Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology.

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.

Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence.

Toxoplasma gondii infects virtually any nucleated cell and resides inside a non-phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of immunity regulated GTPases (IRGs) and guanylate binding proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine-threonine kinases ROP17 and ROP18, and a pseudo-kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM. Here, we characterise GRA60, a novel dense granule effector, which localises to the vacuolar space and PVM and contributes to virulence of RH in mice, suggesting a role in the subversion of host cell defence mechanisms. Members of the host cell IRG defence system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and found associated to ROP18, a member of the virulence complex.

Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases.

The protozoan parasite Toxoplasma gondii lives inside a vacuole in the host cytosol where it is protected from host cytoplasmic innate immune responses. However, IFNγ-dependent cell-autonomous immunity can destroy the vacuole and the parasite inside. Toxoplasma strain differences in susceptibility to human IFNγ exist, but the Toxoplasma effector(s) that determine these differences are unknown. We show that in human primary fibroblasts, the polymorphic Toxoplasma-secreted effector GRA15 mediates the recruitment of ubiquitin ligases, including TRAF2 and TRAF6, to the vacuole membrane, which enhances recruitment of ubiquitin receptors (p62/NDP52) and ubiquitin-like molecules (LC3B, GABARAP). This ultimately leads to lysosomal degradation of the vacuole. In murine fibroblasts, GRA15-mediated TRAF6 recruitment mediates the recruitment of immunity-related GTPases and destruction of the vacuole. Thus, we have identified how the Toxoplasma effector GRA15 affects cell-autonomous immunity in human and murine cells.

Exploring protein myristoylation in Toxoplasma gondii.

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.

High-content imaging assay to evaluate Toxoplasma gondii infection and proliferation: A multiparametric assay to screen new compounds.

Toxoplasma gondii is an intracellular protozoan parasite widely distributed in animals and humans. Infection of host cells and parasite proliferation are essential steps in Toxoplasma pathology. The objective of this study was to develop and validate a novel automatic High Content Imaging (HCI) assay to study T. gondii infection and proliferation. We tested various fluorescent markers and strategies of image analysis to obtain an automated method providing results comparable to those from gold standard infection and proliferation assays. No significant difference was observed between the results obtained from the HCI assay and the standard assays (manual fluorescence microscopy and incorporation of [3H]-uracil). We developed here a robust and time-saving assay. This automated technology was then used to screen a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Inhibition of parasite proliferation and host cell toxicity were measured in the same assay and led to the identification of one hit, a thiosemicarbazone that allows important inhibition of Toxoplasma proliferation while being relatively safe for the host cells.

Microarray analysis of long non-coding RNA expression profiles uncovers a Toxoplasma-induced negative regulation of host immune signaling.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect mammalian cells and thereby regulate host gene expression. The long non-coding RNAs (lncRNAs) have been demonstrated to be an important class of RNA molecules that regulate many biological processes, including host-pathogen interactions. However, the role of host lncRNAs in the response to T. gondii infection remains largely unknown. METHODS: We applied a microarray approach to determine the differential expression profiles of both lncRNAs and mRNAs in the human foreskin fibroblast (HFF) cells after T. gondii infection. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to reveal the potential functions of T. gondii-induced genes. Based on the co-expression networks of lncRNAs and immune-related genes, the role of NONSHAT022487 on the regulation of UNC93B1 related immune signaling was investigated by the knockdown and over-expression of lncRNA in human macrophage derived from the PMA-induced promonocytic cell line THP-1. RESULTS: Our data showed that 996 lncRNAs and 109 mRNAs in HFF cells were significantly and differentially expressed following T. gondii infection (fold change ≥ 5, P < 0.05). The results from the GO and KEGG pathway analyses indicated that the mRNAs with differential expression were mainly involved in the host immune response. Remarkably, we identified a novel lncRNA, NONSHAT022487, which suppresses the expression of the immune-related molecule UNC93B1. After T. gondii infection, NONSHAT022487 impaired the secretion of the cytokines IL-12, TNF-α, IL-1ß and IFN-γ by downregulating UNC93B1 expression in human macrophage cells. CONCLUSIONS: Our study identified infection-induced lncRNA expression as a novel mechanism by which the Toxoplasma parasite regulates host immune signaling, which advances our understanding of the interaction of T. gondii parasites and host cells.

