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Theodor Hiepe (1929-2022) was an outstanding researcher, a world-renowned scientist, a dedicated teacher and a great mentor. During his scientific career, which spanned over 60 years, he made major contributions to many different fields of parasitology. With the passing of Dr. h.c. mult. Theodor Hiepe in September 2022 the scientific community suffered a great loss.
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Successful asexual reproduction of intracellular pathogens depends on their potential to exploit host resources and subvert antimicrobial defense. In this work, we deployed two prevalent apicomplexan parasites of mammalian cells, namely Toxoplasma gondii and Eimeria falciformis, to identify potential host determinants of infection. Expression analyses of the young adult mouse colonic (YAMC) epithelial cells upon infection by either parasite showed regulation of several distinct transcripts, indicating that these two pathogens program their intracellular niches in a tailored manner. Conversely, parasitized mouse embryonic fibroblasts (MEFs) displayed a divergent transcriptome compared to corresponding YAMC epithelial cells, suggesting that individual host cells mount a fairly discrete response when encountering a particular pathogen. Among several host transcripts similarly altered by T. gondii and E. falciformis, we identified cFos, a master transcription factor, that was consistently induced throughout the infection. Indeed, asexual growth of both parasites was strongly impaired in MEF host cells lacking cFos expression. Last but not the least, our differential transcriptomics of the infected MEFs (parental and cFos-/- mutant) and YAMC epithelial cells disclosed a cFos-centered network, underlying signal cascades, as well as a repertoire of nucleotides- and ion-binding proteins, which presumably act in consort to acclimatize the mammalian cell and thereby facilitate the parasite development.
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In fracture healing, skeletal and immune system are closely interacting through common cell precursors and molecular mediators. It is thought that the initial inflammatory reaction, which involves migration of macrophages into the fracture area, has a major impact on the long term outcome of bone repair. Interestingly, macrophages reside during all stages of fracture healing. Thus, we hypothesized a critical role for macrophages in the subsequent phases of bone regeneration. This study examined the impact of in vivo induced macrophage reduction, using clodronate liposomes, on the different healing phases of bone repair in a murine model of a standard closed femoral fracture. A reduction in macrophages had no obvious effect on the early fracture healing phase, but resulted in a delayed hard callus formation, thus severely altering endochondral ossification. Clodronate treated animals clearly showed delayed bony consolidation of cartilage and enhanced periosteal bone formation. Therefore, we decided to backtrack macrophage distribution during fracture healing in non-treated mice, focusing on the identification of the M1 and M2 subsets. We observed that M2 macrophages were clearly prevalent during the ossification phase. Therefore enhancement of M2 phenotype in macrophages was investigated as a way to further bone healing. Induction of M2 macrophages through interleukin 4 and 13 significantly enhanced bone formation during the 3week investigation period. These cumulative data illustrate their so far unreported highly important role in endochondral ossification and the necessity of a fine balance in M1/M2 macrophage function, which appears mandatory to fracture healing and successful regeneration.
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Callo Óseo/metabolismo , Curación de Fractura/fisiología , Fracturas Cerradas/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiología , Osteogénesis/fisiología , Cicatrización de Heridas/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Parasites can either respond to differences in immune defenses that exist between individual hosts plastically or, alternatively, follow a genetically canalized ("hard wired") program of infection. Assuming that large-scale functional plasticity would be discernible in the parasite transcriptome we have performed a dual RNA-seq study of the lifecycle of Eimeria falciformis using infected mice with different immune status as models for coccidian infections. RESULTS: We compared parasite and host transcriptomes (dual transcriptome) between naïve and challenge infected mice, as well as between immune competent and immune deficient ones. Mice with different immune competence show transcriptional differences as well as differences in parasite reproduction (oocyst shedding). Broad gene categories represented by differently abundant host genes indicate enrichments for immune reaction and tissue repair functions. More specifically, TGF-beta, EGF, TNF and IL-1 and IL-6 are examples of functional annotations represented differently depending on host immune status. Much in contrast, parasite transcriptomes were neither different between Coccidia isolated from immune competent and immune deficient mice, nor between those harvested from naïve and challenge infected mice. Instead, parasite transcriptomes have distinct profiles early and late in infection, characterized largely by biosynthesis or motility associated functional gene groups, respectively. Extracellular sporozoite and oocyst stages showed distinct transcriptional profiles and sporozoite transcriptomes were found enriched for species specific genes and likely pathogenicity factors. CONCLUSION: We propose that the niche and host-specific parasite E. falciformis uses a genetically canalized program of infection. This program is likely fixed in an evolutionary process rather than employing phenotypic plasticity to interact with its host. This in turn might limit the potential of the parasite to adapt to new host species or niches, forcing it to coevolve with its host.
