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Persistence of Toxoplasma gondii infections: Mode of actions of drug candidates and the role of physiological heterogeneity
Toxoplasma gondii is an Apicomplexan parasite that infects virtually all warm-blooded animals and causes life-long infections in up to 30% of humans globally and >50% in Germany. With an annual new infection rate of 1% of the German population sustainable reduction of zoonotic transmission of T. gondii remains a public health priority. T. gondii infections are usually self-limiting but severe, and often fatal, symptoms occur during congenital transmission and in immuno-compromised patients. Indeed T. gondii infections impose one of the highest disease burdens among foodborne infections. Chronically persisting parasite tissue cysts are called bradyzoites and are transmitted via undercooked meat products. In contrast to acute forms these quiescent stages adopt heterogeneous phenotypes and resist licensed chemotherapies. The Toxo junior group analyses the metabolic signatures of T. gondii bradyzoites for the evidence-based development of novel chemotherapies. These studies include systematic screening of antiprotozoal compound libraries using a metabolomic approach based on liquid and gas chromatography-coupled mass spectrometry. Identification of molecular targets will facilitate development and optimization of antiprotozoal compounds into much needed drug candidates.
In a complementary approach the group will study the physiological basis of persistence in veterinary isolates. Clonal T. gondii tissue cysts are phenotypically highly heterogeneous and this heterogeneity might be responsible for persistence and resistance to chemotherapies. We create fluorescent parasite reporter cell lines that allow us to monitor key physiological parameters during stress responses at single cell resolution. The obtained data will then be used to calibrate a computational model that allows us to identify the contribution of phenotypic heterogeneity to the persistence of T. gondii.
Taken together, the Toxo group aims to identify strategies to limit T. gondii infections by understanding the fundamentals of persistence on single cell level and facilitate the development of new chemotherapeutic approaches against zoonotic infections.
1. Identification of the mode of action of T. gondii cyst inhibitors
There are currently no medicines that target chronic T. gondii infections. These infections are usually clinically silent; however, they can convert to acute infections and are the basis for a very high infection rate that resulting in a 50% prevalence in Germany.
The discovery of new drugs follows two approaches. The classical forward pharmacological approach that is based on an initial phenotypic screen has been successful in identifying potent drugs. However, their refinement is often hampered by unknown modes of action. Conversely, reverse pharmacological approaches yielded many essential protein target structures. However, the corresponding inhibitory compounds work mostly inefficiently on organismic levels.
To address this gap we established a comprehensive metabolomics platform that allows us to characterize the global metabolic response to candidate antimicrobials and identify their molecular targets. This platform consists of a UHLPC-coupled Orbitrap high resolution mass spectrometer and a gas chromatography-coupled mass spectrometer. Together these instruments allow monitoring of hundreds of small molecules (metabolites) that reflect the activity of metabolic enzymes. This untargeted analysis results fine grained metabolic phenotypes that allow us to identify putative target pathways and enzymes. We then confirm these hits by more targeted approaches that involve stable isotope-resolved analysis of particular pathways and reverse genetic mutation of candidate target genes.
We hope that this untargeted approach will identify novel ways to chemotherapeutically targeted quiescent T. gondii pathogen populations that will be adoptable to other pathogens.
2. Quantifying the contribution of heterogeneity to chronic infections of T. gondii
Persisting forms of T. gondii resist the host immune system and chemotherapies. They differ from easily cleared acute parasite stages by their highly heterogeneous phenotypes that occur even in clonal infections. These are marked by asynchronous cell divisions, varying division rates and differ by size and packaging densities of tissue cysts. Phenotypic heterogeneity of clonal microbial populations is a known bet-hedging strategy for microbial but its role during chronic T. gondii infections has not been studied. To measure these phenotypes on a single cell level we employ fluorescent reporter cell lines that allow us to monitor in key physiological parameters, such as cell division, pH, redox status and particular metabolite levels. The physiological description of dividing and dormant bradyzoites on a single cell level will be used to calibrate an agent-based model that aims to replicate persistence and virulence phenotypes of veterinary isolates based on the newly identified subpopulations.
Dr. Martin Blume
Tel. +49 (0)30 18754 2572
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