Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, remains the world's deadliest infectious disease. Sterilising chemotherapy requires at least six months of multidrug therapy. Difficulty visualising the subcellular localisation of antibiotics in infected host cells means that it is unclear whether antibiotics penetrate into all mycobacteria-containing compartments in the cell. Here, we combine correlated light, electron and ion microscopy to image the distribution of Bedaquiline in infected human macrophages at sub-micrometre resolution. Bedaquiline accumulated primarily in host cell lipid droplets, but heterogeneously in mycobacteria within a variety of intracellular compartments. Furthermore, lipid droplets did not sequester antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Thus, strong lipid binding facilitated drug trafficking by host organelles to an intracellular target during antimicrobial treatment.Mycobacterium tuberculosis (Mtb) can persist in multiple intracellular niches within human * Correspondence to: max.
Mycobacterium tuberculosis triggers macrophage cell death by necrosis, but it is unclear how this affects bacterial replication. Lerner et al. show that this pathogen replicates within necrotic human macrophages before disseminating to other cells upon loss of plasma membrane integrity.
SummaryBacterial nutrition is an essential aspect of host–pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a ‘multi‐omics’ approach consisting of transposon‐directed insertion site sequencing (TraDIS), RNA‐seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry‐based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl‐CoA, the essential precursor for the biosynthesis of the odd‐chain fatty acids.
To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.
18The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to 19 its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis 20 remains obscure. We show that several M. tuberculosis clinical isolates form intracellular 21 cords in primary human lymphatic endothelial cells (hLEC) in vitro and also in the lymph 22 nodes of patients with tuberculosis. We identified via RNA-seq a transcriptional programme 23 in hLEC that activates cellular pro-survival and cytosolic surveillance of intracellular 24 pathogens pathways. Consistent with this, cytosolic access of hLEC is required for 25 intracellular M. tuberculosis cording; and cord formation is dependent on the M. 26 tuberculosis ESX-1 type VII secretion system and the mycobacterial lipid PDIM. Finally, we 27show that M. tuberculosis cording is a novel size-dependent mechanism used by the 28 pathogen to evade xenophagy in the cytosol of endothelial cells. These results provide a 29 mechanism that explains the long-standing association between M. tuberculosis cording and 30 virulence. 31Bacterial xenophagy is the process that regulates the removal of cytosolic bacteria after 64 damage to phagosomal membranes during selective macroautophagy (Galluzzi et al., 2017). 65This pathway constitutes one of the first cell autonomous defence pathways against 66 intracellular pathogens (Deretic and Levine, 2009; Gutierrez et al., 2004). A fraction of the 67 M. tuberculosis population damage phagosomes to access the cytosol and are subsequently 68 recognised by autophagic adaptors and the xenophagy machinery. This process targets M. 69 tuberculosis into autophagosomes and thus the lysosomal degradation pathway (Watson et 70 al., 2012). Whereas there is a large body of literature demonstrating autophagy as an anti-71 mycobacterial pathway (Deretic et al., 2009), recent evidence shows that M. tuberculosis 72 can eventually block the fusion of autophagosomes with lysosomes (Lerner et al., 2016; 73 Romagnoli et al., 2012) and in mice, M. tuberculosis can evade autophagic responses in vivo 74 (Kimmey et al., 2015). 75 76 M. tuberculosis mostly infects macrophages although there is compelling evidence that a 77 minor proportion of M. tuberculosis is found infecting various non-myeloid cells in the lungs 78and lymph nodes in vivo (Ganbat et al., 2016; Lerner et al., 2015; Nair et al., 2016; Randall et 79 al., 2015). The role that these M. tuberculosis subpopulations play in TB pathogenesis in 80 different cell types (e.g. immune vs non-immune) is unclear. We previously showed in 81 extrapulmonary tuberculosis that a subpopulation of M. tuberculosis is found in human 82 lymphatic endothelial cells (hLEC) in lymph node biopsies and these cells could represent a 83 reservoir for M. tuberculosis in infected patients (Lerner et al., 2016). 84 85 Here we discovered that M. tuberculosis forms large intracellular cords consisting of up to 86 thousands of individual bacteria arranged end-to-end in hLEC in vitro and in biopsies of 87 ...
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