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Coccidios/inmunología , Coccidios/parasitología , Eimeria/genética , Eimeria/fisiología , Interacciones Huésped-Parásitos , Análisis de Secuencia de ARN , Eimeria/crecimiento & desarrollo , Evolución Molecular , Perfilación de la Expresión Génica , Reproducción Asexuada/genética , Esporozoítos/genéticaRESUMEN
OBJECTIVE: The aim of this study was to examine whether the natural protease inhibitor Av-cystatin (rAv17) of the parasitic nematode Acanthocheilonema viteae exerts anti-inflammatory effects in an in vitro model of lipopolysaccharide (LPS)-activated microglia. METHODS: Primary microglia were harvested from the brains of 2-day-old Wistar rats and cultured with or without rAv17 (250 nM). After 6 and 24 h the release of nitric oxide (Griess reagent) and TNF-α (ELISA) was measured in the supernatant. Real-time PCR was performed after 2, 6 and 24 h of culture to measure the mRNA expression of IL-1ß, IL-6, TNF-α, COX-2, iNOS and IL-10. To address the involved signaling pathways, nuclear NF-x0138;B translocation was visualized by immunocytochemistry. Morphological changes of microglia were analyzed by Coomassie blue staining. Differences between groups were calculated using one-way ANOVA with Bonferroni's post hoc test. RESULTS: Morphological analysis indicated that LPS-induced microglial transformation towards an amoeboid morphology is inhibited by rAv17. Av-cystatin caused a time-dependent downregulation of proinflammatory cytokines, iNOS and COX-2 mRNA expression, respectively. This was paralleled by an upregulated expression of IL-10 in resting as well as in LPS-stimulated microglia. Av-cystatin reduced the release of NO and TNF-α in the culture supernatant. Immunocytochemical staining demonstrated an attenuated translocation of NF-x0138;B by Av-cystatin in response to LPS. In addition, Western blot analysis revealed a rAv17-dependent reduction of the LPS-induced ERK1/2-pathway activation. CONCLUSION: The parasite-derived secretion product Av-cystatin inhibits proinflammatory mechanisms of LPS-induced microglia with IL-10, a potential key mediator.
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Acanthocheilonema , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Microglía/metabolismo , Fenotipo , Inhibidores de Proteasas/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Helmintos , Mediadores de Inflamación/antagonistas & inhibidores , Microglía/efectos de los fármacos , Inhibidores de Proteasas/aislamiento & purificación , Ratas , Ratas WistarRESUMEN
Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.
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Glucosa/metabolismo , Glutamina/metabolismo , Estadios del Ciclo de Vida , Parásitos/crecimiento & desarrollo , Parásitos/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasma/metabolismo , Acetatos/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Biomasa , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Carbono/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Metabolismo Energético/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Glucólisis/efectos de los fármacos , Humanos , Espacio Intracelular/parasitología , Estadios del Ciclo de Vida/efectos de los fármacos , Lípidos/química , Masculino , Modelos Biológicos , Mutación/genética , Fosforilación Oxidativa/efectos de los fármacos , Parásitos/efectos de los fármacos , Fenotipo , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Toxoplasma/efectos de los fármacos , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitologíaRESUMEN
The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.
