Tuesday, March 21, 2017

Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

2017 Mar 15;2(2). pii: e00054-17. doi: 10.1128/mSphere.00054-17.


Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite.

Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.


ApiAP2; apicomplexan parasites; differentiation; gene expression; intracellular parasites; transcription

Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in Toxoplasma gondii

2017 Mar 17. pii: jbc.M116.765487. doi: 10.1074/jbc.M116.765487. [Epub ahead of print]

Toxoplasma gondii is among the most prevalent protozoan parasites, which infects a wide range of organisms including one-third of the human population. Its rapid intracellular replication within a vacuole requires efficient synthesis of glycerophospholipids. Cytidine diphosphate-diacylglycerol (CDP-DAG) serves as a major precursor for phospholipid synthesis. Given the peculiarities of lipid biogenesis, understanding the mechanism and physiological importance of CDP-DAG synthesis is particularly relevant in T. gondii Here, we report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in the parasite. The eukaryotic-type TgCDS1 and the prokaryotic-type TgCDS2 reside in the endoplasmic reticulum (ER) and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuated the parasite growth and resulted in a nearly complete loss of virulence in a mouse model. Moreover, mice infected with the TgCDS1 mutant became fully resistant to challenge infection with a hyper-virulent strain of T. gondii The residual growth of the TgCDS1 mutant was abolished by consecutive deletion of TgCDS2. Lipidomic analyses of the mutants revealed significant and specific declines in phosphatidylinositol and phosphatidylglycerol levels upon repression of TgCDS1 and following deletion of TgCDS2, respectively. Our data suggest a division of labor model of lipid biogenesis in T. gondii, in which two discrete CDP-DAG pools produced in the ER and apicoplast are subsequently used for the synthesis of phosphatidylinositol in the Golgi bodies and phosphatidylglycerol in the mitochondrion. The essential and divergent nature of CDP-DAG synthesis in the parasite apicoplast offers a potential drug target to inhibit the asexual reproduction of T. gondii.


Toxoplasma gondii; glycerophospholipid; parasite metabolism; phosphatidylglycerol; phosphatidylinositol

Friday, March 17, 2017

Chinese 1 strain of Toxoplasma gondii excreted-secreted antigens negatively modulate Foxp3 via inhibition of the TGFßRII/Smad2/Smad3/Smad4 pathway

2017 Mar 16. doi: 10.1111/jcmm.13115. [Epub ahead of print]

Toxoplasma gondii is an opportunistic intracellular parasite and is considered an important aetiological factor in the process of abortion, especially as occurs in early gestation. Chinese 1 strain of T. gondii is a dominant genotype prevalent in China. Although it is known that early foetal resorption triggered by RH strain of T. gondii is attributable to immune mechanisms rather than its direct effect in uterus, the underlying mechanism of the abortion caused by Chinese 1 strain remains unclear. This study was designed to investigate the effect of excreted-secreted antigens (ESA) of Chinese 1 strain of T. gondii on the expression of forkhead box transcription factor (Foxp3) as it pertains to early pregnancy and abortion. ESA caused a marked inhibition in the expression of Foxp3 both in vivo and in vitro. In addition, ESA negatively modulated Smad2 and Smad3 at the posttranslational level. Smad2 siRNA cooperated with ESA to further suppress the level of Foxp3. This inhibitory effect on Foxp3 expression was partially abrogated by overexpression of Smad2, Smad3 and Smad4. Additionally, ESA attenuated the expression of TGFßRII, whereas TGFßRII agonist could profoundly reversed the decreased Foxp3 triggered by ESA. Collectively, the findings suggested that ESA restricted Foxp3 expression by inhibiting TGFßRII/Smad2/Smad3/Smad4 signalling, ultimately resulting in abortion.


Chinese 1 strain of Toxoplasma gondii; Foxp3; TGFßRII/Smad2/Smad3/Smad4; excreted-secreted antigens

Tuesday, March 14, 2017

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

2017 Mar 13;13(3):e1006272. doi: 10.1371/journal.ppat.1006272. [Epub ahead of print]

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2, which encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

Research advances in interactions related to Toxoplasma gondii microneme proteins

2017 Mar 9. pii: S0014-4894(16)30389-7. doi: 10.1016/j.exppara.2017.03.001. [Epub ahead of print]

Toxoplasma gondii microneme proteins (TgMICs), secreted by micronemes upon contact with host cells, are reported to play important roles in multiple stages of the T. gondii life cycle, including parasite motility, invasion, intracellular survival, and egress from host cells. Meanwhile, during these processes, TgMICs participate in many protein-protein and protein-carbohydrate interactions, such as undergoing proteolytic maturation, binding to aldolase, engaging the host cell receptors and forming the moving junction (MJ), relying on different types of ectodomains, transmembrane (TM) domains and cytoplasmic domains (CDs). In this review, we summarize the research advances in protein-protein and protein-carbohydrate interactions related to TgMICs, and their intimate associations with corresponding biological processes during T. gondii infection, which will contribute to an improved understanding of the molecular pathogenesis of T. gondii infection, and provide a basis for developing effective control strategies against T. gondii.


