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Generation of Mature Toxoplasma gondii Bradyzoites in Human Immortalized Myogenic KD3 Cells
Deborah Maus1, Blake Curtis1,2, David Warschkau3
1Metabolism of Microbial Pathogens (P6), Robert Koch Institute, Berlin, Germany.
Abstract:
is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks.
Insights
This study presents a novel human cell culture system for generating bradyzoites, the chronic form of the parasite, which are crucial for developing new treatments against foodborne illness.
Area of Science:
- Parasitology
- Cell Biology
- Infectious Diseases
Background:
- Toxoplasma gondii bradyzoites are the persistent, chronic form of this zoonotic foodborne pathogen, residing in cysts within host tissues.
- Investigating bradyzoites is challenging due to limited in vitro models, hindering the development of drugs targeting chronic infections.
- Existing treatment options are insufficient for eradicating the chronic stage of infection.
Purpose of the Study:
- To detail a scalable human myotube-based in vitro system for cultivating and studying Toxoplasma gondii bradyzoites.
- To demonstrate the system's ability to generate bradyzoites with in vivo-like characteristics.
- To provide a reliable method for obtaining drug-tolerant bradyzoites for research and drug discovery.
Main Methods:
- Cultivation of terminally differentiated human myotubes.
- Infection of myotubes with tachyzoites, followed by maturation into encysted bradyzoites over four weeks.
- Incubation at ambient CO2 levels, facilitating differentiation without artificial stressors.
Main Results:
- The human myotube system successfully generated encysted bradyzoites with in vivo hallmarks, including pepsin resistance and oral infectivity.
- Bradyzoite differentiation was achieved via CO2 depletion without artificial stress.
- The system is scalable and suitable for experiments requiring large parasite numbers, such as metabolomics.
Conclusions:
- The developed human myotube-based system offers a scalable and effective in vitro model for studying Toxoplasma gondii bradyzoites.
- This model system provides crucial access to drug-tolerant bradyzoites, essential for advancing research into chronic parasitic infections.
- The protocol facilitates functional maturation of bradyzoites, paving the way for improved therapeutic strategies against persistent foodborne pathogens.
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