity of the bradyzoites. This activity was time- and concentration-dependent.

Immunosuppression with cyclophosphamide favors reinfection with recombinantToxoplasma gondiistrains

The aim of this study was to verify the effect of immunosuppression by cyclophosphamide (Cy) on susceptibility of BALB/c mice subjected to challenge with recombinant strains of Toxoplasma gondii. Animals were prime infected with the D8 (recombinant I/III) or the ME49 (type II) non-virulent strains, weekly immunosuppressed with Cy and challenged with the CH3 or EGS virulent strains (I/III). Parasites recovered from surviving mice were submitted to PCR-RFLP analysis to confirm co-infection. Prime-infection with the D8 strain conferred more protection against challenge with the CH3 and EGS strains when compared with ME49 prime infection. Cy treatment caused significant leukopenia in the infected mice, what probably favors reinfection after challenge. Reinfection was associated with increased levels of IgA. Otherwise, Cy-treated mice presented significantly lower IgA levels after challenge, suggesting involvement of this immunoglobulin on protection against reinfection. In conclusion, BALB/c mice susceptibility to reinfection by T. gondii is related to genetic differences among the strains used for primary and challenge infections. Alteration of the host’s immune integrity by Cy probably compromises the protection previously established by primary infection.

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.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-³ and IL-10

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-gamma and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-gamma were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Toxoplasma gondii: In vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1′-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine

The compound 2-hydroxy-3-(1′-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.

Activity of natural and synthetic naphthoquinones against Toxoplasma gondii, in vitro and in murine models of infection.

The effect of 14 natural and synthetic naphthoquinones in the replication of Toxoplasma gondii was evaluated. In vitro studies were accomplished in cultures of 2C4 fibroblasts infected with RH-strain. Enzyme-linked immunosorbent assay was used to quantify parasite growth. For the studies in vivo, mice were infected with tachyzoites of the RH strain or cysts of the T. gondii EGS strain. In vitro, seven naphthoquinones demonstrated significant inhibition of intracellular T. gondii growth at concentrations of 1 and 5 micrograms/ml. Only three compounds were significantly protective when tested in animals: 2-hydroxy-3'-(3'-pentenyl)-1,4-naphthoquinone (PHNQ4), 2-hydroxy-3-(1'-vinylphenyl)-1,4-naphthoquinone (PHNQ5), and 2-hydroxy-3-(1'-propen-3'-phenyl)-1,4-naphthoquinone (PHNQ6). In animals infected with the EGS strain and treated with PHNQ4 (50 mg/kg/day orally), a 7-day prolongation of the time to death was observed. Treatment with 100 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ5 resulted in a 5-day and 16-day prolongation of the time to death, respectively. Treatment with 50 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ6 resulted in a 4-day prolongation of the time to death or up to 30 days after treatment, respectively. Our results suggest that the naphthoquinones may be important therapeutic agents for the treatment of toxoplasmosis.