Temporal genome-wide fitness analysis of Mycobacterium marinum during infection reveals the genetic requirement for virulence and survival in amoebae and microglial cells

• Published in: mSystems Vol. 9; no. 2; p. e0132623
• Main Authors: Lefrançois, Louise H, Nitschke, Jahn, Wu, Huihai, Panis, Gaël, Prados, Julien, Butler, Rachel E, Mendum, Tom A, Hanna, Nabil, Stewart, Graham R, Soldati, Thierry
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 20-02-2024
• Subjects: Amoeba; Bacteria; BV2 microglial cells; Dictyostelium discoideum; DNA damage; DNA repair; Drug resistance; essentiality; Fatty acids; Genetic engineering; Genomes; Glycolysis; Host-Microbial Interactions; Infections; Infectious diseases; Innate immunity; Insertional mutagenesis; Lipid metabolism; Lipids; Localization; Microglial cells; Mutation; Mycobacterium marinum; Next-generation sequencing; Nutrient availability; Pathogenesis; Pathogens; Phagocytes; phenotypic profiling; Reproductive fitness; Research Article; Tn-Seq; Transposons; Tuberculosis; Virulence; Dictyostelium discoideum; essentiality; Mycobacterium marinum; Tn-Seq; BV2 microglial cells; phenotypic profiling
• ISSN: 2379-5077; 2379-5077
• DOI: 10.1128/msystems.01326-23

Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A key feature of pathogenesis, conserved between the human pathogen
and the model pathogen
is the metabolic switch to lipid catabolism and altered expression of virulence genes at different stages of infection. This study aims to identify genes involved in sustaining viable intracellular infection. We applied transposon sequencing (Tn-Seq) to
, an unbiased genome-wide strategy combining saturation insertional mutagenesis and high-throughput sequencing. This approach allowed us to identify the localization and relative abundance of insertions in pools of transposon mutants. Gene essentiality and fitness cost of mutations were quantitatively compared between
growth and different stages of infection in two evolutionary distinct phagocytes, the amoeba
and the murine BV2 microglial cells. In the
genome, 57% of TA sites were disrupted and 568 genes (10.2%) were essential, which is comparable to previous Tn-Seq studies on
and
. Major pathways involved in the survival of
during infection of
are related to DNA damage repair, lipid and vitamin metabolism, the type VII secretion system (T7SS) ESX-1, and the Mce1 lipid transport system. These pathways, except Mce1 and some glycolytic enzymes, were similarly affected in BV2 cells. These differences suggest subtly distinct nutrient availability or requirement in different host cells despite the known predominant use of lipids in both amoeba and microglial cells.IMPORTANCEThe emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system
/
to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for
to identify conservation of virulence pathways between hosts.