IFNs in host defence and parasite immune evasion during Toxoplasma gondii infections.

Interferons (IFNs) are a family of cytokines with diverse functions in host resistance to pathogens and in immune regulation. Type II IFN, i.e. IFN-γ, is widely recognized as a major mediator of resistance to intracellular pathogens, including the protozoan Toxoplasma gondii. More recently, IFN-α/ß, i.e. type I IFNs, and IFN-λ (type III IFN) have been identified to also play important roles during T. gondii infections. This parasite is a widespread pathogen of humans and animals, and it is a model organism to study cell-mediated immune responses to intracellular infection. Its success depends, among other factors, on the ability to counteract the IFN system, both at the level of IFN-mediated gene expression and at the level of IFN-regulated effector molecules. Here, I review recent advances in our understanding of the molecular mechanisms underlying IFN-mediated host resistance and immune regulation during T. gondii infections. I also discuss those mechanisms that T. gondii has evolved to efficiently evade IFN-mediated immunity. Knowledge of these fascinating host-parasite interactions and their underlying signalling machineries is crucial for a deeper understanding of the pathogenesis of toxoplasmosis, and it might also identify potential targets of parasite-directed or host-directed supportive therapies to combat the parasite more effectively.

A comparative pilot study on Gram-negative bacteria contaminating the hands of children living in urban and rural areas of Indonesia versus Germany - A suitable monitoring strategy for diarrhea risk assessment?

Diarrhea is the second leading cause of death mainly effecting young children. Often it is the result of fecal-oral pathogen transmission. We aimed to investigate whether monitoring the prevalence of Gram-negative bacteria on the hands of asymptomatic children is suitable as an indicator of fecal contamination of the environment in their playground. We compared the prevalence of Gram-negative bacteria on the hands of children, who live in the German city of Göttingen, an urban area in a high-income country, with the situation in Medan as an urban area and Siberut as a rural area both in the middle-income country Indonesia. A total of 511 children at the age of 3 months to 14 years were asked to put their thumb print on MacConkey agar, which was used to screen for the presence of Gram-negative bacteria. These were subsequently identified by using MALD-TOF mass spectrometry and classified into the order Enterobacterales, Pseudomonadales, and others. The highest burden of hand contamination was found in children from rural Siberut (66.7%) followed by children from urban Medan (53.9%), and from urban Göttingen (40.6%). In all three study sites, hand contamination was lower in the youngest (<1 year) and oldest age groups (10-14 years) and highest in the age group 5-9 years. Bacteria of the order Enterobacterales possibly indicating fecal contamination were most prevalent in Siberut (85.1%) followed by Medan (62.9%) and Göttingen (21.5%). Most facultative and obligate gastrointestinal pathogens such as Escherichia coli (n = 2) and Providencia rettgeri (n = 7), both being members of the order Enterobacterales, as well as Aeromonas caviae (n = 5), and Vibrio cholerae (n = 1) both belonging to other orders were nearly exclusively identified on the hands of children in Siberut. This result was not surprising, because hygienic conditions were lowest in Siberut. Only one isolate of A. caviae was found in Medan, and no facultative gastrointestinal pathogen was identified on the hands of children from Göttingen. Our pilot study therefore indicates that investigating hands of children for the prevalence of Gram-negative bacteria using selective media are a helpful method to monitor hygienic conditions, and thereby assess the risk for diarrhea-causing bacterial pathogens in the environment.

Assessment of pan-Leishmania detection by recombinase polymerase amplification assay.

