scholarly journals Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

2015 ◽  
Vol 83 (5) ◽  
pp. 2148-2155 ◽  
Author(s):  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Acute Infection ◽  
Cyst Formation ◽  
Type I ◽  
Type Ii ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Monophosphate Decarboxylase ◽  
Uracil Auxotroph

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection byToxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion ofOMPDCinduced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80Δompdcmutants stimulated a fully protective CD8+T cell-dependent immunity that prevented acute infection by type I and type II strains ofT. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdcmutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.

scholarly journals Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Yong Fu ◽  
Xia Cui ◽  
Sai Fan ◽  
Jing Liu ◽  
Xiao Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Metabolism ◽  
Toxoplasma Gondii ◽  
Binding Protein ◽  
Ankyrin Repeat ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
High K ◽  
Acyl Coenzyme A

ABSTRACT Acyl coenzyme A (CoA)-binding protein (ACBP) can bind acyl-CoAs with high specificity and affinity, thus playing multiple roles in cellular functions. Mitochondria of the apicomplexan parasite Toxoplasma gondii have emerged as key organelles for lipid metabolism and signaling transduction. However, the rationale for how this parasite utilizes acyl-CoA-binding protein to regulate mitochondrial lipid metabolism remains unclear. Here, we show that an ankyrin repeat-containing protein, TgACBP2, is localized to mitochondria and displays active acyl-CoA-binding activities. Dephosphorylation of TgACBP2 is associated with relocation from the plasma membrane to the mitochondria under conditions of regulation of environmental [K+]. Under high [K+] conditions, loss of ACBP2 induced mitochondrial dysfunction and apoptosis-like cell death. Disruption of ACBP2 caused growth and virulence defects in the type II strain but not in type I parasites. Interestingly, mitochondrial association factor-1 (MAF1)-mediated host mitochondrial association (HMA) restored the growth ability of ACBP2-deficient type II parasites. Lipidomics analysis indicated that ACBP2 plays key roles in the cardiolipin metabolism of type II parasites and that MAF1 expression complemented the lipid metabolism defects of ACBP2-deficient type II parasites. In addition, disruption of ACBP2 caused attenuated virulence of Prugniuad (Pru) parasites for mice. Taking the results collectively, these data indicate that ACBP2 is critical for the growth and virulence of type II parasites and for the growth of type I parasites under high [K+] conditions. IMPORTANCE Toxoplasma gondii is one of the most successful human parasites, infecting nearly one-third of the total world population. T. gondii tachyzoites residing within parasitophorous vacuoles (PVs) can acquire fatty acids both via salvage from host cells and via de novo synthesis pathways for membrane biogenesis. However, although fatty acid fluxes are known to exist in this parasite, how fatty acids flow through Toxoplasma lipid metabolic organelles, especially mitochondria, remains unknown. In this study, we demonstrated that Toxoplasma expresses an active ankyrin repeat containing protein TgACBP2 to coordinate cardiolipin metabolism. Specifically, HMA acquisition resulting from heterologous functional expression of MAF1 rescued growth and lipid metabolism defects in ACBP2-deficient type II parasites, manifesting the complementary role of host mitochondria in parasite cardiolipin metabolism. This work highlights the importance of TgACBP2 in parasite cardiolipin metabolism and provides evidence for metabolic association of host mitochondria with T. gondii.

scholarly journals The Toxoplasma gondii Rhoptry Kinome Is Essential for Chronic Infection

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Barbara A. Fox ◽  
Leah M. Rommereim ◽  
Rebekah B. Guevara ◽  
Alejandra Falla ◽  
Miryam Andrea Hortua Triana ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Toxoplasma Gondii ◽  
Virulence Factors ◽  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Host Cells ◽  
Type Ii ◽  
Host Manipulation ◽  
Parasite Survival

ABSTRACT    Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δ rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5 , ROP17 , ROP18 , ROP35 , or ROP38 / 29 / 19 ( ROP38 , ROP29 , and ROP19 ) severely reduced cyst burdens. Δ rop5 and Δ rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δ rop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δ rop35 or Δ rop38 / 29 / 19 strain. Resistance to host IRGs was not affected in the Δ rop17 , Δ rop35 , or Δ rop38 / 29 / 19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii . IMPORTANCE Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.

scholarly journals Impact of Engineered Expression of Mitochondrial Association Factor 1b on Toxoplasma gondii Infection and the Host Response in a Mouse Model

