*4.2. FLA Culture*

The amoebae used in this study were *W. magna* C2c Maky ATCC PTA-7824 and *A. castellanii* ATCC 30010. Amoebae were grown at 30 ◦C for 3 days using adhesion culture on CF4 with serum casein glucose yeas<sup>t</sup> extract medium (SCGYEM) [31]. Amoeba cells were maintained in the exponential growth phase by subculturing every 3 days. Quantification of amoeba populations was performed using 0.1 mL of each aliquot utilizing a hemocytometer cell counting chamber method with Trypan blue.

#### *4.3. Coculture of L. pneumophila with Amoebae*

Tubes containing 5 mL of Peptone Yeast Extract Glucose Broth (PYG) [32] were seeded with 5 × 10<sup>5</sup> *W. magna* C2c Maky or *A. castellanii* cells. At Day 0, the different strains of *L. pneumophila*, grown on BCYE plates, were suspended in sterile distilled water at 2.5 × 10<sup>8</sup> cells/mL, and inoculated into the amoebic cultures at a multiplicity of infection (MOI) of 50. Low-speed centrifugation (30 min at 1000× *g*) was used to initiate physical interaction between bacteria and amoebae, and then incubated at 36 ◦C for 1 h. To eliminate extracellular legionella, cocultures were treated for 2 h at 30 ◦C with 0.5 mg/mL of Penicillin and Streptomycin. Cells were then washed twice by, first, centrifuging assay tubes for 10 min at 1000× *g*, second, removing the supernatant, and, finally, adding 5 mL of fresh PYG medium preheated at room temperature. After the second wash, cocultures were incubated at 30 ◦C in PYG medium for 3 days (Figure S13A).

In a second step, coculture solutions were submitted to a mechanical cell lysis using a FastPrep ®-24 instrument (MP Biomedicals, Illkirch-Gra ffenstaden, France) for 2 × 30 s at a speed of 5.0 in order to release legionella from the amoebae. Afterward, 100 μL of each treated coculture were deposited onto BCYE plates and incubated for 3 days at 36 ◦C (Figure S13B).

Control conditions consisted in legionella cultured in PYG medium in the absence of amoebae (FREE condition) for 3 days; for the second step, 100 μL of the latter culture were deposited onto BCYE plates for 3 additional days (Day 6), as described above.

#### *4.4. Preservation of RNA Samples*

Free legionella and cocultures (amoebae with internalized legionella) in liquid medium, or legionella colonies on BCYE plates were collected, rinsed in sterile osmosed water, and centrifuged for 5 min at 4 ◦C at 6000× *g*. The TRIzol ® Max ™ Bacterial RNA Isolation Kit (Thermo Fisher Scientific, Lyon, France) was used to improve the isolation of intact total RNA. The kit utilizes both the Max ™ Bacterial Enhancement Reagent and TRIzol ® Reagent to inactivate endogenous RNases and promote protein denaturing, improving RNA quality and integrity. After removal of the supernatant, 200 μL of pre-heated (95 ◦C) Max Bacterial Enhancement Reagent bu ffer was added on the pelleted cells and incubated for 5 min at 95 ◦C. Afterward, 1 mL of TRIzol Reagent was added, and the tubes were frozen in dry ice before being stored at −80 ◦C.

#### *4.5. Total RNA Extraction*

Biological material in TRIzol ® was used for total RNA extraction according to the manufacturer's instruction and MIQE guidelines [33]. Total nucleic acid extracted was treated with Turbo DNA-free DNAse (Thermo Fisher Scientific, Lyon, France) as recommended by the manufacturer, to remove genomic DNA. Total RNA was then transferred into a nuclease-free tube and stored at −80 ◦C after the concentration was measured using a Biodrop spectrophotometer (Biodrop, Cambridge, UK).

#### *4.6. Calibrated Reverse Transcription (cRT)*

Briefly, 180 ng of purified total RNAs were used for cRT using random hexamers and Multiscribe ™ reverse trancriptase in the presence of RNAse inhibitor (Thermo Fisher Scientific). A synthetic external and non-homologous Standard RNA (SmRNA) was used to control the quality of the RT and to normalize the reverse transcription of mRNAs of biological samples [34]. At the end of the RT, cDNAs were stored at −20 ◦C until further use. It should be noted that the Multiscribe ® reverse transcriptase was not added in the RT master mix as recommended by the manufacturer. Indeed, thanks to the data obtained for the SmRNA, we discerned that the enzyme was rapidly altered in the master mix, thus significantly a ffecting the e fficiency of reverse transcription across samples. This problem was resolved by preparing a master mix without the enzyme and then by adding the enzyme directly in each individual reaction tube.