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

The Redox Homeostasis of Skeletal Muscle Cells Regulates Stage Differentiation of Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasite that causes persistent infections in birds and mammals including ~30% of the world's human population. Differentiation from proliferative and metabolically active tachyzoites to largely dormant bradyzoites initiates the chronic phase of infection and occurs predominantly in brain and muscle tissues. Here we used murine skeletal muscle cells (SkMCs) to decipher host cellular factors that favor T. gondii bradyzoite formation in terminally differentiated and syncytial myotubes, but not in proliferating myoblast precursors. Genome-wide transcriptome analyses of T. gondii-infected SkMCs and non-infected controls identified ~6,500 genes which were differentially expressed (DEGs) in myotubes compared to myoblasts, largely irrespective of infection. On the other hand, genes related to central carbohydrate metabolism, to redox homeostasis, and to the Nrf2-dependent stress response pathway were enriched in both infected myoblast precursors and myotubes. Stable isotope-resolved metabolite profiling indicated increased fluxes into the oxidative branch of the pentose phosphate pathway (OxPPP) in infected myoblasts and into the TCA cycle in infected myotubes. High OxPPP activity in infected myoblasts was associated with increased NADPH/NADP+ ratio while myotubes exhibited higher ROS levels and lower expression of anti-oxidants and detoxification enzymes. Pharmacological reduction of ROS levels in SkMCs inhibited bradyzoite differentiation, while increased ROS induced bradyzoite formation. Thus, we identified a novel host cell-dependent mechanism that triggers stage conversion of T. gondii into persistent tissue cysts in its natural host cell type.

Epigenetic Control of IFN-γ Host Responses During Infection With Toxoplasma gondii.

Host defense against the human pathogen Toxoplasma gondii depends on secretion of interferon (IFN)-γ and subsequent activation of monocytic cells to combat intracellular parasites. Previous studies have shown that T. gondii evades IFN-γ-mediated immunity by secreting the effector TgIST into the host cell where it binds to STAT1, strengthens its DNA binding activity and recruits the Mi-2/NuRD complex to STAT1-responsive promoters. Here we investigated the impact of the host chromatin environment on parasite interference with IFN-γ-induced gene expression. Luciferase reporters under control of primary and secondary IFN-γ response promoters were only inhibited by T. gondii when they were stably integrated into the host genome but not when expressed from a plasmid vector. Absence of CpG islands upstream and/or downstream of the transcriptional start site allowed more vigorous up-regulation by IFN-γ as compared to CpG-rich promoters. Remarkably, it also favored parasite interference with IFN-γ-induced gene expression indicating that nucleosome occupancy at IFN-γ-responsive promoters is important. Promoter DNA of IFN-γ-responsive genes remained largely non-methylated in T. gondii-infected cells, and inhibition of DNA methylation did not impact parasite interference with host responses. IFN-γ up-regulated histone marks H4ac, H3K9ac, and H3K4me3 but down-regulated H3S10p at primary and secondary response promoters. Infection with T. gondii abolished histone modification, whereas total nuclear activities of histone acetyl transferases and histone deacetylases were not altered. Taken together, our study reveals a critical impact of the host chromatin landscape at IFN-γ-activated promoters on their inhibition by T. gondii with a comprehensive blockade of histone modifications at parasite-inactivated promoters.

Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes.

Toxoplasma gondii is a prevalent parasite of mammals and birds including up to 30% of humans world-wide. Primary infection of immunocompetent hosts leads to a robust cell-mediated immune response, which controls but does not clear the infection, thus enabling long-term parasite persistence in brain and muscle tissues. Chronic toxoplasmosis in mice is associated with resistance to heterologous pathogens and this has been related to increased numbers of inflammatory monocytes. Here we have analyzed whether chronic T. gondii infection impacts the subset distribution and the phenotype of peripheral human monocytes in vivo and their responses to parasite infection in vitro. CD14+ monocytes from T. gondii-seropositive blood donors expressed significantly less FcγRIII (CD16) than those from seronegative controls, but they did not show a shift in the distribution of classical, intermediate and non-classical monocyte subpopulations. Percentages of CD62L+ and CD64+ monocytes were however decreased and increased, respectively, in chronically infected individuals as compared to naïve controls. Infection of monocyte-enriched PBMCs from both seropositive and seronegative individuals with T. gondii led to an increase of CD14+CD16- classical monocytes and a decrease of CD14+CD16+ double positive monocytes. Remarkably, after in vitro parasite infection, expression of the chemokine receptor CCR2 was severely impaired in monocytes from both, individuals with chronic toxoplasmosis and seronegative controls. In contrast, only monocytes from chronically infected humans but not those from controls dose-dependently up-regulated HLA-DR, DP, DQ expression following in vitro infection. Furthermore, monocyte-enriched PBMCs from seropositive individuals up-regulated IL-12 mRNA more vigorously after in vitro infection than cells from naïve controls. Collectively, our results establish that infection of humans with T. gondii exerts long-term effects on the phenotype and responsiveness of blood monocytes. This may have important implications for innate immune responses to T. gondii and unrelated pathogens.