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Elizabeth D. English ◽  
Jon P. Boyle
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Acute Infection ◽  
Chronic Phase ◽  
Parasite Burden ◽  
Cytokine Signaling ◽  
Parasite Protein ◽  
Content Type ◽  
Life Threatening ◽  
The Impact

ABSTRACT The opportunistic intracellular parasite Toxoplasma gondii causes a lifelong chronic infection capable of reactivating in immunocompromised individuals, which can lead to life-threatening complications. Following invasion of the host cell, host mitochondria associate with the parasitophorous vacuole membrane. This phenotype is T. gondii strain specific and is mediated by expression of a host mitochondrial association-competent (HMA+) paralog of the parasite protein mitochondrial association factor 1 (MAF1b). Previous work demonstrated that expression of MAF1b in strains that do not normally associate with host mitochondria increases their fitness during acute infection in vivo. However, the impact of MAF1b expression during chronic T. gondii infection is unclear. In this study, we assess the impact of MAF1b expression on cyst formation and cytokine production in mice. Despite generally low numbers of cysts generated by the in vitro culture-adapted strains used in this study, we find that parasites expressing MAF1b have higher numbers of cysts in the brains of chronically infected mice and that MAF1b+ cyst burden significantly increases during the course of chronic infection. Consistent with this, mice infected with MAF1b+ parasites have higher levels of the serum cytokines RANTES and VEGF (vascular endothelial growth factor) at day 57 postinfection, although this could be due to higher parasite burden at this time point rather than direct manipulation of these cytokines by MAF1b. Overall these data indicate that MAF1b expression may also be important in determining infection outcome during the chronic phase, either by directly altering the cytokine/signaling environment or by increasing proliferation during the acute and/or chronic phase. IMPORTANCE The parasite Toxoplasma gondii currently infects approximately one-third of the world’s population and causes life-threatening toxoplasmosis in individuals with undeveloped or weakened immune systems. Current treatments are unable to cure T. gondii infection, leaving infected individuals with slow-growing tissue cysts for the remainder of their lives. Previous work has shown that expression of the parasite protein mitochondrial association factor 1 (MAF1b) is responsible for the association of T. gondii parasites with host mitochondria and provides a selective advantage during acute infection. Here we examine the impact of MAF1b expression during chronic T. gondii infection. We find that mice infected with MAF1b-expressing parasites have higher cyst burden and cytokine levels than their wild-type counterparts. A better understanding of the genes involved in establishing and maintaining chronic infection will aid in discovering effective therapeutics for chronically infected individuals.

scholarly journals Role of Cytokines and Major Histocompatibility Complex Restriction in Mouse Resistance to Infection with a Natural Recombinant Strain (Type I-III) of Toxoplasma gondii

2003 ◽  
Vol 71 (11) ◽  
pp. 6392-6401 ◽  
Author(s):  
Blima Fux ◽  
Cibele V. Rodrigues ◽  
Ricardo W. Portela ◽  
Neide M. Silva ◽  
Chunlei Su ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Toxoplasma Gondii ◽  
Genetic Markers ◽  
Recombinant Strain ◽  
Cyst Formation ◽  
Toxoplasmic Encephalitis ◽  
Type I ◽  
Type Ii ◽  
Histocompatibility Complex ◽  
Mhc Haplotype

ABSTRACT Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain of Toxoplasma gondii (P-Br). From nine genetic markers analyzed, three (B1, ROP1, and SAG1) and three (cS10-A6, GRA6, and SAG3) markers belong to parasites from the type I and type III lineages, respectively. The SAG2 and L363 loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain of T. gondii (ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype “d” were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains of T. gondii.

scholarly journals Toxoplasma gondiiParasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Barbara A. Fox ◽  
Rebekah B. Guevara ◽  
Leah M. Rommereim ◽  
Alejandra Falla ◽  
Valeria Bellini ◽  
...  
Keyword(s):  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Gamma Interferon ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Vacuole Membrane ◽  
Ifn Γ

ABSTRACTToxoplasma gondiievades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronicToxoplasmainfection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cystsin vivowithout affecting the parasite growth rate or the ability to differentiate into cystsin vitro. Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4mutants was reduced but not abolished. In contrast, the PruΔgra12mutant was avirulent in mice and PruΔgra12parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12and type II PruΔgra12parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80and type II PruΔku80strains, respectively. Despite this resistance, Δgra12PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondiicysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cystsin vivo. A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.