Host cells; Microneme proteins; Protein-carbohydrate interactions; Protein-protein interactions; Toxoplasma gondii

Enhanced susceptibility of triple transgenic Alzheimer's disease (3xTg-AD) mice to acute infection

2017 Mar 11;14(1):50. doi: 10.1186/s12974-017-0826-5.


Infection is a recognised risk factor for Alzheimer's disease (AD) and can worsen symptoms in established disease. AD patients have higher rates of infection and are more likely to require hospital admissions due to infections than individuals without dementia. Infections have also been found to increase the risk of those over 84 years of age being diagnosed with dementia. However, few studies have investigated immune responses to infection in AD.


Here, we investigated the immune responses of the triple transgenic Alzheimer's disease (3xTg-AD) mouse model of AD to infection with the parasites Toxoplasma gondii and Trichuris muris. Cytometric bead array, histology, immunohistochemistry and immunofluorescence were used to evaluate immune responses and the effects on the brain of acute infection.


3xTg-AD mice, despite having comparable parasite loads, were more susceptible to infection with more severe morbidity. A worsened outcome to infection can be linked to an exaggerated immune response. 3xTg-AD mice had an increased pro-inflammatory response characterised by the production of pro-inflammatory mediators such as tumour necrosis TNF-α, IL-6, CCL5 and CXCL-1, as well as an increase in immune cell infiltration to the sites of infection. T cell responses to parasite antigen also showed elevated production of the pro-inflammatory cytokines TNF-α (10 fold) and IL-6 (twofold). We investigated whether 3xTg-AD mice had a propensity for a more Th1-dominated response using the T. muris worm infection and showed that akin to T. gondii, there was an enhanced pro-inflammatory response which was associated with retention of worms in the gut and associated pathology. Irrespective of whether the infection was one that could infect the brain or cause a local gut inflammation, 3xTg-AD mice had increased numbers of activated microglia during infection in both the cortex and the hippocampus.


Our findings suggest that in AD, responses to infection are exaggerated outside of the CNS. Additionally, the results presented here indicate that both systemic and localised inflammation caused by an infection exacerbate neuroinflammation in AD.


Alzheimer’s disease; Cytokines; Infection; Inflammation; Microglia; Neuroinflammation; Toxoplasma gondii; Trichuris muris

Friday, March 10, 2017

The human immune response to Toxoplasma: Autophagy versus cell death

2017 Mar 9;13(3):e1006176. doi: 10.1371/journal.ppat.1006176. eCollection 2017.


Genomics of apicomplexan parasites

2017 Feb 22:1-20. doi: 10.1080/10409238.2017.1290043. [Epub ahead of print]

The increasing prevalence of infections involving intracellular apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium (the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, respectively) represent a significant global healthcare burden. Despite their significance, few treatments are available; a situation that is likely to deteriorate with the emergence of new resistant strains of parasites. To lay the foundation for programs of drug discovery and vaccine development, genome sequences for many of these organisms have been generated, together with large-scale expression and proteomic datasets. Comparative analyses of these datasets are beginning to identify the molecular innovations supporting both conserved processes mediating fundamental roles in parasite survival and persistence, as well as lineage-specific adaptations associated with divergent life-cycle strategies. The challenge is how best to exploit these data to derive insights into parasite virulence and identify those genes representing the most amenable targets. In this review, we outline genomic datasets currently available for apicomplexans and discuss biological insights that have emerged as a consequence of their analysis. Of particular interest are systems-based resources, focusing on areas of metabolism and host invasion that are opening up opportunities for discovering new therapeutic targets.


Metabolism; apicomplexan genomics; genomics of apicomplexan parasites; host cell modulation; host invasion; parasite genomics; systems-based approaches

Galectins expressed differently in genetically susceptible C57BL/6 and resistant BALB/c mice during acute ocular Toxoplasma gondii infection

2017 Mar 9:1-9. doi: 10.1017/S0031182017000270. [Epub ahead of print]

Ocular toxoplasmosis (OT) caused by Toxoplasma gondii is a major cause of infectious uveitis, however little is known about its immunopathological mechanism. Susceptible C57BL/6 (B6) and resistant BALB/c mice were intravitreally infected with 500 tachyzoites of the RH strain of T. gondii. B6 mice showed more severe ocular pathology and higher parasite loads in the eyes. The levels of galectin (Gal)-9 and its receptors (Tim-3 and CD137), interferon (IFN)-γ, IL-6 and IL-10 were significantly higher in the eyes of B6 mice than those of BALB/c mice; however, the levels of IFN-α and -β were significantly decreased in the eyes and CLNs of B6 mice but significantly increased in BALB/c mice after infection. After blockage of galectin-receptor interactions by α-lactose, neither ocular immunopathology nor parasite loads were different from those of infected BALB/c mice without α-lactose treatment. Although the expressions of Gal-9/receptor were significantly increased in B6 mice and Gal-1 and -3 were upregulated in both strains of mice upon ocular T. gondii infection, blockage of galectins did not change the ocular pathogenesis of genetic resistant BALB/c mice. However, IFN-α and -β were differently expressed in B6 and BALB/c mice, suggesting that type I IFNs may play a protective role in experimental OT.


