Willaertia magna C2c maky is a thermophilic free-living amoeba strain that showed ability to eliminate Legionella pneumophila, a pathogenic bacterium living in the aquatic environment. The amoeba industry has proposed the use of Willaertia magna as a natural biocide to control L. pneumophila proliferation in cooling towers. Here, transcriptomic and proteomic studies were carried out in order to expand knowledge on W. magna produced in a bioreactor. Illumina RNA-seq generated 217 million raw reads. A total of 8,790 transcripts were identified, of which 6,179 and 5,341 were assigned a function through comparisons with National Center of Biotechnology Information (NCBI) reference sequence and the Clusters of Orthologous Groups of proteins (COG) databases, respectively. To corroborate these transcriptomic data, we analyzed the W. magna proteome using LC–MS/MS. A total of 3,561 proteins were identified. The results of transcriptome and proteome analyses were highly congruent. Metabolism study showed that W. magna preferentially consumed carbohydrates and fatty acids to grow. Finally, an in-depth analysis has shown that W. magna produces several enzymes that are probably involved in the metabolism of secondary metabolites. Overall, our multi-omic study of W. magna opens the way to a better understanding of the genetics and biology of this amoeba.

Legionella pneumophila is a human pathogen responsible for a severe form of pneumonia named Legionnaire disease. Its natural habitat is aquatic environments, being in a free state or intracellular parasites of free-living amoebae, such as Acanthamoeba castellanii. This pathogen is able to replicate within some amoebae. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to resist to L. pneumophila and even to be able to eliminate the L. pneumophila strains Philadelphia, Lens, and Paris. Here, we studied the induction of seven virulence genes of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky in comparison within A. castellanii and with the gene expression level of L. pneumophila strains alone used as controls. We defined a gene expression-based virulence index to compare easily and without bias the transcript levels in different conditions and demonstrated that W. magna C2c Maky did not increase the virulence of L. pneumophila strains in contrast to A. castellanii. These results confirmed the non-permissiveness of W. magna C2c Maky toward L. pneumophila strains.

Free-living amoebae are known to act as replication niches for the pathogenic bacterium Legionella pneumophila in freshwater environments. However, we previously reported that some strains of the Willaertia magna species are more resistant to L. pneumophila infection and differ in their ability to support its growth. From this observation, we hypothesize that L. pneumophila growth in environment could be partly dependent on the composition of amoebic populations and on the possible interactions between different amoebic species. We tested this hypothesis by studying the growth of L. pneumophila and of a permissive free-living amoeba, Vermamoeba vermiformis (formerly named Hartmannella vermiformis), in co-culture with or without other free-living amoebae (Acanthamoeba castellanii and W. magna). We demonstrate the occurrence of inter-amoebic phagocytosis with A. castellanii and W. magna being able to ingest V. vermiformis infected or not infected with L. pneumophila. We also found that L. pneumophila growth is strongly impacted by the permissiveness of each interactive amoeba demonstrating that L. pneumophila proliferation and spread are controlled, at least in part, by inter-amoebic interactions.

Willaertia magna c2c maky is a thermophilic amoeba closely related to the genus Naegleria. This free-living amoeba has the ability to eliminate Legionella pneumophila, which is an amoeba-resisting bacterium living in an aquatic environment. To prevent the proliferation of L. pneumophila in cooling towers, the use of W. magna as natural biocide has been proposed. To provide a better understanding of the W. magna genome, whole-genome sequencing was performed through the study of virulence factors and lateral gene transfers. This amoeba harbors a genome of 36.5 megabases with 18,519 predicted genes. BLASTp analyses reported protein homology between 136 W. magna sequences and amoeba-resistant microorganisms. Horizontal gene transfers were observed based on the basis of the phylogenetic reconstruction hypothesis. We detected 15 homologs of N. fowleri genes related to virulence, although these latter were also found in the genome of N. gruberi, which is a non-pathogenic amoeba. Furthermore, the cytotoxicity test performed on human cells supports the hypothesis that the strain c2c maky is a non-pathogenic amoeba. This work explores the genomic repertory for the first draft genome of genus Willaertia and provides genomic data for further comparative studies on virulence of related pathogenic amoeba, N. fowleri.