The antifungal Aureobasidin A and an analogue are active against the protozoan parasite Toxoplasma gondii but do not inhibit sphingolipid biosynthesis.

Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa, and toxoplasmosis is an important disease of both humans and economically important animals. With a limited array of drugs available there is a need to identify new therapeutic compounds. Aureobasidin A (AbA) is an antifungal that targets the essential inositol phosphorylceramide (IPC, sphingolipid) synthase in pathogenic fungi. This natural cyclic depsipeptide also inhibits Toxoplasma proliforation, with the protozoan IPC synthase orthologue proposed as the target. The data presented here show that neither AbA nor an analogue (Compound 20), target the protozoan IPC synthase orthologue or total parasite sphingolipid synthesis. However, further analyses confirm that AbA exhibits significant activity against the proliferative tachyzoite form of Toxoplasma, and Compound 20, whilst effective, has reduced efficacy. This difference was more evident on analyses of the direct effect of these compounds against isolated Toxoplasma, indicating that AbA is rapidly microbicidal. Importantly, the possibility of targeting the encysted, bradyzoite, form of the parasite with AbA and Compound 20 was demonstrated, indicating that this class of compounds may provide the basis for the first effective treatment for chronic toxoplasmosis.

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication.

Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

Exogenous tumor necrosis factor-alpha could induce egress of Toxoplasma gondii from human foreskin fibroblast cells.

Toxoplasma gondii is an intra-cellular protozoan parasite that can infect almost all nucleated cells, eliciting host immune responses against infection. Host tissue damage is mainly caused by cellular lysis when T. gondii egresses from infected cells. However, the effects of cytokines released by host immune cells on egression of T. gondii remain elusive. This study aimed to investigate the role of tumor necrosis factor-alpha (TNF-α) on the egress of T. gondii from infected human foreskin fibroblast (HFF) cells and to elucidate the underlying mechanisms that regulate TNF-α-induced egress. Using flow cytometry to count tachyzoites of T. gondii released into cell culture medium, we found that egress of T. gondii from infected HFF cells could be induced by 10 ng/mL TNF-α in a time-dependent manner. Pre-treatment of infected HFF cells with BAPTA-AM to chelate intra-parasitic calcium could greatly inhibit TNF-α-induced egress. Similar results were obtained when using cytochalasin D to block parasite motility before the TNF-α-induced egress assay. In addition, blocking host apoptosis by Z-VAD-FMK could decrease TNF-α induced egress, while blocking necroptosis by necrostatin-1 has little impact on TNF-α-induced egress. The egressed tachyzoites displayed a normal growth rate and lost no virulence. Our results suggest that host cytokines could influence the cellular lytic processes of T. gondii, providing new insights into the relationship between host TNF-α and T. gondii pathogenesis.

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii.

Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle-free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.

The brighter (and evolutionarily older) face of the metabolic syndrome: evidence from Trypanosoma cruzi infection in CD-1 mice.

BACKGROUND: Infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, results in chronic infection that leads to cardiomyopathy with increased mortality and morbidity in endemic regions. In a companion study, our group found that a high-fat diet (HFD) protected mice from T. cruzi-induced myocardial damage and significantly reduced post-infection mortality during acute T. cruzi infection. METHODS: In the present study metabolic syndrome was induced prior to T. cruzi infection by feeding a high fat diet. Also, mice were treated with anti-diabetic drug metformin. RESULTS: In the present study, the lethality of T. cruzi (Brazil strain) infection in CD-1 mice was reduced from 55% to 20% by an 8-week pre-feeding of an HFD to induce obesity and metabolic syndrome. The addition of metformin reduced mortality to 3%. CONCLUSIONS: It is an interesting observation that both the high fat diet and the metformin, which are known to differentially attenuate host metabolism, effectively modified mortality in T. cruzi-infected mice. In humans, the metabolic syndrome, as presently construed, produces immune activation and metabolic alterations that promote complications of obesity and diseases of later life, such as myocardial infarction, stroke, diabetes, Alzheimer's disease and cancer. Using an evolutionary approach, we hypothesized that for millions of years, the channeling of host resources into immune defences starting early in life ameliorated the effects of infectious diseases, especially chronic infections, such as tuberculosis and Chagas disease. In economically developed countries in recent times, with control of the common devastating infections, epidemic obesity and lengthening of lifespan, the dwindling benefits of the immune activation in the first half of life have been overshadowed by the explosion of the syndrome's negative effects in later life.