scholarly journals Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections

2015 ◽  
Vol 79 (4) ◽  
pp. 387-401 ◽  
Author(s):  
Kelly J. Pittman ◽  
Laura J. Knoll
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Developmental Stages ◽  
Acute Infection ◽  
The Body ◽  
Sexual Cycle ◽  
Parasitic Infections ◽  
Chronic Infections ◽  
Content Type ◽  
Cyst Form

SUMMARYToxoplasma gondiirepresents one of the most common parasitic infections in the world. The asexual cycle can occur within any warm-blooded animal, but the sexual cycle is restricted to the feline intestinal epithelium.T. gondiiis acquired through consumption of tissue cysts in undercooked meat as well as food and water contaminated with oocysts. Once ingested, it differentiates into a rapidly replicating asexual form and disseminates throughout the body during acute infection. After stimulation of the host immune response,T. gondiidifferentiates into a slow-growing, asexual cyst form that is the hallmark of chronic infection. One-third of the human population is chronically infected withT. gondiicysts, which can reactivate and are especially dangerous to individuals with reduced immune surveillance. Serious complications can also occur in healthy individuals if infected with certainT. gondiistrains or if infection is acquired congenitally. No drugs are available to clear the cyst form during the chronic stages of infection. This therapeutic gap is due in part to an incomplete understanding of both host and pathogen responses during the progression ofT. gondiiinfection. While many individual aspects ofT. gondiiinfection are well understood, viewing the interconnections between host and parasite during acute and chronic infection may lead to better approaches for future treatment. The aim of this review is to provide an overview of what is known and unknown about the complex relationship between the host and parasite during the progression ofT. gondiiinfection, with the ultimate goal of bridging these events.

scholarly journals Human Innate Immunity to Toxoplasma gondii Is Mediated by Host Caspase-1 and ASC and Parasite GRA15

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Lanny Gov ◽  
Alborz Karimzadeh ◽  
Norikiyo Ueno ◽  
Melissa B. Lodoen
Keyword(s):  
Innate Immunity ◽  
Toxoplasma Gondii ◽  
Adaptor Protein ◽  
The Body ◽  
Interleukin 1Β ◽  
Type I ◽  
Type Ii ◽  
Human Monocytes ◽  
Content Type ◽  
Caspase 1

ABSTRACT   Interleukin-1β (IL-1β) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1β; however, the host and parasite factors that mediate IL-1β production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1β transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1β release, suggesting a role for the inflammasome in T. gondii-induced IL-1β production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1β induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1β. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1β mRNA and protein release than did the type I and III strains. Since IL-1β transcript is known to be induced downstream of NF-κB signaling, we investigated a role for the GRA15 protein, which induces sustained NF-κB signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1β induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1β production. IMPORTANCE Monocytes are immune cells that protect against infection by increasing inflammation and antimicrobial activities in the body. Upon infection with the parasitic pathogen Toxoplasma gondii, human monocytes release interleukin-1β (IL-1β), a “master regulator” of inflammation, which amplifies immune responses. Although inflammatory responses are critical for host defense against infection, excessive inflammation can result in tissue damage and pathology. This delicate balance underscores the importance of understanding the mechanisms that regulate IL-1β during infection. We have investigated the molecular pathway by which T. gondii induces the synthesis and release of IL-1β in human monocytes. We found that specific proteins in the parasite and the host cell coordinate to induce IL-1β production. This research is significant because it contributes to a greater understanding of human innate immunity to infection and IL-1β regulation, thereby enhancing our potential to modulate inflammation in the body.

scholarly journals Type II Toxoplasma gondii Induction of CD40 on Infected Macrophages Enhances Interleukin-12 Responses

2014 ◽  
Vol 82 (10) ◽  
pp. 4047-4055 ◽  
Author(s):  
Pedro Morgado ◽  
Dattanand M. Sudarshana ◽  
Lanny Gov ◽  
Katherine S. Harker ◽  
Tonika Lam ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Transcript Level ◽  
Forward Genetics ◽  
Interleukin 12 ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Content Type ◽  
Type Iii ◽  
Infected Cells

