In vitro maturation of Toxoplasma gondii bradyzoites in human myotubes and their metabolomic characterization

The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention.Peer Reviewe

Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species

Characterisation of the replication factors of porcine circovirus regarding initiation and termination of viral replication

Titelblatt und Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung Material Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Publikationsliste Danksagung AnhangUnter DNA-Replikons, welche über rolling circle Replikation (RCR) replizieren, stellen die Bindung an den doppelsträngigen Replikationsorigin (dso) und die Einführung eines strang- und sequenzspezifischen Einzelstrangbruchs essentielle Stufen für den Ablauf der Replikation dar. Während die aus dem Einzelstrangbruch resultierende 3 -Hydroxygruppe als Primer für die nachfolgende DNA-Synthese fungiert, wird die 5 -Phosphatgruppe durch den neu synthetisierten DNA-Strang verdrängt. Nach einer Syntheserunde und zweiter Restriktion innerhalb des regenerierten Replikationsorigins werden die Enden des neu synthetisierten DNA-Moleküls zum zirkulär geschlossenen Genom verknüpft. Aufgrund konservierter Motive sowohl innerhalb der Aminosäuresequenz der Replikationsproteine als auch der Nukleotidsequenz des viralen Replikationsorigins wird die Replikation auch der porcinen Circoviren Typ 1 und Typ 2 (PCV1 und PCV2) über RCR postuliert. In dieser Arbeit wurde erstmals die Restriktions-und Ligationsaktivität der PCV-Replikationsproteine Rep und Rep gegenüber dem viralen Replikationsorigin in vitro demonstriert. Beide Enzyme schneiden den viralen Plusstrang zwischen den Nukleotiden sieben und acht innerhalb des konservierten Nonamers 5 -T1AGTATTAC9-3 und werden nachfolgend kovalent mit dem 3 -Schnittprodukt verknüpft. Für die Einführung des Einzelstrangbruchs wurde das Nonamer als essentielle Sequenzanforderung identifiziert, wohingegen die Ausbildung einer Haarnadelstruktur keine Voraussetzung für die Restriktion in vitro darstellt. Darüber hinaus wurde nachgewiesen, dass Rep und Rep einzelsträngige Originfragmente in vitro ligieren, was eine Funktion von PCV-Rep/Rep hinsichtlich der Termination der viralen Replikation wahrscheinlich erscheinen lässt. Die Ligation ist abhängig von vorangegangener Restriktion und räumlicher Nähe der Substrate. Letzteres wird über Basenpaarung über den Bereich der inversen Repetition gewährleistet. Mit Hilfe von Mutanten der Rep-Proteine wurde die Abhängigkeit der Restriktion von der Integrität der Aminosäuremotive I, II und III verdeutlicht und Tyrosin-93 als das katalytische Zentrum der Reaktion identifiziert. Die GKS- Box wurde erstmals als Voraussetzung für die ATP-Hydrolyse des Rep-Proteins nachgewiesen, diese konservierte Sequenz hat aber keinen Einfluss auf die Restriktion des Replikationsorigins durch Rep und Rep . Durch den Nachweis homologer sowie heterologer Ineraktion der Replikationsproteine Rep und Rep steht zu vermuten, dass die Termination der Replikation durch Tyrosin-93 als Bestandteil eines Rep/Rep -Dimers oder Oligomers höherer Ordnung und nicht durch eine zweite aktive Aminosäure auf dem selben Monomer katalysiert wird. Über einen Replikationsassay wurde die Bedeutung der