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UNLABELLED: In an effort to identify novel compounds with potent inhibition against Toxoplasma gondii, a phenotypic screen was performed utilizing a library of 683 pure compounds derived primarily from terrestrial and marine fungi. An initial screen with a fixed concentration of 5 µM yielded 91 hits with inhibition comparable to an equal concentration of artemisinin. These compounds were then triaged based on known biological and chemical concerns and liabilities. From these, 49 prioritized compounds were tested in a dose response format with T. gondii and human foreskin fibroblasts (HFFs) for cytotoxicity. Ten compounds were identified with an IC50 less than 150 nM and a selectivity index (SI) greater than 100. An additional eight compounds demonstrated submicromolar IC50 and SI values equal to or greater than 35. While the majority of these scaffolds have been previously implicated against apicomplexan parasites, their activities in T. gondii were largely unknown. Herein, we report the T. gondii activity of these compounds with chemotypes including xanthoquinodins, peptaibols, heptelidic acid analogs, and fumagillin analogs, with multiple compounds demonstrating exceptional potency in T. gondii and limited toxicity to HFFs at the highest concentrations tested. IMPORTANCE: Current therapeutics for treating toxoplasmosis remain insufficient, demonstrating high cytotoxicity, poor bioavailability, limited efficacy, and drug resistance. Additional research is needed to develop novel compounds with high efficacy and low cytotoxicity. The success of artemisinin and other natural products in treating malaria highlights the potential of natural products as anti-protozoan therapeutics. However, the exploration of natural products in T. gondii drug discovery has been less comprehensive, leaving untapped potential. By leveraging the resources available for the malaria drug discovery campaign, we conducted a phenotypic screen utilizing a set of natural products previously scre

The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (∆eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in ∆eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

There is a need to reconsider the acceptance of organs from donors considered suboptimal, in the absence of data. Toxoplasma antibody-positive donors (TPD) constitute one such group. The objective of our study was to compare graft survival in deceased donor renal transplant (Tx) recipients, stratified by Toxoplasma IgG status, using the Organ Procurement and Transplantation Network (OPTN) database. A log-linear event history regression model for graft failure categorized by Toxoplasma IgG status, adjusting for confounders was applied to first kidney-only Tx recipients from 2018 to 2022. Of the 51,422 Tx, 4,317 (8.4%) were from TPD. Acute rejection and graft failure (5% each) were similar between groups. Crude graft failure was 7.3 failures per 100 person-years for TPD recipients compared to 6.5 failures per 100 person-years for the Toxoplasma-negative group (p 0.008). The crude failure rate ratio was 1.14 with an adjusted hazard rate ratio of 1.04 (95% CI: 0.94, 1.15, p 0.39). In renal Tx recipients, TPD graft recipients have comparable survival to Tx from Toxoplasma-negative recipients. While caution and close monitoring of recipients post-Tx for surveillance of disseminated toxoplasmosis are still warranted, our study suggests that patients can be successfully managed using TPD organs.

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

Toxoplasma gondii is a single-celled obligate intracellular parasite which is estimated to infect 30% of the global human population. While T. gondii infections are typically asymptomatic in healthy individuals, they can cause life-threatening complications in immunocompromised individuals and congenitally infected neonates. As current treatments are toxic and do not clear the lifelong chronic infection, a deeper understanding of parasite biology is needed to identify novel targets for therapeutic intervention. As a member of the phylum Apicomplexa, T. gondii contains several unique organelles which play critical roles in facilitating the parasite’s lytic cycle, which causes the acute phase of disease. One of these unique apicomplexan organelles is the inner membrane complex (IMC), which lies directly underneath the parasite’s plasma membrane and is composed of a series of flattened vesicles supported by an underlying cytoskeletal network. The IMC plays essential roles throughout the T. gondii lytic cycle by serving as the platform for the molecular machinery that controls parasite motility, stabilizing the apical complex which facilitates host cell invasion, and acting as a scaffold for developing daughter cells during parasite replication.T. gondii replicates using a unique form of internal budding called endodyogeny, in which two daughter buds are formed within the cytoplasm of a single maternal cell. Endodyogeny can be divided into four steps: bud initiation, elongation, constriction, and maturation. During this process, IMC components are added to the developing daughter cell scaffold in a tightly regulated, sequential manner. While many of the components of the IMC are known to be important for parasite fitness, most are maintained in mature parasites and play critical roles in other phases of the lytic cycle besides replication. Several IMC proteins have been identified which are found only in the IMC of daughter buds, but most are dispensable or have only mo

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in a stress-induced model of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16s cyst enhancing abilities are mediated, in part, by phosphorylation-and therefore activation-of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected wild-type (WT) and STAT6KO mice, finding that, compared to WT mice, STAT6KO mice have a decrease in CNS cyst burden but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

The protozoan parasite Toxoplasma gondii infects approximately 2.5 billion people worldwide. Infection induces a rapid dissemination of parasites throughout the body followed by the formation of lifelong cysts within neurons of the host brain. Both stages require a dynamic immune response comprised of both innate and adaptive cells. Neutrophils are a primary responding cell to acute infection and have been observed in the brain during murine chronic infection. Previous studies investigating human neutrophils found that invasion by Toxoplasma tachyzoites inhibits apoptosis of neutrophils, prolonging their survival under inflammatory conditions. Here, we demonstrate the differentiation of two distinct subsets following exposure of human neutrophil-like-cells (HNLC) to Toxoplasma cysts. In vitro stimulation and imaging studies show cyst-specific induction of cytokines and cyst clearance by HNLCs. Further testing demonstrates that aged HNLCs perform less phagocytosis of cysts compared to non-aged HNLCs. In conclusion, this study identifies a novel response of HNLCs to Toxoplasma cysts and may indicate a role for neutrophils in the clearance of cysts during human infection with Toxoplasma.