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Retículo Endoplásmico/enzimología , Glicerofosfolípidos/metabolismo , Proteínas Protozoarias/metabolismo , Treonina/análogos & derivados , Toxoplasma/fisiología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/parasitología , Encéfalo/patología , Células Cultivadas , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Organismos Modificados Genéticamente/inmunología , Organismos Modificados Genéticamente/metabolismo , Enquistamiento de Parásito , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Piel/citología , Piel/inmunología , Piel/metabolismo , Piel/parasitología , Treonina/metabolismo , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Toxoplasmosis/prevención & control , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Vacunas Atenuadas/uso terapéutico , VirulenciaRESUMEN
Immunomodulation is a common feature of chronic helminth infections and mainly attributed to the secretion of bioactive molecules, which target and modify host immune cells. In this study, we show that the helminth immunomodulator AvCystatin, a cysteine protease inhibitor, induces a novel regulatory macrophage (Mreg; AvCystatin-Mreg), which is sufficient to mitigate major parameters of allergic airway inflammation and colitis in mice. A single adoptive transfer of AvCystatin-Mreg before allergen challenge suppressed allergen-specific IgE levels, the influx of eosinophils into the airways, local and systemic Th2 cytokine levels, and mucus production in lung bronchioles of mice, whereas increasing local and systemic IL-10 production by CD4(+) T cells. Moreover, a single administration of AvCystatin-Mreg during experimentally induced colitis strikingly reduced intestinal pathology. Phenotyping of AvCystatin-Mreg revealed increased expression of a distinct group of genes including LIGHT, sphingosine kinase 1, CCL1, arginase-1, and costimulatory molecules, CD16/32, ICAM-1, as well as PD-L1 and PD-L2. In cocultures with dendritic cells and CD4(+) T cells, AvCystatin-Mreg strongly induced the production of IL-10 in a cell-contact-independent manner. Collectively, our data identify a specific suppressive macrophage population induced by a single parasite immunomodulator, which protects against mucosal inflammation.
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Antígenos Helmínticos/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunidad Mucosa/inmunología , Inflamación/prevención & control , Macrófagos/inmunología , Acanthocheilonema/inmunología , Traslado Adoptivo , Animales , Antígenos Helmínticos/farmacología , Colitis/inmunología , Colitis/prevención & control , Modelos Animales de Enfermedad , Femenino , Inmunosupresores/inmunología , Inmunosupresores/farmacología , Inflamación/inmunología , Interleucina-10/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neumonía/inmunología , Neumonía/prevención & controlRESUMEN
BACKGROUND: Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive. AIM: To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses. METHODOLOGY AND PRINCIPAL FINDINGS: Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner. CONCLUSIONS AND SIGNIFICANCE: Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.
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Brugia Malayi/inmunología , Filariasis Linfática/inmunología , Interleucina-10/inmunología , Microfilarias/inmunología , Wuchereria bancrofti/inmunología , Inmunidad Adaptativa/inmunología , Adolescente , Adulto , Anciano , Esclerosis Amiotrófica Lateral , Animales , Linfocitos T CD4-Positivos/inmunología , Femenino , Humanos , Inmunidad Innata/inmunología , Interleucina-13/genética , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Fenotipo , Adulto JovenRESUMEN
BACKGROUND: The phylum Apicomplexa comprises important unicellular human parasites such as Toxoplasma and Plasmodium. Eimeria is the largest and most diverse genus of apicomplexan parasites and some species of the genus are the causative agent of coccidiosis, a disease economically devastating in poultry. We report a complete genome sequence of the mouse parasite Eimeria falciformis. We assembled and annotated the genome sequence to study host-parasite interactions in this understudied genus in a model organism host. RESULTS: The genome of E. falciformis is 44 Mb in size and contains 5,879 predicted protein coding genes. Comparative analysis of E. falciformis with Toxoplasma gondii shows an emergence and diversification of gene families associated with motility and invasion mainly at the level of the Coccidia. Many rhoptry kinases, among them important virulence factors in T. gondii, are absent from the E. falciformis genome. Surface antigens are divergent between Eimeria species. Comparisons with T. gondii showed differences between genes involved in metabolism, N-glycan and GPI-anchor synthesis. E. falciformis possesses a reduced set of transmembrane transporters and we suggest an altered mode of iron uptake in the genus Eimeria. CONCLUSIONS: Reduced diversity of genes required for host-parasite interaction and transmembrane transport allow hypotheses on host adaptation and specialization of a single host parasite. The E. falciformis genome sequence sheds light on the evolution of the Coccidia and helps to identify determinants of host-parasite interaction critical for drug and vaccine development.