Toxoplasma gondii stabilises tetrameric complexes of tyrosine-phosphorylated signal transducer and activator of transcription-1 and leads to its sustained and promiscuous DNA binding.

Toxoplasma gondii is an obligate intracellular parasite that infects up to 30% of humans worldwide. It can lead to severe diseases particularly in individuals with immature or defective immune responses. Control of T. gondii relies on the IFN-γ-induced signal transducer and activator of transcription-1 (STAT1) pathway. T. gondii, however, largely inactivates STAT1-mediated gene transcription by T. gondii inhibitor of STAT1-dependent transcription (TgIST), a parasite effector protein binding to STAT1. Here, we have analysed requirements of STAT1 to bind TgIST and characterised downstream effects on STAT1 signalling. TgIST bound to STAT1 dimers but more efficiently assembled with STAT1 tetramers, which are essential for effective IFN-γ responsiveness. Such binding was abrogated in N-terminal, but not C-terminal deletion mutants of STAT1. Furthermore, TgIST did not bind to the STAT1F77A substitution mutant that cannot form STAT1 tetramers, resulting in a complete unresponsiveness of parasite-infected STAT1F77A -expressing cells to IFN-γ. Remarkably, binding of TgIST considerably increased the affinity of the aberrant STAT1 tetramers for DNA consensus sequence binding motifs and even enabled binding to nonconsensus sequences. Consistent with the increased DNA binding, STAT1 from parasite-infected cells remained phosphorylated at Tyr701 and Ser727 and was retained within the nucleus in a DNA-bound state. The sustained and promiscuous binding activity particularly of STAT1 tetramers to unspecific DNA sites lacking a consensus STAT1-binding motif is an as yet unrecognised mechanism contributing to the defective IFN-γ-mediated signalling in T. gondii-infected cells.

Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions.

The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.

Impact of the host on Toxoplasma stage differentiation.

The unicellular parasite Toxoplasma gondii infects warm-blooded animals and humans, and it is highly prevalent throughout the world. Infection of immunocompetent hosts is usually asymptomatic or benign but leads to long-term parasite persistence mainly within neural and muscular tissues. The transition from acute primary infection towards chronic toxoplasmosis is accompanied by a developmental switch from fast replicating and metabolically highly active tachyzoites to slow replicating and largely dormant bradyzoites within tissue cysts. Such developmental differentiation is critical for T. gondii in order to complete its life cycle and for pathogenesis. Herein, we summarize accumulating evidence indicating a major impact of the host cell physiology on stage conversion between the tachyzoite and the bradyzoite stage of the parasite. Withdrawal from cell cycle progression, proinflammatory responses, reduced availability of nutrients and extracellular adenosine can indeed induce tachyzoite-to-bradyzoite differentiation and tissue cyst formation. In contrast, high glycolytic activity as indicated by increased lactate secretion can inhibit bradyzoite formation. These examples argue for the intriguing possibility that after dissemination within its host, T. gondii can sense its cellular microenvironment to initiate the developmental program towards the bradyzoite stage in distinct cells. This may also explain the predominant localization of T. gondii in neural and muscular tissues during chronic toxoplasmosis.

Histone deacetylase inhibitor MS-275 augments expression of a subset of IFN-γ-regulated genes in Toxoplasma gondii-infected macrophages but does not improve parasite control.