Unicellular parasites of the apicomplexan phylum have a considerable effect on global health and agriculture. Two prominent examples of this phylum include malaria causing parasites of the Plasmodium genus and the widely prevalent parasite Toxoplasma gondii. While sharing a common ancestor, these parasites occupy unique biological niches, follow distinct life cycles, and result in different courses and outcomes of disease. In response to the parasite, the mammalian host has developed efficient and effective defense strategies. However, both Plasmodium and Toxoplasma have evolved strategies to evade the host’s defense response. Plasmodium parasites infect distinct tissues and cell types whereas T. gondii parasites are highly promiscuous and infect all nucleated cells. The identification of key factors involved in the interaction between the host and parasite is crucial for disease intervention, prevention, and eventually eradication efforts. Next-generation sequencing technologies have proven effective tools to investigate the response in a tissue or cell population of an infected organism. Novel genomics methods such as single-cell RNA-seq and spatial transcriptomics have enabled the investigation of heterogeneous transcriptional responses of individual cells in a population as well as heterogeneous expression profiles at spatially distinct tissue positions across entire tissue sections. This thesis pioneers the exploration of these methods in discerning the enormous complexity underlying host-parasite interplay. In Paper I, we determine spatial components of naive mouse liver in its true tissue context. We define gene expression gradients of pericentral and periportal zones in the liver and predict vein types with ambiguous annotations, based on in situ transcriptional profiles. We further identify novel spatial structures with distinct transcriptional profiles, associated with tissue integrity and integrate cell type proportions across the tissue. In Paper II we i

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against dis

Toxoplasma gondii is a ubiquitous zoonotic parasite that can infect warm-blooded vertebrates, including humans. Felids, the definitive hosts, drive T. gondii infections by shedding the environmentally resistant stage of the parasite (oocysts) in their feces. Few studies characterize the role of climate and anthropogenic factors in oocyst shedding among free-ranging felids, which are responsible for the majority of environmental contamination. We determined how climate and anthropogenic factors influence oocyst shedding in free-ranging domestic cats and wild felids using generalized linear mixed models. T. gondii oocyst shedding data from 47 studies were systematically reviewed and compiled for domestic cats and six wild felid species, encompassing 256 positives out of 9,635 total fecal samples. Shedding prevalence in domestic cats and wild felids was positively associated with human population density at the sampling location. Larger mean diurnal temperature range was associated with more shedding among domestic cats and warmer temperature in the driest quarter was associated with lower oocyst shedding in wild felids. Increasing human population density and temperature fluctuation can exacerbate environmental contamination with the protozoan parasite T. gondii. Management of free-ranging domestic cats could lower the burden of environmental oocysts due to their large population sizes and affinity with human settlements.

Toxoplasma gondii is an intracellular protozoan parasite that infects nearly 1 in 3 individuals in the world. In immunocompromised individuals T. gondii infection can disseminate and cause serious disease and even death. Manifestations of the disease caused by T. gondii, toxoplasmosis, can include ocular lesions, vision loss, miscarriage, hydrocephaly, mental retardation, and even death. T. gondii is closely related to the malaria-causing parasite, Plasmodium sp., and both of which are without effective protective vaccines. For the development of an effective treatment or vaccine against parasitic infections, more about the battle between host and pathogen must be understood. What allows for some mouse strains to survive infections, while others succumb? What antigens on T. gondii elicit the strongest antibody response? What receptors and pathways must be triggered to provide protection? The glycosylphosphatidylinositol (GPI)-anchor is a highly conserved glycolipid that anchors proteins to the external membrane of T. gondii and is also found on all protozoan pathogens. Most of the major surface antigens (SAGs) of T. gondii are GPI-anchored proteins, and express GPI without attached proteins on their surface called GIPLs. The GPI is known to be recognized by innate pattern recognition receptors Toll-like Receptors (TLR) -2 and -4 and is robustly targeted by IgM antibodies after infection. While the glycan portion of the GPI consists of a conserved set of 3 mannose sugars, a GlcNAc, and an inositol, species differ in additional sugars or sidechains added to the mannose backbone. The functional significance of GPI sidechains has remained largely unknown because only a few GPI sidechain modifying enzymes have been identified. Since the GPI is such a potent antigen and in high abundance, we sought out to address the role of the GPI sidechain in parasite pathogenesis. We have successfully identified and knocked out both of the enzymes responsible for GPI sidechain modification in T. gondii. Named here PIGJ and PIGE, the former adds the first GalNAc sugar to the core mannose backbone of the GPI, while the latter adds a terminal Glc to the GalNAc as confirmed by mass spectrometry. Parasites lacking the entire GPI sidechain exhibit increased virulence during primary and secondary infections, demonstrating the GPI sidechain as an important pathogenesis factor. To elucidate the mechanism for increased virulence we characterized innate immune responses, parasite burden in various organs, early cytokine responses, antibody recognition, antigen expression, parasite growth and invasion of PIGJ mutant parasites that lack a GPI sidechain. Our findings reveal intact cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants. In contrast, the scavenger receptor CD36 shows enhanced binding to parasites that lack GPI sidechains, potentially explaining the tropism for macrophages observed early in infection. Galecitn-3, which binds to GIPL of T. gondii, exhibits enhanced binding to PIGJ mutants and infection of galectin-3 deficient mice results in their protection from lethal outcomes, suggesting the enhanced virulence of sidechain deficient parasites is galectin-3 dependent. Loss of the sidechain has no bearing on parasite numbers early in the infection, suggest a breakdown of tolerance is occurring, the exact mechanism for which is still unclear. In contrast, we see increased tissue cysts in the brains of PIGJ mutant infected mice, indicating some advantage over the wildtype is involved. Finally, vaccination of TLR2 or TLR4 deficient mice are susceptible to challenge with virulent T. gondii strains. Collectively, our data point to an important role for the GPI and its sidechain in host-parasite interactions, and that the presence of the sidechain prevents T. gondii pathogenesis promoting survival of its host.

