*4.5. Co-Culture Assays*

Amoeba and bacterial working solutions were mixed in 25 cm<sup>3</sup> flasks by adding the required volume according to Table 1. To provide an example, 10 mL of *W. magna* C2c Maky at 3 × 10<sup>5</sup> cells/mL was mixed with 0.1 mL of *L. pneumophila* Lens at 3 × 10<sup>7</sup> CFU / mL. All flasks were left to stand for 2 h at 22 ◦C ± 2 ◦C or at 37 ◦C ± 2 ◦C to allow for amoebae/bacteria contact and the internalization of *L. pneumophila* into amoebae. After the 2-h contact process, each flask was gently shaken 10 times, and the suspension was transferred into a 15 mL Flacon® tube and centrifuged at 3000× *g* for 5 min. This step allowed for the removal of non-internalized (i.e., extracellular) *L. pneumophila* from the co-culture suspensions. The pellet was resuspended in 10 mL of sterile SCYEM, and the suspension was poured into a new 25 cm<sup>3</sup> flask and incubated at 22 ◦C ± 2 ◦C or at 37 ◦C ± 2 ◦C. This time point corresponded to the T0 time point of the assay. Each condition was performed for three independent replicates and repeated three times (n = 9), with the exception of the co-culture with strain Philadelphia that was repeated four times at 22 ◦C (n = 15).

#### *4.6. L. pneumophila and Amoeba Quantifications in Co-Culture Assays from T0 to T0* + *96 h*

Occurring at T0, T0 + 24 h, T0 + 48 h, T0 + 72 h, and T0 + 96 h, a washing step was performed. The culture supernatant was removed from each flask and replaced by 10 mL of sterile SCYEM. This step was intended to remove extracellular *L. pneumophila* to allow for the detection of only intracellular bacteria. Each flask was gently shaken 10 times and an aliquot of 1 mL was sampled. Quantification of amoeba populations was performed using 0.1 mL of each aliquot utilizing a haemocytometer cell counting chamber method with Trypan blue. The remaining 0.9 mL were treated with Triton™ X-100 [31] at 0.02% v/v (final concentration) for 2 min to lyse amoebas and to recover the internal *L. pneumophila*. The sample was then serially 10-fold diluted in SCYEM and plated on BCYE plates in triplicate, with the exception of the undiluted conditions that were spread onto five plates when the number of *L. pneumophila* was intended to decrease below the detection limit. BCYE plates were incubated at 36 ◦C, and CFU were counted after 5 days.

#### *4.7. Microscopic Observations in Co-Culture with L. pneumophila Philadelphia at 37* ◦*C*

Co-cultures of *L. pneumophila* Philadelphia using the three amoeba strains at 37 ◦C were sampled from running experiments and stained by the Gimenez technique [50,51] at T0, T0 + 48 h, and T0 + 96 h. Co-cultures (0.1 mL) were deposited onto glass slides by using a Shandon Cytospin 4 cytocentrifuge (Thermo Scientific, Illkirch-France) at 800× *g* for 10 min and then stained using the Gimenez technique. Briefly, each of the glass slides were stained with fuchsin solution for 3 min and washed with water. Then, the glass slides were stained with malachite green for 5–10 s and washed, and this step was repeated twice. Finally, the glass slides were allowed to dry at room temperature.

The observations were performed using a LEICA DM 2500 LED microscope (Leica Microsystemes SAS, Nanterre-France) under an ×100 oil immersion objective.
