Study of the amoeba Willaertia magna C2c Maky and free-living amoebas belonging to the phylum Amoebozoa by multi-omics approaches

Les amibes libres sont des micro-organismes unicellulaires eucaryotes vivant dans les sols ou les milieux aquatiques. Ces protistes sont des prédateurs dans l’environnement, jouant un rôle important dans la régulation des communautés microbiennes. Cependant, de nombreux microorganismes ont évolué pour résister à la digestion amibienne favorisant ainsi la propagation de bactéries potentiellement pathogènes dans l’environnement telles que les Legionella pneumophila. Willaertia magna C2c Maky s'est illustré par sa capacité à inhiber la multiplication intracellulaire de ce pathogène. Fondée sur cette propriété, la société Amoéba a développé un biocide naturel afin de contrôler la prolifération des légionnelles au sein des réseaux d’eau.Cette thèse se décompose en plusieurs projets. Nous avons dans un premier temps réalisé une analyse approfondie de l’amibe W. magna utilisée comme substance active du biocide naturel « Biomeba ». Notre étude génomique a permis d’améliorer les connaissances sur l’amibe libre W. magna. Les analyses multi-omiques ont apporté des renseignements complémentaires sur le métabolisme et la biologie de l’amibe, fournissant des pistes d’études importantes dans le but d’améliorer les conditions de culture. Dans la seconde partie de cet ouvrage, nos travaux ont porté sur la description et la caractérisation d’amibes libres appartenant à l’embranchement des Amoebozoa sur le plan génomique. L’ensemble de ces travaux a amélioré nos connaissances sur les amibes libres appartenant aux phylums des Excavata et Amoebozoa en apportant des informations essentielles sur l’évolution, le métabolisme et leurs interactions avec le monde microbien.Free-living amoebas are eukaryotic microorganisms living in soil or aquatic environments. These protists are predators in the environment, playing an important role in the regulation of microbial communities. However, many microorganisms have evolved to resist amoebic digestion, thus promoting the spread of potentially pathogenic bacteria in the environment such as Legionella pneumophila. Willaertia magna C2c Maky showed the ability to inhibit the intracellular multiplication of the pathogen. Based on this property, the company Amoéba has developed a natural biocide in order to control the proliferation of legionella bacteria in water networks.This thesis breaks down into several projects. We first carried out an in-depth analysis of the amoeba W. magna used as an active substance in the natural biocide "Biomeba". Our genomic study has improved knowledge about W. magna. The multi-omics analyzes provided additional information on the metabolism and biology of the amoeba, providing important lines of study in order to improve the culture conditions.In the second part of this book, our work focused on the description and characterization of free-living amoebas belonging to the Amoebozoa branch on the genomic level.All of this work has improved our knowledge of free-living amoebas belonging to the phyla of Excavata and Amoebozoa by providing essential information on evolution, metabolism and their interactions with the microbial world

Description of Virulent Factors and Horizontal Gene Transfers of Keratitis-Associated Amoeba Acanthamoeba Triangularis by Genome Analysis

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Confirmatory Virucidal Activity of Ionised Active Water S-100® on the SARS-CoV-2 Virus

International audienceIonised active water S-100® has been proposed as an original solution for use in dermocosmetics and for the treatment of wounds such as burns and atopic dermatitis. Among the mechanisms of action that are not completely understood, an antimicrobial activity would appear to be important. In the context of the COVID-19 pandemic, we assessed the inactivating efficacy of this solution on SARS-CoV-2 based on the recommendations of the NF-EN-14476+A2 standard. The tests carried out demonstrated that ionised active water S-100® 40% has a virucidal activity on SARS-CoV-2 which is at least 3.1 log after a contact time of 30 seconds and 3.5 log after two minutes at 20°C under clean conditions. Assays were also performed at 4°C and 37°C, and the results obtained are identical to those obtained at 20°C. This demonstration of the virucidal effect of ionised water against SARS-CoV-2 paves the way for the development of usage as an alternative disinfectant in SARS-CoV-2 control