Unicellular parasites of the apicomplexan phylum have a considerable effect on global health and agriculture. Two prominent examples of this phylum include malaria causing parasites of the Plasmodium genus and the widely prevalent parasite Toxoplasma gondii. While sharing a common ancestor, these parasites occupy unique biological niches, follow distinct life cycles, and result in different courses and outcomes of disease. In response to the parasite, the mammalian host has developed efficient and effective defense strategies. However, both Plasmodium and Toxoplasma have evolved strategies to evade the host’s defense response. Plasmodium parasites infect distinct tissues and cell types whereas T. gondii parasites are highly promiscuous and infect all nucleated cells. The identification of key factors involved in the interaction between the host and parasite is crucial for disease intervention, prevention, and eventually eradication efforts. Next-generation sequencing technologies have proven effective tools to investigate the response in a tissue or cell population of an infected organism. Novel genomics methods such as single-cell RNA-seq and spatial transcriptomics have enabled the investigation of heterogeneous transcriptional responses of individual cells in a population as well as heterogeneous expression profiles at spatially distinct tissue positions across entire tissue sections. This thesis pioneers the exploration of these methods in discerning the enormous complexity underlying host-parasite interplay. In Paper I, we determine spatial components of naive mouse liver in its true tissue context. We define gene expression gradients of pericentral and periportal zones in the liver and predict vein types with ambiguous annotations, based on in situ transcriptional profiles. We further identify novel spatial structures with distinct transcriptional profiles, associated with tissue integrity and integrate cell type proportions across the tissue. In Paper II we i

Unicellular parasites of the apicomplexan phylum have a considerable effect on global health and agriculture. Two prominent examples of this phylum include malaria causing parasites of the Plasmodium genus and the widely prevalent parasite Toxoplasma gondii. While sharing a common ancestor, these parasites occupy unique biological niches, follow distinct life cycles, and result in different courses and outcomes of disease. In response to the parasite, the mammalian host has developed efficient and effective defense strategies. However, both Plasmodium and Toxoplasma have evolved strategies to evade the host’s defense response. Plasmodium parasites infect distinct tissues and cell types whereas T. gondii parasites are highly promiscuous and infect all nucleated cells. The identification of key factors involved in the interaction between the host and parasite is crucial for disease intervention, prevention, and eventually eradication efforts. Next-generation sequencing technologies have proven effective tools to investigate the response in a tissue or cell population of an infected organism. Novel genomics methods such as single-cell RNA-seq and spatial transcriptomics have enabled the investigation of heterogeneous transcriptional responses of individual cells in a population as well as heterogeneous expression profiles at spatially distinct tissue positions across entire tissue sections. This thesis pioneers the exploration of these methods in discerning the enormous complexity underlying host-parasite interplay. In Paper I, we determine spatial components of naive mouse liver in its true tissue context. We define gene expression gradients of pericentral and periportal zones in the liver and predict vein types with ambiguous annotations, based on in situ transcriptional profiles. We further identify novel spatial structures with distinct transcriptional profiles, associated with tissue integrity and integrate cell type proportions across the tissue. In Paper II we i

