Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis-infected lungs.

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type-specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

Mycobacterium tuberculosis requires glyoxylate shunt and reverse methylcitrate cycle for lactate and pyruvate metabolism.

Bacterial 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.

Phosphorus availability in the source population influences response to dietary phosphorus quantity in a New Zealand freshwater snail.

We investigated whether previously documented variation among populations in availability of dietary phosphorus (P) is linked to heterogeneity in growth rate of the New Zealand freshwater snail Potamopyrgus antipodarum on a P-limited diet. We chose this system because P. antipodarum inhabits water bodies that vary in P availability and because P. antipodarum growth rate varies considerably in response to low P. We quantified specific growth rate and alkaline phosphatase (AP) expression in a diverse array of juvenile P. antipodarum fed high vs. low-P diets. We found strong associations between P content of epilithon in the source lake and P. antipodarum growth rate on high vs. low-P diets, with snails collected from lakes with relatively low-P epilithon showing the greatest increase in growth rate on the high-P relative to low-P diet. We also found substantial intraspecific variation in growth response to P limitation. Expression of AP also varied among lineages and was negatively associated with C: P of lake epilithon but did not explain the relationship between C: P in the lake of origin and sensitivity to P limitation. Together, our results demonstrate a strong signature of the P environment in the lake of origin on how this snail responds to P limitation as well as preliminary evidence for intraspecific variation of AP expression in animals.

Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide.

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

No evidence for a dilution effect of the non-native snail, Potamopyrgus antipodarum, on native snails.

The dilution effect can occur by a range of mechanisms and results in reduced parasite prevalence in host taxa. In invaded ecosystems, the dilution effect can benefit native species if non-native species, acting as resistant or less competent hosts, reduce rates of parasitic infections in native species. In field experiments, we assessed whether manipulating biomass of the non-native snail, Potamopyrgus antipodarum, caused a dilution effect by reducing trematode infections in three taxa of native snails. In contrast to many studies showing resistant or less competent non-native hosts can "dilute" or reduce infection rates, we found no evidence for a dilution effect reducing infection rates of any of the native snails. We suggest that a dilution effect may not have occurred because most trematode taxa are highly host specific, and thus the trematode transmission stages did not recognize the invasive snail as a possible host. In this case, community composition appears to be important in influencing the dilution effect.

Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity.

The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. Here, we show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLECs) in vitro and in the lymph nodes of patients with tuberculosis. We identified via RNA-Seq a transcriptional program that activated, in infected-hLECs, cell survival and cytosolic surveillance of pathogens pathways. Consistent with this, cytosolic access was required for intracellular M. tuberculosis cording. Mycobacteria lacking ESX-1 type VII secretion system or phthiocerol dimycocerosates expression, which failed to access the cytosol, were indeed unable to form cords within hLECs. Finally, we show that M. tuberculosis cording is a size-dependent mechanism used by the pathogen to avoid its recognition by cytosolic sensors and evade either resting or IFN-γ-induced hLEC immunity. These results explain the long-standing association between M. tuberculosis cording and virulence and how virulent mycobacteria use intracellular cording as strategy to successfully adapt and persist in the lymphatic tracts.

Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected macrophages.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, remains the world's deadliest infectious disease. Sterilizing chemotherapy requires at least 6 months of multidrug therapy. Difficulty visualizing the subcellular localization of antibiotics in infected host cells means that it is unclear whether antibiotics penetrate all mycobacteria-containing compartments in the cell. Here, we combined correlated light, electron, and ion microscopy to image the distribution of bedaquiline in infected human macrophages at submicrometer 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 replicates within necrotic human macrophages.

Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) integrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor-differentiated macrophages more than in granulocyte-macrophage colony-stimulating factor-differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial replication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host.

TransCelerate's Clinical Quality Management System: Issue Management.

Issue management is one of the elements of the TransCelerate clinical quality management (QMS) conceptual framework. Effective issue management benefits clinical development organizations by allowing them to focus efforts on those issues that materially impact patient safety, rights, and well-being; data integrity and/or scientific rigor; compliance with regulatory requirements; or trust in the clinical research enterprise, which have been defined as "Issues that Matter." Issue management begins with a triage of issues to identify those issues that materially impact as defined above, which are then handled by an end-to-end corrective and preventive action (CAPA) process. An added benefit of a well-defined issue management framework is the capability for trending and analytics designed to provide data and insights into risk management and knowledge management efforts.