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Fig. S1. Co-infection of epithelial cells by Toxoplasma and C. trachomatis for 24 h. Immunofluorescence microscopy of HeLa cells or HCT-8 cells co-infected with RFP-expressing Toxoplasma and C. trachomatis revealed by EF-Tu staining, showing poor inclusion development. Scale bars are 10 μm.

Fig. S2. Production of PmpB by C. trachomatis in our experimental model. Fluorescence microscopy of C. trachomatis infecting fibroblasts. The bacteria were doubly stained for EF-Tu and PmpB. Panel a displays an inclusion from a mono-culture in our in vitro system, which undergoes normal development. Indeed, RBs that are associated with the inclusion membrane do not produce PmpB whereas those that are in the middle of the inclusion and therefore in the process of late differentiation to EBs, do produce PmpB. In contrast, PmpB expression is blocked under stress conditions. Panel b shows no labelling for PmpB in inclusions exposed to penicillin (+pen) compared with unexposed inclusions that produce both PmpB and ET-Tu. Scale bars are 10 μm.

Fig. S3. Influence of PV number and distance from the inclusion on PmpB production by Chlamydia in co-infected cells.

A and B. Immunofluorescence microscopy of Toxoplasma and Chlamydia co-infecting fibroblasts.

A. A single PV (panel a) or seven PVs (panel b) are present in a fibroblast containing an inclusion, and in both situations, PmpB production by Chlamydia was dramatically reduced.

B. Panels a and b show an inclusion close to two PVs (< 0.5 μm) and another distant from a PV (∼ 50 μm) respectively, revealing a decline in PmpB production more pronounced as the PV is closer to the inclusion. Arrows show the PV. Scale bars are 10 μm.

Fig. S4. Morphology of the PV and inclusions in pyrimethamine-treated cells. Mono-infected cells with either RFP-expressing T. gondii or C. trachomatis were incubated with 10 μM pyrimethamine for 24 h before examination by fluorescence microscopy. The morphology of the parasites is dramatically altered whereas the inclusions contain actively multiplying RBs of C. trachomatis as in untreated cells. Scale bars are 10 μm.

Fig. S5. Superinfection of Toxoplasma-containing cells by Chlamydia. HFF were infected for 1 day (A) or 2 days (B) with RFP-expressing T. gondii before adding C. trachomatis for an additional day. The bacteria were able to invade all parasitized cells but could neither develop nor replicate efficiently, more especially in cells occupied by 48 h PV. Scale bars are 10 μm.

Fig. S6. Effect of pyrimethamine on chlamydial development in co-infected cells. Fluorescence microscopy of Toxoplasma and Chlamydia infecting fibroblasts upon pyrimethamine treatment from exp. C shown in Fig. 4. A co-infected cell that contains two PVs show large inclusions made of normal size RBs that are actively dividing. Scale bars are 10 μm.

Fig. S7. Distribution of [NBD]cholesterol-LDL in intravacuolar Toxoplasma visualized by live fluorescence microscopy. T. gondii-infected HFF incubated for 2 h with fluorescent cholesterol display staining concentrated at the plasma membrane (PM) and the perinuclear area (n) of the parasites. Scale bar is 5 μm.

Fig. S8. Co-infection of fibroblasts with C. trachomatis and cystogenic strains of T. gondii, or Toxoplasma deleted for gra7 or gra2/6. Immunofluorescence microscopy of fibroblasts co-infected with Chlamydia stained for EF-Tu and different strains of Toxoplasma: the cystogenic Prugniaud (Pru) or 76 K-GFP strains; the parasite strain lacking gra7 (RHΔgra7); or the double knockout parasite deleted for gra2 and gra6 (RHΔgra2/6). For RHΔgra7, the infection assay has been performed in DMEM supplemented with 2.5% FBS as opposed to the normal 10% FBS. The left image shows the abnormal morphology of the mutant parasite grown for 24 h in 2.5% FBS during mono-infection. The right image shows a co-infected cell containing a small size PV and an aberrant inclusion (arrow), which is rarely found under these co-infection conditions. For RHΔgra2/6) stained for GRA7, the left image displays a mono- and a co-infected cell (delineated by white and yellow dashed lines respectively) allowing a direct comparison of inclusion sizes; the right figure illustrates a cell harbouring several inclusions and PVs). Scale bars are 10 μm.

Fig. S9. Co-infection of fibroblasts by Toxoplasma and C. trachomatis serovar L2 for 24 h. Immunofluorescence microscopy of RFP-expressing T. gondii- and C. trachomatis-infected cells 24 h p.i. In mono- and co-cultures, intravacuolar bacteria were stained for EF-Tu 24 h p.i. Scale bars are 10 μm.

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