Unicellular parasites of the apicomplexan phylum have a considerable effect on global health and agriculture. Two prominent examples of this phylum include malaria causing parasites of the Plasmodium genus and the widely prevalent parasite Toxoplasma gondii. While sharing a common ancestor, these parasites occupy unique biological niches, follow distinct life cycles, and result in different courses and outcomes of disease. In response to the parasite, the mammalian host has developed efficient and effective defense strategies. However, both Plasmodium and Toxoplasma have evolved strategies to evade the host’s defense response. Plasmodium parasites infect distinct tissues and cell types whereas T. gondii parasites are highly promiscuous and infect all nucleated cells. The identification of key factors involved in the interaction between the host and parasite is crucial for disease intervention, prevention, and eventually eradication efforts. Next-generation sequencing technologies have proven effective tools to investigate the response in a tissue or cell population of an infected organism. Novel genomics methods such as single-cell RNA-seq and spatial transcriptomics have enabled the investigation of heterogeneous transcriptional responses of individual cells in a population as well as heterogeneous expression profiles at spatially distinct tissue positions across entire tissue sections. This thesis pioneers the exploration of these methods in discerning the enormous complexity underlying host-parasite interplay. In Paper I, we determine spatial components of naive mouse liver in its true tissue context. We define gene expression gradients of pericentral and periportal zones in the liver and predict vein types with ambiguous annotations, based on in situ transcriptional profiles. We further identify novel spatial structures with distinct transcriptional profiles, associated with tissue integrity and integrate cell type proportions across the tissue. In Paper II we i

Parasites require a hospitable organism to reproduce and spread and have evolved multiple strategies to subvert their hosts. Parasites scavenge nutrients directly from host cells, evade the host immune system and even modify host behavior to increase their transmission. This course will explore the strategies used by a ubiquitous and harmful class of parasites to hijack the biology of their host cells. We will discuss pathogens such as Plasmodium and Toxoplasma, responsible for some of the deadliest and most pervasive infectious diseases on the planet.By exploring how these pathogens invade a host cell and replicate while evading the immune system, students will gain a broad understanding of basic cell biology, biochemistry and immunology, as well as learn techniques commonly used in cell biology. Students will be challenged to think creatively and flexibly to understand, critique, interpret, and design scientific experiments in the field of host-pathogen interactions.

Parasites require a hospitable organism to reproduce and spread and have evolved multiple strategies to subvert their hosts. Parasites scavenge nutrients directly from host cells, evade the host immune system and even modify host behavior to increase their transmission. This course will explore the strategies used by a ubiquitous and harmful class of parasites to hijack the biology of their host cells. We will discuss pathogens such as Plasmodium and Toxoplasma, responsible for some of the deadliest and most pervasive infectious diseases on the planet.By exploring how these pathogens invade a host cell and replicate while evading the immune system, students will gain a broad understanding of basic cell biology, biochemistry and immunology, as well as learn techniques commonly used in cell biology. Students will be challenged to think creatively and flexibly to understand, critique, interpret, and design scientific experiments in the field of host-pathogen interactions.

Objective: in this work, the level of knowledge about toxoplasmosis and its risk factors in pregnant women who attend prenatal care was investigated because the low level of knowledge of this infection can be directly related to the increase in its incidence due to poor perception of the population regarding the risk and consequences in the future.Methodology: a random sample of 93 pregnant women who were taking their prenatal check-ups at a Clinic in Barranquilla, during the second trimester of 2021, was adminis-tered a survey with binary questions related to the understanding about toxoplasmosis and risk factors for contracting the infection. The survey was divided into two part: one part before and the other, after an informative talk about Toxoplasmosis, risk factors, health consequence and prevention methods. The clinical history, prenatal controls and socioeconomic level of the participant were taken into account.Results: it was found that 64.5 % of pregnant women did not know what Toxoplasmosis is and 66.6 % the risk factors and prevention methods.Conclusions: the level of knowledge about Toxoplasma gondii infection found in the pregnant women who attend prenatal control at the San Diego General Clinic, during the study period, is low.infección por Toxoplasma gondii; zoonosis; toxoplasmosis congénita, Objetivo: en este estudio, se investigó el nivel de conocimiento acerca de la toxoplasmosis y sus factores de riesgo en mujeres gestantes que asisten a control prenatal. Esto se debió a que el bajo nivel de conocimiento sobre esta infección podría relacionarse directamente con un aumento en su incidencia, debido a la pobre percepción de la población respecto a los riesgos y consecuencias a futuro. Metodología: se aplicó una encuesta aleatoria a 93 mujeres embarazadas que estaban realizando sus controles prenatales en una Clínica de Barranquilla, durante el segundo trimestre de 2021. La encuesta consistió en preguntas de tipo binario relacionadas con el entendimiento sobre la toxoplasmosis y los factores de riesgo para contraer la infección. La encuesta se dividió en dos partes: una antes y otra después de una charla informativa sobre la toxoplasmosis, sus factores de riesgo, las consecuencias para la salud y los métodos de prevención. Además, se tuvieron en cuenta los antecedentes clínicos, los controles prenatales y el nivel socioeconómico de las participantes. Resultados: se encontró que el 64,5 % de las embarazadas desconocía qué es la toxoplasmosis, y el 66,6 % desconocía los factores de riesgo y los métodos de prevención. Conclusiones: el nivel de conocimiento sobre la infección causada por el Toxoplasma gondii que se encontró en las gestantes que asistieron a control prenatal en la Clínica General San Diego durante el período de estudio es bajo.