konservierten Motive innerhalb der Replikationsproteine für die virale Replikation in PK15-Zellen demonstriert. Weiterhin wurde die Rekrutierung von PCV-Rep/Rep an den dso über Interaktion mit den Hexameren H1 und H2 bestätigt. Die Ausbildung einer Haarnadelstruktur als Voraussetzung für die Initiation innerhalb des einzelsträngigen Bogenbereichs sowie eine Abhängigkeit der Termination von der Sequenzspezifität upstream des Nonamers wird vermutet.DNA cleavage and ligation are pivotal steps for initiation and termination of genome replication via the rolling circle mechanism. After recruitment of the replicon-encoded initiator protein, replication is initiated by introduction of a strand- and site-specific nick within the double-stranded origin of replication (dso). The resulting free 3 -hydroxyl group serves as a primer for DNA synthesis, while the 5 -phosphate group is displaced during DNA replication. After at least one round of replication, the nascent strand is cleaved again within the regenerated origin and the 5 -phosphate end is ligated to the newly created 3 -hydroxyl group resulting in release of unit- length monomers. Due to characteristic sequences found within the origin of replication and the replication proteins, genome amplification of porcine circoviruses type 1 and type 2 (PCV1 and PCV2) is assumed to be mediated by rolling circle replication. This study demonstrated for the first time the ability of Rep and Rep of PCV to introduce and reseal strand discontinuities within the origin of replication in vitro. Rep and Rep cleave the viral strand between nucleotides 7 and 8 within the conserved nonamer 5 -T1AGTATTAC9-3 and become covalently attached to the 3 -cleavage product. The conserved nonamer is the only sequence element identified necessary for cleavage. Since PCV Rep and Rep are also capable of resealing the viral single-stranded DNA, participation of these proteins in termination of replication is suggested. In this context, joining was strictly dependent on preceding substrate cleavage as well as close proximity of origin fragments presumably accomplished by base pairing. Cleavage of origin fragments in vitro depends on conserved motives I, II and III. Mutation analysis identified tyrosine-93 as the catalytic amino acid. In contrast, the GKS box is not essential for DNA cleavage, although PCV Rep was proven to hydrolyse ATP in vitro. Formation of homo- and heterocomplexes of the replication proteins was observed. This supports the hypothesis that Rep/Rep form a dimer or multimer for initiating and terminating RCR, thereby providing the second catalytic center essential for termination. The impact of the motives conserved in the replication proteins as well as in the origin of replication was tested in a cell culture-based replication asay. Integrity of conserved motives I, II, III as well as the GKS box and the ability of of PCV Rep/Rep to promote replication are linked. Recruitment of Rep and Rep to the viral origin by binding to hexamers H1/H2 of the minimal binding site was demonstrated to be a prerequisite for replication. Cruciform extrusion for providing the single- stranded DNA conformation of the nonamer indispensable for cleavage and dependency of termination on sequences flanking the nonamer is suggested

