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

ABSTRACT: Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple or even fully drug-resistant strains increases the risk and emphasizes the need for more efficient and better drug treatments. A key feature of mycobacteria pathogenesis, conserved between the human pathogen Mycobacterium tuberculosis and the model pathogen Mycobacterium marinum, is the metabolic switch to lipid catabolism during infection and altered expression of virulence genes in coordination with different stages of infection. This study aims at identifying genes that are involved in the establishment and maintenance of the infection. To achieve this, we have applied Transposon Sequencing (Tn-Seq) to M. marinum, an unbiased genome-wide strategy that combines saturation insertional mutagenesis and high throughput sequencing. This approach allowed us to precisely identify the localization and relative abundance of insertions in pools of transposon mutants. The essentiality of genes and the positive and negative fitness cost of mutations were quantitatively compared between in vitro growth and different stages of infection in two evolutionary distinct host phagocytes, the amoeba Dictyostelium discoideum and the murine BV2 microglial cells. We found that 57% of TA sites in the M. marinum genome were disrupted and that 568 genes (10.2%) were essential for M. marinum, which is comparable to previous Tn-Seq studies on M. tuberculosis and M. bovis. Major pathways involved in the survival of M. marinum during infection of D. discoideum were related to DNA damage repair, vitamin metabolism, the type VII secretion system (T7SS) ESX-1 and the Mce1 lipid transport system.

ORGANISM(S): Mycobacterium marinum

PROVIDER: GSE249096 | GEO | 2023/12/01

REPOSITORIES: GEO

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Project description:Background During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum recapitulates what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and to a large extent recapitulate the regulation in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

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

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