The gene RARRES3 uses an unexpected strategy to eliminate the parasite Toxoplasma gondii from human cells.

Toxoplasma gondii (T. gondii) is one of the most prolific parasites in the world where it is estimated that 30% of the human population is infected. The encystment of the parasite in CNS-neurons results in a lifelong chronic infection. Within the brain, a pro-inflammatory response is essential to prevent parasite reactivation, however, an anti-inflammatory response is also required to prevent inflammation-induced mortality. This suggests a balanced immune response to the pathogen. Infection in the immunocompetent experience asymptomatic infection, further suggesting a tightly regulated immune response in the CNS. However, it is unclear what are the signals or cell-mediators that regulate this delicately balanced immune response. In addition, parasite recrudescence leads to lethal toxoplasmic encephalitis if left untreated in immunocompromised individuals. However, it is unclear what developmental pathways the parasite undergoes upon reawakening from its dormant state. This dissertation investigates these unknowns in the follow ways:Chapter 1 is an introduction to T. gondii infection and the effects the parasite has on the host. Topics covered in this section include the life cycle and the immune response to T. gondii. This encompasses evasion strategies by the parasite, and signals that are required for protective immune cells to traverse the blood brain barrier. This chapter ends on describing the role astrocytes play in the CNS, and the implications of T. gondii infection on glutamate regulation.Chapter 2 describes the attempts that have been made to study recrudescence of tissue cysts. This chapter poses a new ex vivo model of bradyzoite-initiated development that better preserves the key attributes of the parasite’s intermediate life cycle. In chapter 3, we discuss elements found in the CNS-microenvironment during chronic T. gondii infection, and consider how tissue-derived signals, such as high extracellular glutamate, influence protective CD8 T cells. Chapter

The gene RARRES3 uses an unexpected strategy to eliminate the parasite Toxoplasma gondii from human cells.

The phylum Apicomplexa contains several parasitic species of medical and agricultural importance, including Plasmodium falciparum and Toxoplasma gondii. Apicomplexan parasites have complex life cycles involving several morphological stages across multiple hosts, and rely on extensive signaling networks to sense their environment, transduce external signals and coordinate appropriate responses. Currently, there are few specific and effective therapeutics to treat parasitic infections and great efforts have been made to understand the signaling pathways and downstream post-translational modifications that regulate crucial parasite life cycle transitions. In the current literature (reviewed in Chapter 1), phosphorylation and the upstream pathways that mediate this post-translational modification is well described in apicomplexan parasites, while ubiquitination is less well understood due to a lack of extensive characterisation of the ubiquitination machinery. Intracellular Ca2+ signaling and the downstream activity of Ca2+-dependent protein kinases (CDPKs) is paramount to parasite motility, and therefore facilitates host cell invasion and egress. In recent years the function of cyclic nucleotide signaling in regulation of Ca2+ mobilisation and motility has come to light. cGMP signaling and its downstream effector kinase, protein kinase G (PKG) is crucial for activating parasite motility, while cAMP signaling via protein kinase A (PKA) has been shown to be a negative regulator of motility in Toxoplasma tachyzoites. PKA has been implicated in Plasmodium merozoite invasion as the kinase responsible for phosphorylation of the key invasion ligand apical membrane antigen 1 (AMA1), however it is unclear how Plasmodium PKA functions in motility. Chapter 3 explores the function of PKA in Plasmodium falciparum through conditional knockdown of the PKA catalytic subunit (PfPKAc) in asexual stage parasites. I show this kinase to be essential in merozoite invasion of erythrocytes, a

Toxoplasmosis is a worldwide disease affecting all warm-blooded animals, including humans. Vaccination strategies aimed at inducing an efficient immune response while preventing transmission have been attempted in the past. While many different approaches can partially protect immunized animals against subsequent infections, full and lasting protection is rarely attained and only with live-attenuated vaccines. In addition, vaccines based on mutant strains that are deficient in forming the chronic phase of the parasite (such as Toxovax™) cannot be extensively used due to their zoonotic potential and the possibility of reversion to virulent phenotypes. An increasing number of studies using emerging genetic-engineering tools have been conducted to design novel vaccines based on recombinant proteins, DNA or delivery systems such as nanoparticles. However, these are usually less efficient due to their antigenic simplicity. In this perspective article we discuss potential target genes and novel strategies to generate live-attenuated long-lasting vaccines based on tissue cysts and oocysts, which are the environmentally resistant chronic forms of Toxoplasma. By selectively disrupting genes important for parasite dissemination, cyst formation and/or sporozoite invasion, alone or in combination, a vaccine based on a live-attenuated strain that elicits a protective immune response while preventing the transmission of Toxoplasma could be created. Finally, further improvements of protocols to generate Toxoplasma sexual stages in vitro might lead to the production of oocysts from such a strain without the need for using mice or cats.