Toxoplasma gondii is a ubiquitous apicomplexan parasite of mammals and birds and an important pathogen of humans. IFN-γ is the major mediator of host resistance against T. gondii but intriguingly, parasite-infected host cells including macrophages are severely impaired to respond to IFN-γ due to defective transcriptional activation of target genes. Here, we tested the possibility that the impaired responsiveness of T. gondii-infected macrophages to IFN-γ can be restored by inhibiting histone deacetylases (HDACs) using the class I-specific inhibitor MS-275. Treatment of RAW264.7 cells with MS-275 indeed increased MHC class II surface expression in infected and non-infected cells and largely abolished the inhibition of IFN-γ-regulated MHC class II expression exerted by T. gondii. Genome-wide transcriptome profiling revealed that MS-275 increased mean mRNA levels of IFN-γ-regulated genes particularly in non-infected macrophages. Transcript levels of 33% of IFN-γ secondary response genes but only those of a few primary response genes were also increased by MS-275 in T. gondii-infected cells. Importantly, the unresponsiveness of parasite-infected cells to IFN-γ was however not abolished by MS-275. Furthermore, MS-275 also up-regulated several anti-inflammatory cytokines or signaling molecules in T. gondii-infected macrophages. It additionally regulated expression of more than 2500 genes in non-infected macrophages expression of which was surprisingly counteracted by prior infection with T. gondii. FACS analysis and immunofluorescence microscopy revealed that MS-275 did not considerably diminish the number of parasite-positive cells or the intracellular replication in macrophages stimulated or not with IFN-γ. Thus, a supportive therapy using MS-275 appears inappropriate for treatment of toxoplasmosis.

Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.

Survivin inhibits apoptosis in numerous tumor cell lines and has emerged as promising target for cancer therapy. The anti-apoptotic effect of survivin was attributed to a direct interaction with XIAP (X-linked inhibitor of apoptosis) and to an indirect effect, where survivin antagonizes the anti-XIAP action of Smac. The direct interaction is thought to lead to synergistic inhibition of caspase-9 and, at the same time, to enhanced stability of XIAP by reducing its auto-ubiquitination. Using recombinant proteins, we have investigated the influence of survivin on the inhibition of caspase-9 by XIAP in vitro. With a fluorescence-based assay for the apoptosome-stimulated activity of caspase-9, we show that survivin has no effect on the inhibition of caspase-9 by XIAP, neither in the presence nor in the absence of Smac. Employing analytical size exclusion chromatography (SEC) and analytical ultracentrifugation, we show that survivin does not physically interact with XIAP. We confirm in vitro that XIAP ubiquitinates itself in the presence of the appropriate recombinant enzymes and Mg2+-ATP and could show that survivin neither influences the kinetics nor the extent of XIAP's self-ubiquitination. Our results call for a revision of the current view of how survivin interferes with the mitochondrial pathway of apoptosis.

Correction: Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major.

[This corrects the article DOI: 10.1371/journal.pntd.0004205.].

Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major.

Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.

Releasing the Brake on IFN-γ Signaling on Infection.

Toxoplasma gondii effectively inhibits the responsiveness of its host cell to interferon gamma (IFN-γ). Using a genome-wide genetic screen, Beiting and colleagues have recently identified coactivators of the transcription factor STAT1 that can diminish this inhibitory effect. One of these coactivators, TLX, enhances type 1 helper (Th1) immune responses and restricts parasite replication during chronic toxoplasmosis.

Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage.

Toxoplasma gondii is a widespread intracellular parasite of mammals and birds and an important opportunistic pathogen of humans. Following primary infection, fast-replicating tachyzoites disseminate within the host and either are subsequently eliminated by the immune system or transform to latent bradyzoites which preferentially persist in brain and muscle tissues. The factors which determine the parasites' tissue distribution during chronic toxoplasmosis are unknown. Here we show that mouse skeletal muscle cells (SkMCs) after differentiation to mature, myosin heavy chain-positive, polynucleated myotubes, significantly restrict tachyzoite replication and facilitate expression of bradyzoite-specific antigens and tissue cyst formation. In contrast, proliferating mononuclear myoblasts and control fibroblasts enable vigorous T. gondii replication but do not sustain bradyzoite or tissue cyst formation. Bradyzoite formation correlates with upregulation of testis-specific Y-encoded-like protein-2 gene expression (Tspyl2) and p21(Waf1/Cip1 as well as downregulation of cyclin B1 and absence of DNA synthesis, i.e. a cell cycle arrest of syncytial myotubes. Following infection with T. gondii, myotubes but not myoblasts or fibroblasts further upregulate the negative cell cycle regulator Tspyl2. Importantly, RNA interference-mediated knock-down of Tspyl2 abrogates differentiation of SkMCs to myotubes and enables T. gondii to replicate vigorously but abolishes bradyzoite-specific gene expression and tissue cyst formation. Together, these data indicate that Tspyl2-mediated host cell cycle withdrawal is a physiological trigger of Toxoplasma stage conversion in mature SkMCs. This finding might explain the preferred distribution of T. gondii tissue cysts in vivo.

Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly.

Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome cin vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway.

Toxoplasma gondii within skeletal muscle cells: a critical interplay for food-borne parasite transmission.

Toxoplasma gondii infects virtually any nucleated cell type of warm-blooded animals and humans including skeletal muscle cells (SkMCs). Infection of SkMCs by T. gondii, differentiation from the highly replicative tachyzoites to dormant bradyzoites and tissue cyst formation are crucial for parasite persistence in muscle tissue. These processes are also prerequisites for one of the major routes of transmission to humans via undercooked or cured meat products. Evidence obtained in vitro and in vivo indicates that SkMCs are indeed a preferred cell type for tissue cyst formation and long-term persistence of T. gondii. This raises intriguing questions about what makes SkMCs a suitable environment for parasite persistence and how the SkMC-T. gondii interaction is regulated. Recent data from our laboratory show that differentiation of SkMCs from myoblasts to syncytial myotubes, rather than the cell type itself, is critical for parasite growth, bradyzoite formation and tissue cyst maturation. Myotube formation is accompanied by a permanent withdrawal from the cell cycle, and the negative cell cycle regulator cell division autoantigen (CDA)-1 directly or indirectly promotes T. gondii stage conversion in SkMCs. Moreover, host cell cycle regulators are specifically modulated in mature myotubes, but not myoblasts, following infection. Myotubes also up-regulate the expression of various pro-inflammatory cytokines and chemokines after T. gondii infection and they respond to IFN-γ by exerting potent anti-parasitic activity. This highlights that mature myotubes are active participants rather than passive targets of the local immune response to T. gondii which may also govern the interaction between SkMCs and the parasite.

The Campylobacter jejuni Cj0268c protein is required for adhesion and invasion in vitro.

Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.

Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents: a proof-of-concept study.

Ancient pathways of an apoptosis-like cell death have been identified in unicellular eukaryotes including protozoan parasites. Here, we examined programmed cell death in the apicomplexan Toxoplasma gondii which is a common intracellular pathogen of humans and warm-blooded animals. Treatment of extracellular T. gondii with various pro-apoptotic stimuli significantly induced DNA strand breaks as revealed by TUNEL and flow cytometry. Using staurosporine or miltefosine as pro-apoptotic stimuli, parasites also presented a reduced cell size, i.e. pyknosis and externalized phosphatidylserine while the plasma membrane remained intact. Importantly, staurosporine also induced DNA strand breaks in intracellular T. gondii. Data mining of the Toxoplasma genome resource identified 17 putative cell death-associated genes encoding proteases, a nuclease and several apoptosis regulators. Staurosporine-treated parasites but not controls strongly up-regulated several of these genes in a time-dependent fashion with a putative PDCD2 protein being more than 100-fold up-regulated. However, the mitochondrial membrane potential (ΔΨ(m)) remained intact and caspase-like activity increased only slightly during staurosporine-triggered cell death. As compared to staurosporine, the transcriptional response of parasites to miltefosine was more restricted but PDCD2 was again strongly induced. Furthermore, T. gondii lost their ΔΨ(m) and rapidly presented strong caspase-like activity during miltefosine treatment. Consequently, protease inhibitors abrogated miltefosine-induced but not staurosporine-induced Toxoplasma cell death. Finally, toxoplasmacidal drugs triggered DNA strand breaks in extracellular T. gondii. Interestingly, clindamycin also induced markers of an apoptosis-like cell death in intracellular parasites. Together, the data indicate that T. gondii possesses ancient apoptosis-like cell death machinery which can be triggered by chemotherapeutic agents.