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Eimeria/genética , Genoma de Protozoos , Interacciones Huésped-Parásitos/genética , Parásitos/genética , Animales , Antígenos de Superficie/metabolismo , Composición de Base/genética , Secuencia de Bases , Mapeo Cromosómico , Análisis por Conglomerados , Coccidiosis/parasitología , Eimeria/crecimiento & desarrollo , Eimeria/metabolismo , Femenino , Ontología de Genes , Tamaño del Genoma , Humanos , Estadios del Ciclo de Vida , Ratones , Anotación de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta/genética , Parásitos/crecimiento & desarrollo , Parásitos/metabolismo , Filogenia , Proteoma/metabolismoRESUMEN
Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (â¼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.
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Carboxiliasas/metabolismo , Mitocondrias/metabolismo , Fosfatidiletanolaminas/biosíntesis , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Carboxiliasas/clasificación , Carboxiliasas/genética , Supervivencia Celular , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Diglicéridos/metabolismo , Etanolaminas/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
Intracellular parasites reprogram host functions for their survival and reproduction. The extent and relevance of parasite-mediated host responses in vivo remains poorly studied, however. We utilized Eimeria falciformis, a parasite infecting the mouse intestinal epithelium, to identify and validate host determinants of parasite infection. Most prominent mouse genes induced during the onset of asexual and sexual growth of parasite comprise interferon γ (IFNγ)-regulated factors, e.g., immunity-related GTPases (IRGA6/B6/D/M2/M3), guanylate-binding proteins (GBP2/3/5/6/8), chemokines (CxCL9-11), and several enzymes of the kynurenine pathway including indoleamine 2,3-dioxygenase 1 (IDO1). These results indicated a multifarious innate defense (tryptophan catabolism, IRG, GBP, and chemokine signaling), and a consequential adaptive immune response (chemokine-cytokine signaling and lymphocyte recruitment). The inflammation- and immunity-associated transcripts were increased during the course of infection, following influx of B cells, T cells, and macrophages to the parasitized caecum tissue. Consistently, parasite growth was enhanced in animals inhibited for CxCr3, a major receptor for CxCL9-11 present on immune cells. Interestingly, despite a prominent induction, mouse IRGB6 failed to bind and disrupt the parasitophorous vacuole, implying an immune evasion by E. falciformis. Furthermore, oocyst output was impaired in IFNγ-R(-/-) and IDO1(-/-) mice, both of which suggest a subversion of IFNγ signaling by the parasite to promote its growth.
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Ciego/metabolismo , Ciego/parasitología , Coccidiosis/metabolismo , Coccidiosis/parasitología , Eimeria , Interferón gamma/metabolismo , Transducción de Señal , Animales , Análisis por Conglomerados , Coccidiosis/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/metabolismo , Receptores CXCR3/metabolismo , Receptores de Interferón/genética , Triptófano/metabolismo , Receptor de Interferón gammaRESUMEN
BACKGROUND: cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation. RESULTS: Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP. CONCLUSIONS: Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes. SIGNIFICANCE: The method is applicable to other gene-tractable intertwined systems. Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.