The obligate intracellular parasite Toxoplasma gondii makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has remained unresolved. Here, we addressed the effects of T. gondii infection on the trans-endothelial migration (TEM) of dendritic cells (DCs) across polarised brain endothelial monolayers. We report that upregulated expression of leukocyte ICAM-1 is a feature of the enhanced TEM of parasitised DCs. The secreted parasite effector GRA15 induced an elevated expression of ICAM-1 in infected DCs that was associated with enhanced cell adhesion and TEM. Consequently, gene silencing of Icam-1 in primary DCs or deletion of parasite GRA15 reduced TEM. Further, the parasite effector TgWIP, which impacts the regulation of host actin dynamics, facilitated TEM across polarised endothelium. The data highlight that the concerted action of the secreted effectors GRA15 and TgWIP modulate the leukocyte-endothelial interactions of TEM in a parasite genotype-related fashion to promote dissemination. In addition to the canonical roles of endothelial ICAM-1, this study identifies a previously unappreciated role for leukocyte ICAM-1 in infection-related TEM.

Upon pathogen detection, macrophages normally stay sessile in tissues while dendritic cells (DCs) migrate to secondary lymphoid tissues. The obligate intracellular protozoan Toxoplasma gondii exploits the trafficking of mononuclear phagocytes for dissemination via unclear mechanisms. We report that, upon T. gondii infection, macrophages initiate the expression of transcription factors normally attributed to DCs, upregulate CCR7 expression with a chemotactic response, and perform systemic migration when adoptively transferred into mice. We show that parasite effector GRA28, released by the MYR1 secretory pathway, cooperates with host chromatin remodelers in the host cell nucleus to drive the chemotactic migration of parasitized macrophages. During in vivo challenge studies, bone marrow-derived macrophages infected with wild-type T. gondii outcompeted those challenged with MYR1- or GRA28-deficient strains in migrating and reaching secondary organs. This work reveals how an intracellular parasite hijacks chemotaxis in phagocytes and highlights a remarkable migratory plasticity in differentiated cells of the mononuclear phagocyte system.

This work is intended to provide basic information and numerical experimentation of biological models focusing on how computation can be used to generate results via software R. In addition, this work enriches the scientific literature in Spanish that links mathematics and computational biology. It also provides detailed information on the analysis of Predator-prey, Toxoplasma gondii, and Common influency models. For the development of this article we will speak in the first instance of the dynamics of the predator-prey model. In the last two models, it is solved numerically for a range of values of a given parameter. This in order to show deductions that contribute to a deeper investigation of the data involved, and even to the analysis of a professional specialized in the model., Este trabajo esta destinado a proporcionar información básica y experimentación numérica de modelos biológicos centrándose en cómo la computación se puede utilizar para generar resultados a través del software R. Además, este trabajo enriquece la literatura científica en español que vincula las matemáticas y la biología computacional. También proporciona información detallada sobre el análisis de los modelos depredador-presa, toxoplasma gondii e influencia común. Para el desarrollo de este artículo hablaremos en primera instancia de la dinámica del modelo depredador-presa. En los dos últimos modelos, se resuelve numéricamente para un rango de valores de un parámetro dado. Esto con el fin de mostrar deducciones que contribuyan a una investigación más profunda de los datos involucrados, e incluso al análisis de un profesional especializado en el modelo.

Background: Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1. Methods: One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1. Results: Detection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg of T. gondii genomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection of Toxoplasma gondii DNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1. Conclusions: Data from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis o

In humans, clinically relevant disease involving Toxoplasma gondii, an obligate intracellular parasite, results from T. gondii’s life-long persistence in the central nervous system (CNS). Successful T. gondii persistence is driven by the ability of the parasite to evade the immune response during acute infection and disseminate throughout the body. Once the parasite has disseminated to the CNS it switches from a fast-replicating form, the tachyzoite, to a slow-growing encysted form, the bradyzoite. Additionally, an emerging body of studies indicates that the genotype of the infecting T. gondii strain can influence CNS disease outcomes. These differences are thought to be driven by polymorphic effector proteins that can modulate different immune responses in a strain-specific manner. The work in this dissertation will address the role of one such polymorphic effector protein, rhoptry protein 16 (ROP16). Most work on ROP16 has been done in a hypervirulent strain of T. gondii where it has little effect on acute virulence in mice. In addition, the hypervirulent nature of the strain means studies on chronic infection cannot be done. Recent work revealed a role for ROP16 in a less virulent strain during in vivo infection. To further address the role of ROP16 in this less virulent strain in both tachyzoites and bradyzoites, we used a panel of ROP16 mutants and infected cells cultured in both tachyzoite and bradyzoite growth conditions. We have found that during tachyzoite growth ROP16 facilitates parasite growth and survival in a strain-specific manner by reducing host cell reactive oxygen species (ROS) through the activation of a host cell transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). We also observed that under bradyzoite-inducing conditions ROP16 facilitates encystment through the activation of STAT6 in vitro and that, compared to wild-type mice, STAT6 knockout (STAT6KO) mice have fewer CNS cysts despite having similar parasite burdens

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient tools for genetic manipulation. To enable efficient homologous recombination in N. caninum, we targeted the Ku heterodimer DNA repair mechanism in the genomic reference strain, Nc-Liverpool (NcLiv), and show that deletion of Ku80 results in a destabilization and loss of its partner Ku70. Disruption of Ku80 generated parasites in which genes are efficiently epitope tagged and only short homology regions are required for gene knockouts. We used this improved strain to target novel nonessential genes encoding dense granule proteins that are unique to N. caninum or conserved in T. gondii. To expand the utility of this strain for essential genes, we developed the auxin-inducible degron system for N. caninum using parasite-specific promoters. As a proof of concept, we knocked down a novel nuclear factor in both N. caninum and T. gondii and showed that it is essential for survival of both parasites. Together, these efficient knockout and knockdown technologies will enable the field to unravel specific gene functions in N. caninum, which is likely to aid in the identification of targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. IMPORTANCE Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii. To enable this comparison, it is important to develop efficient molecular tools for N. c

Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.