Rep and Rep′ Protein of Porcine circovirus Type 1 Bind to the Origin of Replication in Vitro

AbstractGenome replication of Porcine circovirus type 1 (PCV1) relies upon expression of the full-length protein Rep and a spliced isoform (Rep′), and the presence of a 111-bp genomic fragment comprising the origin of replication. Using an electrophoretic mobility shift assay (EMSA), the capability of both Rep proteins to bind to partial fragments of the origin of replication of PCV1 was investigated in vitro. Both proteins formed complexes with double-stranded DNA origin fragments containing a stem-loop structure with a conserved nonamer and four hexamer repeats (5′-CGGCAG; H1 to H4). Use of truncated EMSA substrates identified minimal binding sites (MBS) for Rep and Rep′ protein: The Rep binding site was mapped to the right leg of the stem-loop and the two inner hexamer repeats H1/H2, while binding of Rep′ required only the presence of two hexamer repeats. Two differentially retarded complexes were observed with Rep protein, which presumably result from alternative binding to the MBS or to H3/4

Functional Analysis of cis- and trans-Acting Replication Factors of Porcine Circovirus Type 1▿

The replication proteins Rep and Rep′ of porcine circovirus type 1 (PCV1) are both capable of introducing and resealing strand discontinuities at the viral origin of DNA replication in vitro underlying genome amplification by rolling-circle replication. The PCV1 origin of replication encompasses the minimal binding site (MBS) of the Rep and Rep′ proteins and an inverted repeat with the potential to form a stem-loop. In this study, both elements of the PCV1 origin were demonstrated to be essential for viral replication in transfected cells. Furthermore, investigation of conserved amino acid motifs within Rep and Rep′ proteins revealed that the mutation of motifs I, II, and III and of the GKS box interfered with viral replication. In vitro studies demonstrated that motifs I to III were essential for origin cleavage, while the GKS box was dispensable for the initiation of viral replication. A covalent link between Rep/Rep′ and the DNA after origin cleavage was demonstrated, providing a mechanism for energy conservation for the termination of replication

New Reporter Gene-Based Replication Assay Reveals Exchangeability of Replication Factors of Porcine Circovirus Types 1 and 2

Two types of porcine circovirus (PCV), which differ in their pathogenicity, are known. PCV type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome in swine, while PCV1 has not yet been linked to a disease. Corroborating earlier observations in PCV1, transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep′ and that the simultaneous expression of Rep and Rep′ proteins is essential for initiation of replication of PCV2. The interchangeability of the replication factors of PCV1 and PCV2 was examined. The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa. To investigate the competence of the Rep/Rep′ proteins to initiate replication at the heterologous origin, a new replication assay was developed. It measures the expression of a luc reporter gene present on a plasmid carrying the origin of the investigated replicon. Replication is initiated by expression of the appendant replicase from a second plasmid and results in replication of the origin plasmid coupled with an increase in the Luc activity. Using this method to compare replication of PCV1 and PCV2 in cell culture, it was shown that the Rep/Rep′ protein of PCV2 initiated replication at the origin of PCV1, as did the reciprocal combination. Our results indicate that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable

High-quality recycling through self-learning and resilient recycling networks using a combination of agent-based modelling and life cycle assessment

Especially in the case of long-lived products, the crucial questions about the proper implementation and assurance of high-quality recycling targets often only arise after decades. Furthermore, information about material composition is often not sufficiently known and communicated to the end user. With the presented extended socio-technical approach of a self-learning and resilient recycling network, which should include manufacturers and operators of wind farms, dismantlers, waste processors and recyclers, as well as authorities and players from research and development, such problems can be adequately addressed. On the one hand, this requires knowledge tools to ensure a high-quality material cycle, such as databases in which installed products and their characteristic values for the masses and materials used are documented. In addition, material flow modeling to track material flows generated for the end-of-life (EoL) of products including life cycle assessments of recycling and disposal routes, as well as forecasting tools for expected waste volumes are needed. On the other hand, a simulation tool such as agent-based modeling (ABM) is also needed to map courses of action and their impacts, taking into account stakeholders’ interests in terms of target formulation of the recycling network. The example of wind turbine rotor blades is used to show how such an approach can be used for a meaningful recycling network, which supports the operator responsibility of wind farms as well as the extended producer responsibility of wind turbines with regard to sustainable recycling of long-lived products. The developed tools and especially their active combination are presented. In addition, the example of rotor blades is used to present the concrete possibilities for resource-saving control of the material flows

The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii

Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver virulence determinants into the host cell during parasite invasion. One such determinant called rhoptry protein 18 (ROP18) is a polymorphic serine/threonine kinase that phosphorylates host targets to modulate acute virulence. Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this hostpathogen interface. However, the functional consequences of PVM association are not known. In this report, we show that ROP18 mutants altered in an arginine-rich domain upstream of the kinase domain fail to associate to the PVM following secretion from rhoptries. During infection, host cells upregulate immunity-related GTPases that localize to and destroy the PVM surrounding the parasites. ROP18 disarms this host innate immune pathway by phosphorylating IRGs in a critical GTPase domain and preventing loading on the PVM. Vacuole-targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. As a consequence, these mutants were avirulent in a mouse model of acute toxoplasmosis. Thus, the association of ROP18 with the PVM, mediated by its N-terminal arginine-rich domain, is critical to its function as a virulence determinant