#### *4.7. Quantitative PCR (qPCR)*

qPCR was performed using a Rotorgene Q (Qiagen, Courtaboeuf, France) and the Rotor-Gene SYBR Green PCR kit (Qiagen). All qPCR assays were run under the following conditions: 95 ◦C for 5 min, followed by 40 cycles of 5 s at 95 ◦C and 20–45 s (depending on the size of the amplicons) at 60 ◦C. The melting curve analysis was performed to evaluate the specificity of the DNA amplified during the PCR. The cycle of quantification Cq was determined at the intersection between the threshold

line and the amplification curves when data were displayed as semi-logarithmic representation of the accumulated fluorescence versus cycle number. Cq was then transformed into a number of cDNA copies, according to standard curves composed of standards with known copies of cDNAs (1 to 10<sup>8</sup> copies). These standard curves reliably indicated the method's detection sensitivity to targeted cDNAs (1 copy detected in 25% of cases) and the linear range of the quantification (from 10 to 10<sup>8</sup> copies). Primer sequences, the slope of the standard curve established between the Cq and the initial cDNA concentration, as well as the e fficiency of the qPCR of each targeted cDNAs, are presented in Table 1.

#### *4.8. Calculation of the Virulence Index*

As mentioned above (Section 2.3), the cDNA copy number contained in a given sample has been expressed in percent of the averaged copy number measured in all samples: 36 samples in each series of Task-1 (Lp Paris + Lp Philadelphia + Lp Lens) and 30 samples in Task-2 (Lp Paris + Lp Lens). Thus, at the end of each experiment series, and for each gene, the averaged "balanced" cDNA copy number calculated from all samples equaled 100. Finally, for a given sample, Gene expression-based Virulence Index was calculated as follows:

$$\text{Virulence} - \text{Index for sample A} = \sum\_{\mathbf{n}=1}^{7} \frac{\text{(cDNA copy nbr for gene}\_{(\mathbf{n})}) \text{in sample A} \times 100}{\text{average cDNA copy nbr for gene}\_{(\mathbf{n})} \text{ in all samples}}$$

#### *4.9. Data and Statistical Analysis*

For each group of the first step, experiments were replicated once with *n* = 3 in each replicate, and the data obtained in each replicate were pooled; therefore, *n* = 6 in each group. For the second step, experiments were performed once with *n* = 3 in each group. For each group, data are expressed as mean ± SD of either the number of copies of the di fferent cDNAs analyzed or the "gene expression"-based virulence index. Statistical significance for within-group comparisons was calculated by a two-way analysis of variance (ANOVA 2) with Tukey's post hoc test, using XLSTAT Software (version 19.4). The *p* value of 0.05 defined the significance cut-o ff.

When the *p* value for Factor 1 was below 0.05, meaning that di fferences between *L. pneumophila* strains were not statistically di fferent, data for *L. pneumophila* strains were pooled together with "*n* = n L. pneumophila Paris + n L. pneumophila Philadelphia + n L. pneumophila Lens."

For the first step, Factor 1 equals "L. pneumophila strain (Paris, Philadelphia, and Lens)" and Factor 2 equals "culture condition (T0, T3D-FREE, T3D-WILL, T3D-ACANTH)." For the virulence index, because ANOVA 2 showed no statistical di fference between the three strains (*p* = 0.832), values for the three strains were pooled together (*n* = 18).

For the second step, Factor 1 equals "L. pneumophila strain (Paris and Lens)" and Factor 2 equals "culture condition (T0, T3D-FREE, T6D-FREE, T6D-WILL, T6D-ACANTH)." Strain Philadelphia was not analyzed in this second step because after internalization into both amoebae, it did not grow on BCYE plates in 2 out of 3 samples; because *n* = 1 for this condition, data from strain Philadelphia were not considered for the statistical analysis. Because ANOVA 2 showed no statistical di fference between the two other strains (*p* = 0.566), values for both strains were pooled together (*n* = 6).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-0817/9/6/447/s1, Figure S1: Level of*rsp<sup>L</sup>* transcript in the different conditions, expressed as the number of copies in 10<sup>6</sup> *L. pneumophila* (Lp) ± SD, Figure S2: *ccmF* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S3: *gacA* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S4: *htpX* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S5: *icmE* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S6: *lirR* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S7: *lvrE* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S8: *tatB* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S9: *ccmF* and *gacA* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S10: *htpX* and *icmE* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S11: *lirR* and *lvrE* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S12: *tatB* cDNA copy number measured in 10<sup>6</sup> *L. pneumophila* for each strain and each culture condition, Figure S13: Synoptic diagram of the experiments, Table S1: Fold changes of gene expression after 3-48h exposure of *L. pneumophila* Philadelphia to CuO nanoparticles (CuO-NPs) or synthetic Gray water (Gw).

**Author Contributions:** Conceptualization, R.M.M., J.B. and L.B.; methodology, R.M.M.; formal analysis, L.B.; writing—original draft preparation; R.M.M., S.D., and L.B.; writing—review and editing, S.D. and L.B.; supervision, L.B.; L.B. and S.D. contributed equally to the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors are grateful to Florian Lanet, Béatrice Georges and Florian Bénitière for their technical assistance in RT-qPCR and to Benjamin Quelard for his technical assistance for the coculture experiments

**Conflicts of Interest:** J.B. is the co-founder of Amoeba SA.