α-lactose; BALB/c mice; C57BL/6 mice; galectins; ocular toxoplasmosis; type I interferons

Monday, March 06, 2017

A Forward-genetic Screen Identifies a Negative Regulator of Rapid Ca2+-dependent Cell Egress in the Intracellular Parasite Toxoplasma gondii

2017 Mar 3. pii: jbc.M117.775114. doi: 10.1074/jbc.M117.775114. [Epub ahead of print]

Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+signalling pathways to activate gliding motility, which drives tissue dissemination, host cell invasion and egress. A group of plant-like Ca2+-dependent protein kinases (CDPKs), transduce cytosolic Ca2+ flux into enzymatic activity, but the molecular details of their activity are poorly understood. To investigate how Ca2+ signalling activates egress through CDPKs, we performed a forward-genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3-knockout strain. We recovered mutants that regained the ability to egress from host cells. These harboured mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near-wild type levels. We also show that phosphorylation of SCE1 is required to relieve its suppressive activity to potentiate egress, and that CDPK3 may regulate this phosphorylation. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during the T. gondii lytic growth.


Ca2+ signalling; Toxoplasma gondii; apicomplexa; forward genetic screen; host cell egress; molecular genetics; parasite; proteomics; signaling

Toxoplasma growth in vitro is dependent on exogenous tyrosine and is independent of AAH2 even in tyrosine-limiting conditions

2017 Feb 28. pii: S0014-4894(16)30401-5. doi: 10.1016/j.exppara.2017.02.018. [Epub ahead of print]

Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all nucleated cell types in almost all warm-blooded animals. Interestingly, Toxoplasma has a relatively full repertoire of amino acid biosynthetic machinery, perhaps reflecting its broad host range and, consequently, its need to adapt to a wide array of amino acid resources. Although Toxoplasma has been shown to be auxotrophic for tryptophan and arginine, it has not previously been determined if Toxoplasma is also auxotrophic for tyrosine. Toxoplasma tachyzoites and bradyzoites were recently found to express an amino acid hydroxylase (AAH2) that is capable of synthesizing tyrosine and dihydroxyphenylalanine (DOPA) from phenylalanine; however, the role of AAH2 in tachyzoite and bradyzoite infection has not yet been identified. To determine if Toxoplasma requires exogenous tyrosine for growth, we performed growth assays on tachyzoites and bradyzoites in nutrient-rich media titrated with varying amounts of tyrosine. We found that Toxoplasma tachyzoites form significantly smaller plaques in tyrosine-limiting media in a dose-dependent manner and that this phenotype is not affected by deletion of TgAAH2. To determine if bradyzoites require exogenous tyrosine for growth, we induced differentiation from tachyzoites in vitro in tyrosine-limiting media and found that replication and vacuole number are all decreased in tyrosine-deficient media. Importantly, culture of confluent human fibroblasts in tyrosine-deficient media does not affect their viability, indicating that, at least in vitro, the need for tyrosine is at the level of Toxoplasma, not the host cell supporting its growth.


Amino acid hydroxylase; Apicomplexan; Metabolism
[PubMed - as supplied by publisher]

Friday, March 03, 2017

Opposing Transcriptional Mechanisms Regulate Toxoplasma Development

2017 Feb 22;2(1). pii: e00347-16. doi: 10.1128/mSphere.00347-16.

The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst.

IMPORTANCEToxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.


Toxoplasma gondii; apicomplexan parasites; development; gene expression; transcription factors

Thursday, March 02, 2017

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

2017 Feb 28;8(1). pii: e02189-16. doi: 10.1128/mBio.02189-16.

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

Recent developments in drug discovery against the protozoal parasites Cryptosporidium and Toxoplasma

2017 Jan 17. pii: S0960-894X(17)30059-8. doi: 10.1016/j.bmcl.2017.01.046. [Epub ahead of print]

Apicomplexan parasites cause some of the most devastating human diseases, including malaria, toxoplasmosis, and cryptosporidiosis. New drug discovery is imperative in light of increased resistance. In this digest article, we briefly explore some of the recent and promising developments in new drug discovery against two apicomplexan parasites, Cryptosporidium and Toxoplasma.
[PubMed - as supplied by publisher]