Detection of Leishmania infantum in the smegma of infected dogs / Detecção de Leishmania infantum no esmegma de cães infectados

Considering the venereal transmission of visceral leishmaniasis from dogs to bitches, the aim of this study was to verify if the penile surface and smegma from infected dogs can be the source of parasites in bitches. Twelve Leishmania infantum infected dogs had semen and smegma samples collected for submission to PCR identification of the DNA of the parasite. Semen (41.7 percent) and smegma (50.0 percent) have similar positive incidence (P>0.05; Fisher's exact test), with 58.3 percent of the dogs positive for semen and/or smegma samples. The proportion of positivity for both semen and smegma was 33.3 percent, but 8.3 percent was positive only for semen, and 16.7 percent only for smegma, revealing a moderate agreement between tests (K=0.5; Kappa index). It was concluded that Leishmania infantum is present in the smegma of contaminated dogs and it can be a source of parasites for the semen and the bitch...

Tendo em vista a transmissão venérea da leishmaniose visceral do cão para a cadela, o objetivo deste estudo foi verificar se a superfície peniana e o esmegma de cães infectados poderiam ser a fonte de parasitas para a fêmea. Amostras de sêmen e esmegma de 12 cães infectados com Leishmania infantum foram submetidas à identificação do DNA do parasita por PCR. As incidências de positividade no sêmen (41,7 por cento) e no esmegma (50,0 por cento) foram semelhantes (P>0,05; teste exato de Fisher), sendo 58,3 por cento dos cães positivos para sêmen e/ou esmegma. A positividade para sêmen e esmegma juntos ocorreu em 33,3 por cento, mas em 8,3 por cento dos casos apenas no sêmen, e em 16,7 por cento apenas no esmegma, o que revela uma concordância moderada entre os testes (K=0,5; índice Kappa). Conclui-se que a Leishmania infantum está presente no esmegma de cães contaminados, podendo ser a fonte de parasitas para o sêmen e a cadela...

Use of pooled protozoal cultures of preputial scraping samples obtained from bulls for the detection of Tritrichomonas foetus by means of a real-time polymerase chain reaction assay.

OBJECTIVE: To determine the sensitivity of a real-time PCR assay for the detection of Tritrichomonas foetus in protozoal cultures of preputial scraping samples pooled from up to 25 bulls and to determine the specificity of that assay for detection of T foetus in cultures for individual animals. DESIGN: Cross-sectional study. ANIMALS: 188 bulls and 150 steers. PROCEDURES: Preputial scraping samples were collected, placed in a culture kit, and incubated at 37°C for 7 days. Cultures for individual animals were tested for T foetus by means of a real-time PCR assay. Pools of protozoal cultures were made by including fixed aliquots of samples with known positive and negative results in ratios of 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25. Specificities of the real-time PCR assay and culture for detection of T foetus in samples obtained from individual animals and sensitivity of real-time PCR assay for each evaluated pool ratio were determined. RESULTS: Specificity estimates for culture and the real-time PCR assay for detection of T foetus in preputial scraping samples for individual animals were not significantly different (98.8% and 100%, respectively). Sensitivities of the real-time PCR assay for the various pooled samples with known positive and negative T foetus results were not significantly different; overall sensitivity of the assay was 94%. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated the evaluated real-time PCR assay had high specificity and good sensitivity for the detection of T foetus in pooled protozoal cultures of preputial scraping samples obtained from up to 25 animals.

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

A novel family of Toxoplasma IMC proteins displays a hierarchical organization and functions in coordinating parasite division.

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.