86 research outputs found
Drug susceptibility testing in microaerophilic parasites: Cysteine strongly affects the effectivities of metronidazole and auranofin, a novel and promising antimicrobial
No full textThe microaerophilic parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia lamblia annually cause hundreds of millions of human infections which are treated with antiparasitic drugs. Metronidazole is the most often prescribed drug but also other drugs are in use, and novel drugs with improved characteristics are constantly being developed. One of these novel drugs is auranofin, originally an antirheumatic which has been relabelled for the treatment of parasitic infections. Drug effectivity is arguably the most important criterion for its applicability and is commonly assessed in susceptibility assays using in vitro cultures of a given pathogen. However, drug susceptibility assays can be strongly affected by certain compounds in the growth media. In the case of microaerophilic parasites, cysteine which is added in large amounts as an antioxidant is an obvious candidate because it is highly reactive and known to modulate the toxicity of metronidazole in several microaerophilic parasites.In this study, it was attempted to reduce cysteine concentrations as far as possible without affecting parasite viability by performing drug susceptibility assays under strictly anaerobic conditions in an anaerobic cabinet. Indeed, T. vaginalis and E. histolytica could be grown without any cysteine added and the cysteine concentration necessary to maintain G. lamblia could be reduced to 20%. Susceptibilities to metronidazole were found to be clearly reduced in the presence of cysteine. With auranofin the protective effect of cysteine was extreme, providing protection to concentrations up to 100-fold higher as observed in the absence of cysteine. With three other drugs tested, albendazole, furazolidone and nitazoxanide, all in use against G. lamblia, the effect of cysteine was less pronounced. Oxygen was found to have a less marked impact on metronidazole and auranofin than cysteine but bovine bile which is standardly used in growth media for G. lamblia, displayed a marked synergistic effect with metronidazole. Keywords: Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia, Drugs, Susceptibility testing, Cystein
Metabolomic Profiling of Wildtype and Transgenic Giardia lamblia Strains by 1H HR-MAS NMR Spectroscopy.
No full textGiardia lamblia, a causative agent of persistent diarrhea in humans, domestic animals, and cattle, is usually treated with nitro compounds. Consequently, enzymes involved in anaerobic nitro reduction have been investigated in detail as potential targets. Their role within the normal metabolic context is, however, not understood. Using 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, we analyzed the metabolomes of G. lamblia trophozoites overexpressing three nitroreductases (NR1-NR3) and thioredoxin reductase (TrxR), most likely a scavenger of reactive oxygen species, as suggested by the results published in this study. We compared the patterns to convenient controls and to the situation in the nitro drug resistant strain C4 where NR1 is downregulated. We identified 27 metabolites in G. lamblia trophozoites. Excluding metabolites of high variability among different wildtype populations, only trophozoites overexpressing NR1 presented a distinct pattern of nine metabolites, in particular arginine catabolites, differing from the respective controls. This pattern matched a differential pattern between wildtype and strain C4. This suggests that NR1 interferes with arginine and thus energy metabolism. The exact metabolic function of NR1 (and the other nitroreductases) remains to be elucidated
Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp.
No full textAcanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference genes (RGs) for RT-qPCR in Acanthamoeba were comprehensively evaluated, comparing different Acanthamoeba strains and employing four different algorithms (NormFinder, GeNorm, BestKeeper and RefFinder). Expression stability was assessed under various conditions and the potentials of the most promising RGs for accurate normalization of target genes were evaluated. Expression stability of RGs varied depending on conditions and employed algorithms, however, the genes for the 18S rRNA and the hypoxanthine phosphoribosyl transferase seem to be widely suitable RGs. Normalization with a combination of two carefully chosen RGs resulted in reliable expression data for target genes, while normalization with unsuitable RGs led to significant misinterpretation of expression profiles. Thus, a careful evaluation of RGs prior to expression studies is essential
Neue Ansaetze in der schematischen Schnittelimination