ABSTRACTToxoplasma gondiiis an obligate intracellular parasite that can cause severe neurological disease in infected humans. CD40 is a receptor on macrophages that plays a critical role in controllingT. gondiiinfection. We examined the regulation of CD40 on the surface ofT. gondii-infected bone marrow-derived macrophages (BMdMs).T. gondiiinduced CD40 expression both at the transcript level and on the cell surface, and interestingly, the effect was parasite strain specific: CD40 levels were dramatically increased in type IIT. gondii-infected BMdMs compared to type I- or type III-infected cells. Type II induction of CD40 was specific to cells harboring intracellular parasites and detectable as early as 6 h postinfection (hpi) at the transcript level. CD40 protein expression peaked at 18 hpi. Using forward genetics with progeny from a type II × type III cross, we found that CD40 induction mapped to a region of chromosome X that included the gene encoding the dense granule protein 15 (GRA15). Using type I parasites stably expressing the type II allele ofGRA15(GRA15II), we found that type I GRA15IIparasites induced the expression of CD40 on infected cells in an NF-κB-dependent manner. In addition, stable expression of hemagglutinin-tagged GRA15IIin THP-1 cells resulted in CD40 upregulation in the absence of infection. Since CD40 signaling contributes to interleukin-12 (IL-12) production, we examined IL-12 from infected macrophages and found that CD40L engagement of CD40 amplified the IL-12 response in type II-infected cells. These data indicate that GRA15IIinduction of CD40 promotes parasite immunity through the production of IL-12.

scholarly journals A Family of Toxoplasma gondii Genes Related to GRA12 Regulate Cyst Burdens and Cyst Reactivation

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Chronic Infection ◽  
Acute Infection ◽  
Dense Granule ◽  
Toxoplasmic Encephalitis ◽  
Content Type ◽  
The Central Nervous System ◽  
Related Proteins ◽  
Cyst Development

ABSTRACT Toxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. The dense granule protein GRA12 is a major parasite virulence factor required for parasite survival during acute infection. Here, we characterized the role of four GRA12-related genes in acute and chronic stages of infection. While GRA12A, GRA12B, and GRA12D were highly expressed in asexual stage tachyzoites and bradyzoites, expression of GRA12C appeared to be restricted to the sexual stages. In contrast to deletion of GRA12 (Δgra12), no major defects in acute virulence were observed in Δgra12A, Δgra12B, or Δgra12D parasites, though Δgra12B parasites exhibited an increased tachyzoite replication rate. Bradyzoites secreted GRA12A, GRA12B, and GRA12D and incorporated these molecules into the developing cyst wall, as well as the cyst matrix in distinct patterns. Similar to GRA12, GRA12A, GRA12B, and GRA12D colocalized with the dense granules in extracellular tachyzoites, with GRA2 and the intravacuolar network in the tachyzoite stage parasitophorous vacuole and with GRA2 in the cyst matrix and cyst wall. Chronic stage cyst burdens were decreased in mice infected with Δgra12A parasites and were increased in mice infected with Δgra12B parasites. However, Δgra12B cysts were not efficiently maintained in vivo. Δgra12A, Δgra12B, and Δgra12D in vitro cysts displayed a reduced reactivation efficiency, and reactivation of Δgra12A cysts was delayed. Collectively, our results suggest that a family of genes related to GRA12 play significant roles in the formation, maintenance, and reactivation of chronic stage cysts. IMPORTANCE If host immunity weakens, Toxoplasma gondii cysts recrudesce in the central nervous system and cause a severe toxoplasmic encephalitis. Current therapies target acute stage infection but do not eliminate chronic cysts. Parasite molecules that mediate the development and persistence of chronic infection are poorly characterized. Dense granule (GRA) proteins such as GRA12 are key virulence factors during acute infection. Here, we investigated four GRA12-related genes. GRA12-related genes were not major virulence factors during acute infection. Instead, GRA12-related proteins localized at the cyst wall and cyst matrix and played significant roles in cyst development, persistence, and reactivation during chronic infection. Similar to GRA12, the GRA12-related proteins selectively associated with the intravacuolar network of membranes inside the vacuole. Collectively, our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection.

scholarly journals Functional Analysis of the Role of Toxoplasma gondii Nucleoside Triphosphate Hydrolases I and II in Acute Mouse Virulence and Immune Suppression

2016 ◽  
Vol 84 (7) ◽  
pp. 1994-2001 ◽  
Author(s):  
Philipp Olias ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Specific Effect ◽  
Nucleoside Triphosphate ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Raw264.7 Macrophages ◽  
Ifn Γ ◽  
Fluc Activity ◽  
Strain Dependent

Bioluminescent reporter assays have been widely used to study the effect ofToxoplasma gondiion host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages,T. gondiitype I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of theT. gondiienzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growthin vitroor virulence in mice. Our study reveals that althoughT. gondiiNTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independentGaussialuciferase, which provides a more appropriate reporter for use withT. gondiiinfection studies.

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