Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner.

Autophagy is a eukaryotic catabolic process also participating in cell-autonomous defence. Infected host cells generate double-membrane autophagosomes that mature in autolysosomes to engulf, kill and digest cytoplasmic pathogens. However, several bacteria subvert autophagy and benefit from its machinery and functions. Monitoring infection stages by genetics, pharmacology and microscopy, we demonstrate that the ESX-1 secretion system of Mycobacterium marinum, a close relative to M. tuberculosis, upregulates the transcription of autophagy genes, and stimulates autophagosome formation and recruitment to the mycobacteria-containing vacuole (MCV) in the host model organism Dictyostelium. Antagonistically, ESX-1 is also essential to block the autophagic flux and deplete the MCV of proteolytic activity. Activators of the TORC1 complex localize to the MCV in an ESX-1-dependent manner, suggesting an important role in the manipulation of autophagy by mycobacteria. Our findings suggest that the infection by M. marinum activates an autophagic response that is simultaneously repressed and exploited by the bacterium to support its survival inside the MCV

Contribution à l'étude de l'altération Crippled Growth chez le champignon filamenteux Podospora anserina (implication de voies de signalisation MAP kinases)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Setting up and monitoring an infection of Dictyostelium discoideum with mycobacteria.

Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. Therefore, M. marinum is used as a model for the study of mycobacterial infection in various host organisms. Recently, the Dictyostelium-M. marinum system has been shown to be a valuable model that recapitulates the main features of the intracellular fate of M. marinum including phagosome maturation arrest, as well as its particular cell-to-cell dissemination mode. We present here a "starter kit" of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantitatively the intracellular bacterial growth

Survey on medicinal plants traditionally used in Senegal for the treatment of tuberculosis (TB) and assessment of their antimycobacterial activity

In West Africa, populations are used to taking traditional medicine as a first aid against common health problems. In this aspect, many plants are claimed to be effective in the treatment of Tuberculosis (TB), which according to the World Health Organization (WHO) remains one of the world's deadliest communicable diseases

Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model

Tuberculosis remains one of the major threats to public health worldwide. Given the prevalence of multi drug resistance (MDR) in Mycobacterium tuberculosis strains, there is a strong need to develop new anti-mycobacterial drugs with modes of action distinct from classical antibiotics. Inhibitors of mycobacterial virulence might target new molecular processes and may represent a potential new therapeutic alternative. In this study, we used a Dictyostelium discoideum host model to assess virulence of Mycobacterium marinum and to identify compounds inhibiting mycobacterial virulence. Among 9995 chemical compounds, we selected 12 inhibitors of mycobacterial virulence that do not inhibit mycobacterial growth in synthetic medium. Further analyses revealed that 8 of them perturbed functions requiring an intact mycobacterial cell wall such as sliding motility, bacterial aggregation or cell wall permeability. Chemical analogs of two compounds were analyzed. Chemical modifications altered concomitantly their effect on sliding motility and on mycobacterial virulence, suggesting that the alteration of the mycobacterial cell wall caused the loss of virulence. We characterized further one of the selected compounds and found that it inhibited the ability of mycobacteria to replicate in infected cells. Together these results identify new antimycobacterial compounds that represent new tools to unravel the molecular mechanisms controlling mycobacterial pathogenicity. The isolation of compounds with anti-virulence activity is the first step towards developing new antibacterial treatments

Identification of Anti-Mycobacterium and Anti-Legionella Compounds With Potential Distinctive Structural Scaffolds From an HD-PBL Using Phenotypic Screens in Amoebae Host Models

Tubercular Mycobacteria and Legionella pneumophila are the causative agents of potentially fatal respiratory diseases due to their intrinsic pathogenesis but also due to the emergence of antibiotic resistance that limits treatment options. The aim of our study was to explore the antimicrobial activity of a small ligand-based chemical library of 1255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and one assessing virulence of M. marinum. We set up these assays using two amoeba strains, the genetically tractable social amoeba Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, 64 (5.1%) compounds showed anti-infective/anti-virulence activity in at least one of the three assays. The intracellular assays hit rate varied between 1.7% (n = 22) for M. marinum and 2.8% (n = 35) for L. pneumophila with seven compounds in common for both pathogens. In parallel, 1.2% (n = 15) of the tested compounds were able to restore D. discoideum growth in the presence of M. marinum spiked in a lawn of food bacteria. We also validated the generality of the hits identified in the A. castellanii-M. marinum anti-infective screen using the D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 μM. Moreover, the chemical space and physico-chemical properties of the anti-M. marinum hits were compared to standard and candidate Mycobacterium tuberculosis (Mtb) drugs using ChemGPS-NP. A principle component analysis identified separate clusters for anti-M. marinum and anti-L. pneumophila hits unveiling the potentially new physico-chemical properties of these hits compared to standard and candidate M. tuberculosis drugs. Our studies underscore the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance in concert with a rationalized focused library of compounds to identify new chemical scaffolds and to dissect some of their properties prior to taking further steps toward compound development

Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drug-resistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba was performed to decipher the different host pathways impacted during infection. We investigated how fine-tunes its gene expression in response to infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport)

Adrenergic antagonists restrict replication of Legionella

Legionella pneumophila is a facultative intracellular bacterium, which upon inhalation can cause a potentially fatal pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in environmental amoebae and mammalian macrophages within a unique membrane-bound compartment, the 'Legionella-containing vacuole'. Bacteria are exposed to many environmental cues including small signalling molecules from eukaryotic cells. A number of pathogenic bacteria sense and respond to catecholamine hormones, such as adrenalin and noradrenalin, a process mediated via the QseBC two-component system in some bacteria. In this study, we examined the effect of adrenergic compounds on L. pneumophila, and discovered that the adrenergic receptor antagonists benoxathian, naftopidil, propranolol and labetalol, as well as the QseC sensor kinase inhibitor LED209, reduced the growth of L. pneumophila in broth or amoebae, while replication in macrophages was enhanced. Growth restriction was common to members of the genus Legionella and Mycobacterium, and was observed for L. pneumophila in the replicative but not stationary phase of the biphasic life cycle. Deletion of the L. pneumophila qseBC genes indicated that growth inhibition by adrenergics or LED209 is mediated only to a minor extent by this two-component system, implying the presence of other adrenergic sensing systems. This study identifies adrenergic molecules as novel inhibitors of extra- and intracellular growth of Legionella and reveals LED209 as a potential lead compound to combat infections with Legionella or Mycobacterium spp

Exploring anti-bacterial compounds against intracellular Legionella.

Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target