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Adenilil Ciclasas/biosíntesis , AMP Cíclico/fisiología , Toxoplasma/metabolismo , Adenilil Ciclasas/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/parasitología , Regulación Enzimológica de la Expresión Génica , Interacciones Huésped-Parásitos , Humanos , Optogenética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Sistemas de Mensajero Secundario , Toxoplasma/genética , Toxoplasma/fisiologíaRESUMEN
Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins (TgDLC8a-d), of which only TgDLC8a clusters in the mainstream LC8 class. TgDLC8b-d proteins form a divergent and alveolate-specific clade. TgDLC8b-d proteins are largely cytosolic, whereas TgDLC8a resides in the conoid at the apical end of T. gondii. The apical location of TgDLC8a is also not shared by its nearly identical Eimeria (EtDLC8a), Plasmodium (PfDLC8), or human (HsDLC8) orthologs. Notwithstanding an exclusive conoid targeting, TgDLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the ß strands that interact with the antiparallel ß' strands of the opposing monomers. The TgDLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported PfDLC8 homodimer is shaped by binding of the ß strand with the parallel ß' strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the TgDLC8a-S88E mutant monomeric and cytosolic in T. gondii, and its overexpression inhibits the parasite growth in human fibroblasts.
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Dineínas/metabolismo , Multimerización de Proteína , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/crecimiento & desarrollo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Células Cultivadas , Dineínas/genética , Fibroblastos/parasitología , Fibroblastos/patología , Humanos , Datos de Secuencia Molecular , Mutación Missense , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
Helminth parasites modulate the immune system by complex mechanisms to ensure persistence in the host. Released immunomodulatory parasite components lead to a beneficial environment for the parasite by targeting different host cells and in parallel to a modulation of unrelated inflammatory responses in the host, such as allergy. The aim of this study was to investigate the effect of the potent helminth immunomodulator, filarial cystatin, in a murine model of airway inflammation and hyperreactivity induced by a clinically relevant aeroallergen (timothy grass (Phleum pratense) pollen) and on the function of peripheral blood mononuclear cells (PBMCs) from timothy grass pollen allergic patients. BALB/c mice were systemically sensitised with a recombinant major allergen of timothy grass pollen (rPhl p 5b) and then challenged with timothy grass pollen extract (GPE) via the airways. Filarial cystatin was applied i.p. during the sensitisation phase. Airway hyperresponsiveness to methacholine challenges, inflammation of airways, inflammatory cell recruitment, cytokine production and lung histopathology were investigated. In a translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with cystatin prior to stimulation with GPE. Administration of filarial cystatin suppressed rPhl p 5b-induced allergen-specific Th2-responses and airway inflammation, inhibited local recruitment of eosinophils, reduced levels of allergen-specific IgE and down-regulated IL-5 and IL-13 in the bronchoalveolar lavage (BAL). Ex vivo restimulation with cystatin of spleen cells from cystatin-treated mice induced the production of IL-10, while cystatin inhibited allergen-specific IL-5 and IL-13 levels. Human PBMCs from timothy grass pollen allergic patients displayed a shift towards a Th1 response after treatment with cystatin. These results show that filarial cystatin ameliorates allergic inflammation and disease in a clinically relevant model of allergy. This data indicate that filarial cystatin has a modulatory effect on grass pollen-specific responses warranting further investigation of potential preventive and therapeutic options in the treatment of allergies.
Asunto(s)
Cistatinas/uso terapéutico , Proteínas del Helminto/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Phleum/inmunología , Polen/inmunología , Células Th2/inmunología , Adulto , Animales , Células Cultivadas , Cistatinas/inmunología , Regulación hacia Abajo , Femenino , Proteínas del Helminto/inmunología , Humanos , Hipersensibilidad/inmunología , Interleucina-10/inmunología , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Adulto JovenRESUMEN
Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K(m) of 67 µM. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for â¼20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted â¼20% of its total PSD activity at 37 °C, and the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.
