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C3 Comparative analysis of intestinal innate immune responses from wild rodents upon infection: Challenging the T. gondii – lab mouse model (Seeber)

Research Group: Parasitology unit of the Robert Koch-Institute
Address: Robert Koch-Institute

Division 16, Mycotic and Parasitic Agents and Mycobacteria
Nordufer 20
13353 Berlin

Supervisors: Prof. Dr. Frank Seeber
Doctoral Researcher: Benedikt Fabian, Estefanía Delgado Betancourt

Project Description

State of the art:
Toxoplasma gondii’s (Tg) route of infection is via the intestine, which is also the habitat of
Giardia sp. Almost all studies dealing with intestinal Tg infections were done in inbred
laboratory Mus musculus (Mm) with conflicting results. Wild rodents (voles, field mice) are
much more preyed by cats (the definitive host for Tg) in Europe than Mm, and also show a
higher Tg seroprevalence. Thus, they play a more important role as intermediate hosts than
mice when Tg has to reach a cat to complete its sexual cycle, but its relevance has been
largely ignored so far. Accordingly, studying intestinal innate immune factors requires careful
consideration of rodent models, and lab Mm might not provide the same insights as wild
rodents with regard to intestinal innate immune factors influencing a Tg infection. Moreover,
given the high overall prevalence of Giardia sp. infection in wild rodent’s intestines in Germany
it is likely that co-infection affects the immediate infection event of Tg to some extent.

Previous own work:
In the 1st funding period essential groundwork for the proposed project has provided the
following: (i) wild rodent cell culture models and cell lines for infection studies; (ii) generation
of species-specific recombinant IFNγ from the bank vole Myodes glareolus (Mg) and a reporter
cell line for it (3); (iii) transcriptomes of a Mg cell line in dependence on IFN-γ stimulation; (iv)
a sequence space of IFNγ-inducible immunity-related GTPases (IrgB2-B1-like) implicated in
the resistance to virulent Tg strains from several wild rodents for further analysis; (v)
importantly, the 2nd generation PhD student has already established stem cell-derived
intestinal organoids (IOs) from several animals from our in-house Mg colony and started to
characterize them (in addition to previously established lab mice IOs).

Hypotheses and work plan:
We hypothesize that intestinal innate immune factors, including immune cells and microbiota,
determine the success of infection of Tg alone and in co-infections with Giardia, in relevant
natural hosts like voles, and that in this respect differences exist to the lab mouse model.
(1) We will colonize IOs with select microbiota and compare them with “naïve/sterile IOs”. This
will be approached by co-culture of infected IOs with select bacteria (based on published
microbiomes of wild mice and voles and/or own microbiome data generated from locally caught
bank voles) and studying their influence on the course of infection by Fluorescence (Live Cell)
Microscopy (F(LC)M) microscopy, cytokine assays etc. (2) We will then analyze upon IFNγ
stimulation innate immune responses, including IFN-γ-inducible IRGs, by transcriptomics and
F(LC)M, following infection of above IO’s from Mg and Mm with Tg and compare them to a
co-infection with Giardia sp. (3) We will further analyze by FLCM whether the reported death
of Paneth cells in Tg-infected C57/BL6 mice is also seen in wild rodents. For this we will
colonize and complement IOs with bacteria and immune cells, respectively, derived from our
Mg colony, and studying their effect(s) by F(LC)M, cytokine assays etc. (4) Based on
transcriptome analyses from 2nd period select genes involved in innate immune responses that
have the largest impact on parasite infection, IOs will be deleted/manipulated via
CRISPR/Cas9 in stem cells of Mg and Mm IOs and resulting infection phenotypes in organoids
derived thereof will be analyzed largely by F(LC)M and if appropriate by transcriptome
analyses.

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