No full textIn dieser Dissertation untersuchen wir Formel- und Beweisschemata in der Aussagen- und in der Prädikatenlogik. Mit -schematischmmeinen wir dass Formeln und Beweise durch einen freien numerischen Parameter indiziert sind. Im propositionalen Fall untersuchen wir eine Erweiterung eines Entscheidungsverfahrens für die Erfüllbarkeit regulärer aussagenlogischer Formelschemata (definiert in [4]) auf mehrere freie Parameter. Der Hauptteil der Arbeit besteht in der Untersuchung von Schnittelimination in prädikatenlogischen Beweisschemata mit Hilfe der CERES-Methode für Beweisschemata defininiert in [31]. Diese Beweisschemata haben eine zyklische Form mit einem freien numerischen Parameter. Die Schnitteliminationsmethode CERES definiert in [14] extrahiert eine sogenannte charakteristische Klauselmenge aus einem Beweis im Sequentialkalkül und widerlegt diese mit Resolution. Die charakteristische Klauselmenge repräsentiert die Schnittstruktur des Beweises und ist immer unerfüllbar. Im Gegensatz zur Methode in [14] welche eine vollständige Schnitteliminationsmethode für die Logik erster Stufe liefert ist die Methode in [31] inhärent unvollständig. Das Hauptproblem besteht darin dass Beweischemata unendliche Folgen von Beweis-Schemata repräsentieren und viele theoretische Resultate in [14] nicht für Schemata gelten. Insbesondere ist auch unklar wie und ob eine reduktive Schnittelimination für Schemata definiert werden kann. Auch die schematische Resolution ist nicht mehr vollständig für unerfüllbare Klausel-Schemata. Das Problem der Spezifikation schematischer Resolutionsbeweise zur Widerlegung der schematischen charakteristischen Klauselmengen nimmt einen zentralen Platz in der Untersuchung ein. Da es dazu keinen theoretischen Hintergrund gibt untersuchen wir das Problem anhand typischer Fallstudien der Beweistheorie (wie das unendliche Schubfachprinzip). An Hand dieser Fallstudien untersuchen wir die Expressivität des Formalismus in [31] und zeigen seine Grenzen auf. Im Zuge dieser Untersuchung verallgemeinern wir den schematischen Resolutionskalkül und entwickeln eine Methode der Herbrand-Extraktion (welche aber relativ eng auf spezifische Schemata beschränkt ist). Abschließend ist zu sagen, dass automatische Beweiser in der Auffindung der Resolutions-Widerlegungs-Schemata eine wichtige Rolle gespielt haben und geben einen Hinweis auf potentielle künftige Verwendung von automatischen Beweisern. viiAbstract In this dissertation we study schematic formulae and proofs from both the propositional and first-order perspective. By schematic we mean that the formulae have a free numeric parameter indexing the formulae. In the propositional case we study an extension of a decision procedure for satisfiability of regular propositional schematic formulae, defined in [4], allowing for multiple free parameters within a single formula. Our work concerning first-order schematic formulae makes up the major of the dissertation and is mainly concerned with cut-elimination for proof schemata using the CERES method [31]. By proof schemata, we are referring to a restricted form of cyclic proofs with a free indexing parameter. The CERES method of cut elimination introduced in [14] extracts what is referred to as the characteristic clause set from a sequent calculus proof and uses resolution to refute the clause set. The characteristic clause set represents the cut structure of the sequent calculus proof it is derived from and is always unsatisfiable. Unlike the work of [14], which introduces a complete method for cut elimination in first order logic, [31] only introduces a framework which can be used to define proof schemata and possibly eliminate the cuts within a given proof schemata. The problem with proof schemata is that they essentially define an infinite sequence of proofs and many theoretic results used in [14] no longer hold for proof schemata. For example, reductive cut elimination is not as straight forward as it was for first-order sequent calculus proofs and it is unclear if it is even possible to define a reductive method for proof schemata. Also, most importantly, resolution is on longer a complete method for unsatisfiable clause sets, specifically, it is not complete for unsatisfiable schematic clause sets. This issue with resolution being one of the main issues addressed in this thesis. We approach this problem of the lack of a theoretic backbone for cut-elimination on proof schemata by constructing several formal proofs in a schematic version of the standard sequent calculus, each of which is based on a well studied problem (the infinitary pigeonhole principle) often used in the analysis of proof complexity within a given proof system. Using these formal proofs, we apply the schematic version of the CERES method to each of the proofs to see if the method is expressive enough to handle cut elimination for the given proof schema, and if it is not, we ask if we can generalize certain parts of the method to handle the given proof schema and allow for cut elimination in this more general framework. In the process, we develop a generalized version of the resolution calculus introduced in [31], a possible method for Herbrand sequent extraction, though the method is quite restrictive and relies a lot on the structure of the proof schema, and combinatorial results pertaining to the structure of the characteristic clause sets of the given proof schemata. As a final remark, theorem provers where heavily used in the application of the schematic CERES method to the formalized proof schemata used in this dissertation. We also provide an outline as to how this was done and what results where attained with the aid of automated theorem provers, as well as, idea on how automated theorem provers might be beneficial to future research in this subject
Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target
No full textAbstractThe antioxidative enzyme thioredoxin reductase (TrxR) has been suggested to be a drug target in several pathogens, including the protist parasite Giardia lamblia. TrxR is also believed to catalyse the reduction of nitro drugs, e.g. metronidazole and furazolidone, a reaction required to render these compounds toxic to G. lamblia and other microaerophiles/anaerobes. It was the objective of this study to assess the potential of TrxR as a drug target in G. lamblia and to find direct evidence for the role of this enzyme in the activation of metronidazole and other nitro drugs.TrxR was overexpressed approximately 10-fold in G. lamblia WB C6 cells by placing the trxR gene behind the arginine deiminase (ADI) promoter on a plasmid. Likewise, a mutant TrxR with a defective disulphide reductase catalytic site was strongly expressed in another G. lamblia WB C6 cell line. Susceptibilities to five antigiardial drugs, i.e. metronidazole, furazolidone, nitazoxanide, albendazole and auranofin were determined in both transfectant cell lines and compared to wildtype. Further, the impact of all five drugs on TrxR activity in vivo was measured.Overexpression of TrxR rendered G. lamblia WB C6 more susceptible to metronidazole and furazolidone but not to nitazoxanide, albendazole, and auranofin. Of all five drugs tested, only auranofin had an appreciably negative effect on TrxR activity in vivo, albeit to a much smaller extent than expected. Overexpression of TrxR and mutant TrxR had hardly any impact on growth of G. lamblia WB C6, although the enzyme also exerts a strong NADPH oxidase activity which is a source of oxidative stress.Our results constitute first direct evidence for the notion that TrxR is an activator of metronidazole and furazolidone but rather question that it is a relevant drug target of presently used antigiardial drugs
Effect of various stress factors on mitochondrial processes of pathogenic protists
No full textMitochondria perform various important functions in cells. They are the main site of energy metabolism, biosynthetic and regulatory processes, and the center of iron metabolism. Additionally, mitochondria are also the central organelle responsible for the production of potentially dangerous reactive oxygen species and possess a self-destructive arsenal capable of inducing whole-cell apoptosis. This single organelle thus controls the fate of the entire cell. Given these facts, this organelle has become the focus of interest for many scientists and pharmaceutical companies developing drugs targeting mitochondria. During evolution, unicellular parasites have evolved several mechanisms to survive, defend themselves and reproduce in the hostile environment of their host. One of these mechanisms is the ability to adapt its mitochondrial metabolism to maintain the viability of the whole cell. This work focuses on mitochondria from two different perspectives: First, concerning the phenomenon of nutritional immunity, we studied the effect of iron and copper deprivation on the mitochondria of the "brain-eating" amoeba Naegleria fowleri. Proteomic analysis of cells pre-incubated with specific chelators, together with the determination of several enzyme activities and measurements of oxygen consumption,..
Giardia lamblia: missing evidence for a canonical thioredoxin system
No full textAbstract
The microaerophilic protozoan parasite Giardia lamblia occurs globally and causes dysentery in humans and animals. Since it is very sensitive to oxygen and reactive oxygen species, G. lamblia disposes over several enzymatic pathways to counter oxidative stress. One of the enzymes involved is thioredoxin reductase (TrxR), a central redox regulator that indirectly reduces peroxiredoxins via thioredoxin, an electron shuttle protein. Interestingly, the components of the TrxR-mediated redox system, including functional thioredoxins, have so far not been described despite their surmised importance for parasite survival. We aimed at filling this gap and attempted to identify functional thioredoxins and other interaction partners of TrxR in G. lamblia. To this end, we conducted database searches and expressed three recombinant candidate thioredoxins in Escherichia coli for ensuing enzyme assays. Further, co-immunoprecipitation experiments were conducted in order to identify further components of the thioredoxin redox network. Finally, the cellular localization of TrxR and peroxiredoxin 1 was determined by immunofluorescence microscopy. Surprisingly, our endeavours did not result in the identification of a functional thioredoxin or other credible interaction partners of TrxR. We, therefore, conclude that there is currently no evidence for a canonical thioredoxin redox network in G. lamblia.</jats:p
Faculty Opinions recommendation of Giardia Alters Commensal Microbial Diversity throughout the Murine Gut.
No full textFaculty Opinions recommendation of Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis.
No full textFaculty Opinions recommendation of Epigenetics regulates transcription and pathogenesis in the parasite Trichomonas vaginalis.
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