Asunto(s)
Carboxiliasas/genética , Carboxiliasas/metabolismo , Fosfatidilserinas/metabolismo , Toxoplasma/enzimología , Toxoplasmosis/metabolismo , Toxoplasmosis/microbiología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Carboxiliasas/química , Catálisis , Células Cultivadas , Fibroblastos/citología , Fibroblastos/parasitología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células Jurkat , Metabolismo de los Lípidos/fisiología , Liposomas/metabolismo , Datos de Secuencia Molecular , Fagocitosis/fisiología , Filogenia , Estructura Terciaria de Proteína , Solubilidad , Temperatura , Vacuolas/enzimologíaRESUMEN
The obligate intracellular apicomplexan parasites, e.g. Toxoplasma gondii and Plasmodium species, induce an IFNγ-driven induction of host indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of tryptophan catabolism in the kynurenine pathway. Induction of IDO1 supposedly depletes cellular levels of tryptophan in host cells, which is proposed to inhibit the in vitro growth of auxotrophic pathogens. In vivo function of IDO during infections, however, is not clear, let alone controversial. We show that Eimeria falciformis, an apicomplexan parasite infecting the mouse caecum, induces IDO1 in the epithelial cells of the organ, and the enzyme expression coincides with the parasite development. The absence or inhibition of IDO1/2 and of two downstream enzymes in infected animals is detrimental to the Eimeria growth. The reduced parasite yield is not due to a lack of an immunosuppressive effect of IDO1 in the parasitized IDO1(-/-) or inhibitor-treated mice because they did not show an accentuated Th1 and IFNγ response. Noticeably, the parasite development is entirely rescued by xanthurenic acid, a by-product of tryptophan catabolism inducing exflagellation in male gametes of Plasmodium in the mosquito mid-gut. Our data demonstrate a conceptual subversion of the host defense (IFNγ, IDO) by an intracellular pathogen for progression of its natural life cycle. Besides, we show utility of E. falciformis, a monoxenous parasite of a well appreciated host, i.e. mouse, to identify in vivo factors underlying the parasite-host interactions.
Asunto(s)
Coccidiosis/metabolismo , Eimeria/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Células TH1/metabolismo , Triptófano/metabolismo , Animales , Coccidiosis/genética , Coccidiosis/inmunología , Culicidae/parasitología , Eimeria/genética , Eimeria/inmunología , Inhibidores Enzimáticos/farmacología , Hipolipemiantes/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células TH1/inmunología , Triptófano/genética , Triptófano/inmunología , Xanturenatos/farmacologíaRESUMEN
The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (â¼70-kDa) TgCK displayed a low affinity for choline (K(m) â¼0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by â¼60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.
Asunto(s)
Colina Quinasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Mutagénesis , Fosfatidilcolinas/biosíntesis , Proteínas Protozoarias/biosíntesis , Toxoplasma/enzimología , Secuencia de Bases , Colina Quinasa/genética , Deanol/metabolismo , Datos de Secuencia Molecular , Mutación , Fosfatidilcolinas/genética , Multimerización de Proteína/fisiología , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
The roles of Th1 and Th17 responses as mediators of host protection and pathology in the intestine are the subjects of intense research. In this study, we investigated a model of intestinal inflammation driven by the intracellular apicomplexan parasite Eimeria falciformis. Although IFN-γ was the predominant cytokine during E. falciformis infection in wild-type mice, it was found to be dispensable for host defense and the development of intestinal inflammation. E. falciformis-infected IFN-γR(-/-) and IFN-γ(-/-) mice developed dramatically exacerbated body weight loss and intestinal pathology, but they surprisingly harbored fewer parasites. This was associated with a striking increase in parasite-specific IL-17A and IL-22 production in the mesenteric lymph nodes and intestine. CD4(+) T cells were found to be the source of IL-17A and IL-22, which drove the recruitment of neutrophils and increased tissue expression of anti-microbial peptides (RegIIIß, RegIIIγ) and matrix metalloproteinase 9. Concurrent neutralization of IL-17A and IL-22 in E. falciformis-infected IFN-γR(-/-) mice resulted in a reduction in infection-induced body weight loss and inflammation and significantly increased parasite shedding. In contrast, neutralization of IL-22 alone was sufficient to increase parasite burden, but it had no effect on body weight loss. Treatment of an E. falciformis-infected intestinal epithelial cell line with IFN-γ, IL-17A, or IL-22 significantly reduced parasite development in vitro. Taken together, to our knowledge these data demonstrate for the first time an antiparasite effect of IL-22 during an intestinal infection, and they suggest that IL-17A and IL-22 have redundant roles in driving intestinal pathology in the absence of IFN-γ signaling.