**1. Introduction**

The genus *Legionella* includes more than 60 species, but human infections that progress to a severe pneumonia, known as Legionnaires' disease, are most often caused by *Legionella pneumophila* [1,2]. This Gram-negative bacterium especially a ffects immunocompromised individuals after inhalation of Legionella-contaminated aerosols. *L. pneumophila* invades and replicates within alveolar macrophages and epithelial cells of the lungs, inducing a severe respiratory infection [1,3].

*L. pneumophila* is ubiquitous in natural, artificial, and industrial aquatic environments and is mostly nested in intracellular hosts, such as free-living amoebae (FLA). In 1980, Rowbotham first demonstrated the intracellular multiplication of *L. pneumophila* within *Acanthamoeba* spp. and *Naegleria* spp. [4]. Following this study, several reports described the replication of *Legionella* isolates from clinical samples within protozoa isolated from the presumed source of infection. Intracellular growth within protozoa was shown to increase the ability of *L. pneumophila* to infect human monocytes and to resist to chemical disinfectants, biocides, and antibiotics [5–7]. Inhalation of legionellae packaged in amoebae is associated with the induction of more severe clinical cases of legionellosis. The speculated link between the homing of legionella in amoebae and the increased virulence of legionella is supported by the publication of a mouse model of co-inhalation of *L. pneumophila* and *Hartmannella vermiformis*. It was shown to significantly enhance the intrapulmonary growth of *L. pneumophila*, with a greater mortality than that observed from inhalation of legionellae alone [8]. The intra-amoebae growth was

demonstrated to enhance the ability of *L. pneumophila* to infect epithelial cells (100- to 1000-fold), murine macrophages (10- to 100-fold), human monocytes (100- to 1000-fold), and *Acanthamoeba castellanii* (10- to 100-fold) [9]. Moreover, *L. pneumophila* grown in *A. castellanii* displays enhanced infection in monocytes compared to buffered charcoal yeas<sup>t</sup> extract (BCYE)-grown bacteria [10]. Within FLA and human macrophages, *L. pneumophila* cells are able to reroute the phagosome thanks to a functional Dot/Icm type 4 secretion system (T4SS) and the approximately 300 proteins it secretes and to induce the biogenesis of a legionella-containing vacuole (LCV) [11,12]. Bacterial replication occurs in LCV, evades fusion with lysosomes, and associates intimately with the host endoplasmic reticulum (ER), inducing the lysis of the host cells. The induction of apoptosis in the host cell is induced by a type IV-like secretion machinery [13]. The internalization of *L. pneumophila* into host cells, such as FLA, promotes not only its ability to survive and multiply but also to acquire and increase its virulence [14]. The *L. pneumophila* Dot/Icm-secreted effector PlcC/CegC1 was demonstrated to promote virulence [15], and the translocated Dot/Icm type IVB secretion system effector SdhA was demonstrated to be of crucial importance in infection processes [16].

Genes of the T4SS system have also been identified, with several other genes, as being responsible for the increased virulence in *L. pneumophila* once internalized by FLA, such as *A. castellanii* and *Vermamoeba vermiformis* [17]. In two studies from NJ Ashbolt's laboratory, about 30 transcripts of genes involved in bacterial metabolism, replication, and virulence have been investigated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in *L. pneumophila* Philadelphia after exposure to CuO nanoparticles (CuO-NPs) or synthetic gray water (Gw) for a period ranging from 3 to 48 h [18,19].

Here, we investigated the expression level by RT-qPCR of 7 genes (*htpX*, *icmE*, *lirR*, *ccmF*, *gacA*, *tatB*, and *lvrE*) that we have shown to be expressed by all three reference strains, *L. pneumophila* Paris, Philadelphia, and Lens, after co-incubation with two FLA: *A. castellanii* known to be permissive to the legionella multiplication [10], and *Willaertia magna* C2c Maky considered as non-permissive to the legionella multiplication [20]. We also evaluated transcript levels of these genes in *L. pneumophila* cultivated alone and harvested at the end of the growth exponential phase corresponding to the end of the replicative phase and the beginning of the virulence phase. The aim of this work was to evaluate the evolution of virulence of *L. pneumophila* strains after internalization into a non-permissive amoeba, such as *W. magna* C2c Maky. To facilitate the interpretation and comparison of gene expression, a GENE EXPRESSION-based index was developed.