Interest of bacterial pangenome analyses in clinical microbiology

International audienceThanks to the progress and decreasing costs in genome sequencing technologies, more than 250,000 bacterial genomes are currently available in public databases, covering most, if not all, of the major human-associated phylogenetic groups of these microorganisms, pathogenic or not. In addition, for many of them, sequences from several strains of a given species are available, thus enabling to evaluate their genetic diversity and study their evolution. In addition, the significant cost reduction of bacterial whole genome sequencing as well as the rapid increase in the number of available bacterial genomes have prompted the development of pangenomic software tools. The study of bacterial pangenome has many applications in clinical microbiology. It can unveil the pathogenic potential and ability of bacteria to resist antimicrobials as well identify specific sequences and predict antigenic epitopes that allow molecular or serologic assays and vaccines to be designed. Bacterial pangenome constitutes a powerful method for understanding the history of human bacteria and relating these findings to diagnosis in clinical microbiology laboratories in order to optimize patient management

Whole-Genome Sequence of French Clinical Peptoniphilus catoniae Strain P8546

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Vermamoeba vermiformis CDC-19 draft genome sequence reveals considerable gene trafficking including with candidate phyla radiation and giant viruses

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Proteomics of the Oomycete <i>Phytophthora parasitica</i> Strain INRA 310

The phytopathogen Phytophthora parasitica, from the Oomycetes class, known to be the tobacco black shank agent, can induce devastating diseases in various crop, plant and forest ecosystems. The genus Phytophthora has been studied at the cellular level, suggesting that different developmental steps are induced by the expression of some specific genes. However, these studies have only been carried out on certain species, such as Phytophthora infestans and Phytophthora cactorum. As for Phytophthora parasitica, which can be considered as one of the top ten oomycete pathogens due to the economic impact and effect it has on food security, even less functional analyses and transcriptomics data are available. To date, little is known about the protein expression of Phytophthora parasitica, information that is essential for achieving a better understanding of this species. In this study, we aimed to gain insight into the proteomics of the mycelium of the Phytophthora parasitica strain INRA 310 by addressing the following questions: (i) how many predicted proteins can be detected on the mycelium of P. parasitica INRA 310, and (ii) what proteins can be detected? The proteomics experiments were performed on the mycelium of the strain Phytophthora parasitica INRA310, using the nanoliquid chromatography-MS/MS technique. A total of 219 proteins were identified, including ten unknown proteins and 209 proteins involved in lipid, carbohydrate, nucleotide, energy production and other metabolic pathways. This proteomics study is, to our knowledge, the first to be performed on the mycelium of Phytophthora parasitica INRA 310. It gives a brief first insight into its in vitro-expressed proteins. This work may be the first step before further, more comprehensive studies are undertaken with the aim of better understanding the biology of this species and its pathogenicity

Intracellular Behaviour of Three Legionella pneumophila Strains within Three Amoeba Strains, Including Willaertia magna C2c Maky

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Insight into the Lifestyle of Amoeba Willaertia magna during Bioreactor Growth Using Transcriptomics and Proteomics

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 8790 transcripts were identified, of which 6179 and 5341 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 3561 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

Genomic analysis of a Raoultella ornithinolytica strain causing prosthetic joint infection in an immunocompetent patient

International audienceWe sequenced the genome of Raoultella ornithinolytica strain Marseille-P1025 that caused a rare case of prosthetic joint infection in a 67-year-old immunocompetent male. The 6.7-Mb genome exhibited a genomic island (RoGI) that was unique among R. ornithinolytica strains. RoGI was likely acquired by lateral gene transfer from a member of the Pectobacterium genus and coded for a type IVa secretion system found in other pathogenic bacteria and that may have conferred strain Marseille-P1025 an increased virulence. Strain Marseille-P1025 was also able to infect, multiply within, and kill Acanthamoaeba castellanii amoebae