Establishment of stable Flp-In-3T3 cell lines with wild rodents-derived Irg-like genes

proceeded with wild rodents-derived Irg-like sequences. I first transfected tandem Irg-like genes from the available wild rodents-derived cell lines, and in particular the identified tandem from M. glareolus BVK168 cells and tandem 1 from A. agrarius AAL-R cells (visible in Fig. 9). The cloning strategy used

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for Irgb2-b1BL6 and Irgb2-b1CIM and explained in previous chapters was used to clone these wild rodents’

sequences in pFS-1, resulting in pFS-BVK and pFS-AAL transfected in Flp-In-3T3 cells. Surprisingly, I observed an HA-tagged protein of 50 kDa instead of the expected 100 kDa, via Western Blot of cellular lysates from the created Flp-BVK and Flp-AAL cell lines (Fig. 15a). Nevertheless, these smaller IRG-like proteins loaded on the PVM of virulent parasites, as seen via immunofluorescent detection with an anti-HA-tag antibody (Fig 15b). I then PCR-amplified the full lenght genes from the plasmids used for cloning, to investigate the reason behind the detected smaller proteins. Full lenght genes were observed in pES51 cloned plasmids. However, pFS-BVK and pFS-AAL had a shorter inserted gene compared to positive control cells (Fig. 15c). A closer look to the full lenght sequences identified two additional SapI sites in both tandem Irg-like genes, the first exactly at the end of the Irgb2-like exon and the second 50 bp prior Irgb1-like C-terminal (Fig. 15d). Sequencing of both pFS-BVK and pFS-AAL plasmids confirmed sequence digestion at these sites during Golden Gate Assembly and ligation of the two fragments creating a shorter version of the gene with a gap of 1,256 bp in the Irgb1-like exon. The use of a reverse primer binding the Irgb2-like exon for colony PCR on cloned pFS plasmids did not allow identification of these truncated Irg-like sequences before establishment of the cell lines. However, this mistake allowed me to observe that the IRGb2-subunit only –together with the last residues of IRGb1–

is sufficient for binding to the PVM of virulent parasites.

Fig. 15. The IRGb2 subunit is sufficient for binding to virulent parasites vacuole membranes. (a) DB71 total protein staining (left) and Western Blot (right) with an anti-HA-tag antibody. First lane with molecular weight marker (with sizes in kDa given to the left). A representative experiment of two replicates is shown. (b) Immunofluorescence assay of AAL-IRGb2-like cell line (left) and Flp-BVK-IRGb2-like cell line (right) expressing an HA-tagged IRGb2-b1. Cells were infected for 1 h with a virulent T. gondii strain expressing a green fluorescent protein in the mitochondria, RHβGFPmt.

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Following staining is shown: IRGb2-b1 (red), T. gondii mitochondria (green), DAPI staining for nuclei (blue). A representative image is shown. The scale bar represents 20 µm. (c) 1 % agarose gels loaded with PCR-amplified Irg aand Irg-like tandem genes cloned in the pES51- bl plasmid (upper gel) and in the pFS-1 plasmid (lower gel). Red boxes highlight full length BVK and AAL sequences cloned in pES51- bl and truncated BVK and AAL sequences cloned in pFS-1. Positive and negative control sequences (-CIM and –BL6) and empty plasmids are indicated for comparison. First lane with DNA ladder (with sizes in kb given to the left). (d) Alignment of the full (pES-BVK) and truncated (pFS-BVK) versions of BVK Irg-like tandem genes. Red boxes highlight SapI sites within the sequence.

Nucleotides positions relative to the full lenght sequence are reported above the sequences. Nucleotide sequences are reported, and for the full lenght pES-BVK also the translated sequence.

6.1.5.5 T. gondii infection phenotype in established positive and negative control cell lines

Host-mediated T. gondii killing is followed by programmed death of the cell itself (109). An association between this phenotype and host resistance was suggested, since the parasite niche gets disrupted (7, 150). On the contrary, susceptible cells allow parasite proliferation until active egress of the same around 48 hpi. Resistant M. musculus castaneus CIM cells display an IFN-γ-dependent increase in necrotic cell death for both type I and type II infection, whereas susceptible BL6 cells display this phenotype only following avirulent infection (7). I will then refer to the IFN-γ-mediated cell death as the resistance phenotype.

Necrotic death results in permeabilization of both plasma and nuclear membranes. I thus performed a double staining with both a nuclear impermeable dye, Propidium Iodide (PI), and a permeable one, Hoechst 33342, to quantify the ratio of necrotic cells during T. gondii infection and IFN-γ stimulation, similarly to published protocols (150). As shown in Fig. 16, an increase in necrotic murine Flp-In-3T3 cells was observed during type II infection following stimulation with IFN-γ, however not to the extent expected from literature (7). A similar increase in PI⁺ cells increase was observed following type I infection of IFN-γ-treated cells, suggesting that cells are not completely susceptible to infection.

We expected Flp-CIM positive cells to show an increase in necrotic cell death following IFN-γ treatment and T. gondii infection. Surprisingly, a rather decreased amount of necrotic cells was observed for both Flp-CIM and Flp-BL6 compared to the wild-type (wt) cell line (Fig. 16). No obvious control of parasite proliferation was observed at the microscope in positive control Flp-CIM cells compared to other cell lines. Indeed, preliminary experiments confirmed that IFN-γ treated Flp-CIM cells did not display a reduced number of parasites per vacuole compared to Flp-BL6 cells (Appendix Fig. 38).

Results suggest that our cell culture-based system does not reproduce results published in literature.

Despite the identical Irgb2-b1 genotype, Flp-In-3T3 cells display a different phenotype during T. gondii infection compared to other inbred mouse cells. A difference between the derived positive Flp-CIM and negative control Flp-BL6 cell lines is not obvious in regard to T. gondii infection.

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Fig. 16. The positive control cell line CIM does not display the host resistance phenotype. Flp-In-3T3 (Flp) parental strain, negative control Flp-BL6 and positive control Flp-CIM cell lines were treated with 200 U/ml mouse IFN- γ or left untreated 24 h prior infection with avirulent (type II) and virulent (type I) T. gondii. 24 hpi a double staining with Propidium Iodide (PI) and Hoechst 33342 on living cells was performed. Double stained nuclei were quantified at the fluorescent microscope. Mean

± sd (n = 3 experiments, each with 2 replicates).

6.1.5.6 Flp-In-3T3 cells express higher levels of endogenous Irgb2-b1 compared to primary