The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites.IMPORTANCE The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the Toxoplasma gondii IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of dr

Toxoplasma gondii is an obligatory intracellular pathogen that causes life threatening illness in immunodeficient individuals, miscarriage in pregnant woman, and blindness in newborn children. Similar to any other eukaryotic cell, protein kinases play critical and essential roles in the Toxoplasma life cycle. Accordingly, many studies have focused on identifying and defining the mechanism of function of these signalling proteins with a long-term goal to develop anti-Toxoplasma therapeutics. In this review, we briefly discuss classification and key components of the catalytic domain which are critical for functioning of kinases, with a focus on domains, families, and groups of kinases within Toxoplasma. More importantly, this article provides a comprehensive, current overview of research on kinase groups in Toxoplasma including the established eukaryotic AGC, CAMK, CK1, CMGC, STE, TKL families and the apicomplexan-specific FIKK, ROPK and WNG family of kinases. This work provides an overview and discusses current knowledge on Toxoplasma kinases including their localization, function, signalling network and role in acute and chronic pathogenesis, with a view towards the future in probing kinases as viable drug targets.

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.

The virulence of eukaryotic parasites like Toxoplasma gondii depends on their capacity to escape from the host immune response and disseminate throughout the host organism. However, Toxoplasma gene products essential for its in vivo pathogenesis remain uncharacterized. Here, we present the complete workflow of a CRISPR-Cas9 in vivo loss-of-function screen to identify Toxoplasma fitness-conferring genes. This protocol can be used to uncover gene products that play a role in Toxoplasma immune evasion, nutrient acquisition, dissemination, and tissue colonization. For complete details on the use and execution of this protocol, please refer to Sangaré et al. (2019).

The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites.IMPORTANCE The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the Toxoplasma gondii IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of dr

Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.

Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.

Toxoplasma gondii is a neurotropic protozoan parasite, which is linked to neurological manifestations in immunocompromised individuals as well as severe neurodevelopmental sequelae in congenital toxoplasmosis. While the complement system is the first line of host defense that plays a significant role in the prevention of parasite dissemination, Toxoplasma artfully evades complement-mediated clearance via recruiting complement regulatory proteins to their surface. On the other hand, the details of Toxoplasma and the complement system interaction in the brain parenchyma remain elusive. In this study, infection-induced changes in the mRNA levels of complement components were analyzed by quantitative PCR using a murine Toxoplasma infection model in vivo and primary glial cells in vitro. In addition to the core components C3 and C1q, anaphylatoxin C3a and C5a receptors (C3aR and C5aR1), as well as alternative complement pathway components properdin (CFP) and factor B (CFB), were significantly upregulated 2 weeks after inoculation. Two months post-infection, CFB, C3, C3aR, and C5aR1 expression remained higher than in controls, while CFP upregulation was transient. Furthermore, Toxoplasma infection induced significant increase in CFP, CFB, C3, and C5aR1 in mixed glial culture, which was abrogated when microglial activation was inhibited by pre-treatment with minocycline. This study sheds new light on the roles for the complement system in the brain parenchyma during Toxoplasma infection, which may lead to the development of novel therapeutic approaches to Toxoplasma infection-induced neurological disorders., Frontiers in Immunology, 11, art.no.603924; 2021

Traditionally used as a broad-spectrum antibiotic, doxycycline is frequently used for malaria prophylaxis and treatment - the latter in combination with artemisinin derivatives. Its mechanism of action in Plasmodium spp. has not yet been fully elucidated, though there is substantial evidence that ribosomes in the apicoplast - a relict plastid - are the primary target, with doxycycline causing delayed death (a phenotype associated with inhibitors of apicoplast housekeeping). Inhibition of the apicoplast depletes isoprenoids, synthesised via a pathway housed in the organelle, perturbing the prenyl-dependent trafficking mechanism for haemoglobin uptake and trafficking. This same uptake of haemoglobin is required for activation of artemisinin derivatives. Here, we show that apicoplast-targeting antibiotics, such as doxycycline, reduce the abundance of the catalyst of artemisinin activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between dihydroartemisinin and these antibiotics, likely because apicoplast inhibitors reduce artemisinin activation. Separately, we identify a secondary, more immediate target of doxycycline that exists at clinically relevant concentrations. We show that supplementation with the apicoplast-derived isoprenoid precursor, isopentenyl pyrophosphate, only rescues parasites from delayed death, demonstrating independence of the first cycle target from the relict plastid. Instead, we show that doxycycline depletes mitochondrial electron transport and selectively reduces the abundance of proteins encoded by the mitochondrion in the related apicomplexan parasite, Toxoplasma gondii, suggesting that inhibition of mitochondrial protein synthesis could underpin the immediate death phenotype caused by doxycycline. These data have potential clinical significance when considering the reliance on - and widespread use of - doxycycline and other apicoplast-targeting antibiotics in malaria endemic reg

Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.

Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.

Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.

The severity of toxoplasmosis depends on a combination of host and parasite factors. Among them, the Toxoplasma strain causing the infection is an important determinant of the disease outcome. Type 2 strains dominate in Europe, whereas in North America type 2, followed by type 3 and 12 strains are commonly isolated from wildlife and patients. To identify the strain type a person is infected with, serological typing provides a promising alternative to the often risky and not always possible biopsy-based DNA methods of genotyping. However, despite recent advances in serotyping, improvements in the sensitivity and specificity are still needed, and it does not yet discriminate among the major Toxoplasma lineages infecting people. Moreover, since infections caused by non-1/2/3 strains have been associated with more severe disease, the ability to identify these is critical. In the present study we investigated the diagnostic potential of an ELISA-based assay using 28 immunogenic Toxoplasma peptides derived from a recent large-scale peptide array screen. Our results show that a discrete number of peptides, derived from Toxoplasma dense granule proteins (GRA3, GRA5, GRA6, and GRA7) was sufficient to discriminate among archetypal strains that infect mice and humans. The assay specifically relies on ratios that compare individual serum reactivities against GRA-specific polymorphic peptide variants in order to determine a "reactivity fingerprint" for each of the major strains. Importantly, nonarchetypal strains that possess a unique combination of alleles, different from types 1/2/3, showed either a non-reactive, or different combinatorial, mixed serum reactivity signature that was diagnostic in its own right, and that can be used to identify these strains. Of note, we identified a distinct "HG11/12" reactivity pattern using the GRA6 peptides that is able to distinguish HG11/12 from archetypal North American/European strain infections.

Toxoplasma gondii is a useful model for intracellular parasitism given its ease of culture in the laboratory and genomic resources. However, as for many other eukaryotes, the T. gondii genome contains hundreds of sequence gaps owing to repetitive and/or unclonable sequences that disrupt the assembly process. Here, we use the Oxford Nanopore Minion platform to generate near-complete de novo genome assemblies for multiple strains of T. gondii and its near relative, N. caninum We significantly improved T. gondii genome contiguity (average N50 of ∼6.6 Mb) and added ∼2 Mb of newly assembled sequence. For all of the T. gondii strains that we sequenced (RH, ME49, CTG, II×III progeny clones CL13, S27, S21, S26, and D3X1), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome, and found to be due to a consistent fusion of Chromosomes VIIb and VIII. These data were validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. By using this technology, we also resolved hundreds of tandem repeats of varying lengths, including in well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38 Finally, when we compared T. gondii with N. caninum, we found that although the 13-chromosome karyotype was conserved, extensive, previously unappreciated chromosome-scale rearrangements had occurred in T. gondii and N. caninum since their most recent common ancestry.

BackgroundClinically useful predictors for fatal toxoplasmosis are lacking. We investigated the value of serological assays for antibodies to whole Toxoplasma antigens and to peptide antigens of the Toxoplasma cyst matrix antigen 1 (MAG1), for predicting incident toxoplasmic encephalitis (TE) in people living with human immunodeficiency virus (HIV; PLWH).MethodsWe performed a nested case control study, conducted within the Multicenter AIDS Cohort Study (MACS), using serum samples obtained 2 years prior to diagnosis of TE from 28 cases, and 37 HIV disease-matched Toxoplasma seropositive controls at matched time-points. Sera were tested for Toxoplasma antibodies using a commercial assay and for antibodies to MAG1_4.2 and MAG1_5.2 peptides in enzyme-linked immunosorbent assay (ELISA).ResultsTwo years prior to clinical diagnosis, 68% of TE cases were MAG1_4.2 seropositive compared with 16% of controls (odds ratio [OR] 25.0, 95% confidence interval [CI] 3.14-199.18). Corresponding results for MAG1_5.2 seropositivity were 36% and 14% (OR 3.6, 95% CI .95-13.42). Higher levels of antibody to MAG1_4.2 (OR 18.5 per doubling of the optical density [OD] value, 95% CI 1.41-242) and to Toxoplasma (OR 2.91 for each OD unit increase, 95% CI 1.48-5.72) were also associated with the risk of TE. When seropositivity was defined as the presence of MAG1 antibody or relatively high levels of Toxoplasma antibody, the sensitivity was 89% and specificity was 68% for subsequent TE.ConclusionsAntibodies to MAG1 showed predictive value on the occurrence of TE in PLWH, and the predictive performance was further improved by adding the levels of Toxoplasma antibody. These measures could be clinically useful for predicting subsequent diseases in multiple at-risk populations.

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection.

Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.

Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.