One size does not fit all: Lysosomes exist in biochemically and functionally distinct states.

Single-organelle resolution approaches have the potential to advance our knowledge of the heterogeneity of lysosome function. Challenging population-based models, we propose a "lysosome states" concept that links single lysosomes to function.

Increased dosage of DYRK1A leads to congenital heart defects in a mouse model of Down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21). DS is a gene dosage disorder that results in multiple phenotypes including congenital heart defects. This clinically important cardiac pathology is the result of a third copy of one or more of the approximately 230 genes on Hsa21, but the identity of the causative dosage-sensitive genes and hence mechanisms underlying this cardiac pathology remain unclear. Here, we show that hearts from human fetuses with DS and embryonic hearts from the Dp1Tyb mouse model of DS show reduced expression of mitochondrial respiration genes and cell proliferation genes. Using systematic genetic mapping, we determined that three copies of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1a) gene, encoding a serine/threonine protein kinase, are associated with congenital heart disease pathology. In embryos from Dp1Tyb mice, reducing Dyrk1a gene copy number from three to two reversed defects in cellular proliferation and mitochondrial respiration in cardiomyocytes and rescued heart septation defects. Increased dosage of DYRK1A protein resulted in impairment of mitochondrial function and congenital heart disease pathology in mice with DS, suggesting that DYRK1A may be a useful therapeutic target for treating this common human condition.

MptpB Inhibitor Improves the Action of Antibiotics against Mycobacterium tuberculosis and Nontuberculous Mycobacterium avium Infections.

Treatment of Mycobacterium tuberculosis and Mycobacterium avium infections requires multiple drugs for long time periods. Mycobacterium protein-tyrosine-phosphatase B (MptpB) is a key M. tuberculosis virulence factor that subverts host antimicrobial activity to promote intracellular survival. Inhibition of MptpB reduces the infection burden in vivo and offers new opportunities to improve current treatments. Here, we demonstrate that M. avium produces an MptpB orthologue and that the MptpB inhibitor C13 reduces the M. avium infection burden in macrophages. Combining C13 with the antibiotics rifampicin or bedaquiline showed an additive effect, reducing intracellular infection of both M. tuberculosis and M. avium by 50%, compared to monotreatment with antibiotics alone. This additive effect was not observed with pretomanid. Combining C13 with the minor groove-binding compounds S-MGB-362 and S-MGB-363 also reduced the M. tuberculosis intracellular burden. Similar additive effects of C13 and antibiotics were confirmed in vivo using Galleria mellonella infections. We demonstrate that the reduced mycobacterial burden in macrophages observed with C13 treatments is due to the increased trafficking to lysosomes.

Stress granules plug and stabilize damaged endolysosomal membranes.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

ER-dependent membrane repair of mycobacteria-induced vacuole damage.

IMPORTANCE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

Host cell environments and antibiotic efficacy in tuberculosis.

The aetiologic agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), can survive, persist, and proliferate in a variety of heterogeneous subcellular compartments. Therefore, TB chemotherapy requires antibiotics crossing multiple biological membranes to reach distinct subcellular compartments and target these bacterial populations. These compartments are also dynamic, and our understanding of intracellular pharmacokinetics (PK) often represents a challenge for antitubercular drug development. In recent years, the development of high-resolution imaging approaches in the context of host-pathogen interactions has revealed the intracellular distribution of antibiotics at a new level, yielding discoveries with important clinical implications. In this review, we describe the current knowledge regarding cellular PK of antibiotics and the complexity of drug distribution within the context of TB. We also discuss the recent advances in quantitative imaging and highlight their applications for drug development in the context of how intracellular environments and microbial localisation affect TB treatment efficacy.

Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.

Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria.

Quantitative Spatio-temporal Analysis of Phagosome Maturation in Live Cells.

Phagocytosis and phagosome maturation are central processes to the development of the innate and adaptive immune response. Phagosome maturation is a continuous and dynamic process that occurs rapidly. In this chapter we describe fluorescence-based live cell imaging methods for the quantitative and temporal analysis of phagosome maturation of beads and M. tuberculosis as two phagocytic targets. We also describe simple protocols for monitoring phagosome maturation: the use of the acidotropic probe LysoTracker and analyzing the recruitment of EGFP-tagged host proteins by phagosomes.

ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages.

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR-Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.

A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages.

Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves the transformation of infected phagolysosomes from a site of killing into a nutrient-rich replicative niche. Here, we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis, 1-tuberculosinyladenosine (1-TbAd), caused lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing M. tuberculosis, caused intralysosomal and peribacillary lipid storage patterns that matched both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters and that 1-TbAd increased M. tuberculosis growth under conditions of restricted lipid access in macrophages. Furthermore, lipidomics identified 1-TbAd-induced lipid substrates that define Gaucher's disease, Wolman's disease, and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of M. tuberculosis-induced lysosomal failure, leading to successful testing of an agonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.

High content quantitative imaging of Mycobacterium tuberculosis responses to acidic microenvironments within human macrophages.

Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane-bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy-based approaches to investigate Mtb responses to acidic environments in cellulo. We report high-content imaging modalities to probe Mtb sensing of external pH or visualise in real-time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step-by-step analyses that enable robust image-based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb-host cell interactions. These methods can be adapted to study host-pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival.

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism.

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required leakage of lysosomal cathepsins and was independent of mitophagy, mitoproteases and proteasome degradation. In an in vivo mouse model of endomembrane damage, live lung macrophages that internalised crystals displayed impaired mitochondrial function. Single-cell RNA-sequencing revealed that lysosomal damage skewed metabolic and immune responses in alveolar macrophages subsets with increased lysosomal content. Functionally, drug modulation of macrophage metabolism impacted host responses to Mycobacterium tuberculosis infection in an endomembrane damage dependent way. This work uncovers an inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

Intracellular niche switching as host subversion strategy of bacterial pathogens.

Numerous bacterial pathogens "confine" themselves within host cells with an intracellular localization as main or exclusive niche. Many of them switch dynamically between a membrane-bound or cytosolic lifestyle. This requires either membrane damage and/or repair of the bacterial-containing compartment. Niche switching has profound consequences on how the host cell recognizes the pathogens in time and space for elimination. Moreover, niche switching impacts how bacteria communicate with host cells to obtain nutrients, and it affects the accessibility to antibiotics. Understanding the local environments and cellular phenotypes that lead to niche switching is critical for developing new host-targeted antimicrobial strategies, and has the potential to shed light into fundamental cellular processes.

Visualizing Pyrazinamide Action by Live Single-Cell Imaging of Phagosome Acidification and Mycobacterium tuberculosis pH Homeostasis.

Mycobacterium tuberculosis segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and M. tuberculosis intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that M. tuberculosis can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH), or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by disrupting M. tuberculosis intrabacterial pH homeostasis in cellulo. By using M. tuberculosis mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. IMPORTANCE We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug. Our observations can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies for tuberculosis that target heterogenous bacterial populations within the host.

A sandPIT for Salmonella to play with efferosomes.

In this issue of Cell Host and Microbe, Hiyoshi et al. show that Salmonella uses a type III secretion system (T3SS-2) to damage the Salmonella-containing vacuole, leading to complement deposition on intracellular bacteria followed by neutrophil efferocytosis that protects intracellular bacteria from the respiratory burst.

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.

Progress in robotics for combating infectious diseases.

The world was unprepared for the COVID-19 pandemic, and recovery is likely to be a long process. Robots have long been heralded to take on dangerous, dull, and dirty jobs, often in environments that are unsuitable for humans. Could robots be used to fight future pandemics? We review the fundamental requirements for robotics for infectious disease management and outline how robotic technologies can be used in different scenarios, including disease prevention and monitoring, clinical care, laboratory automation, logistics, and maintenance of socioeconomic activities. We also address some of the open challenges for developing advanced robots that are application oriented, reliable, safe, and rapidly deployable when needed. Last, we look at the ethical use of robots and call for globally sustained efforts in order for robots to be ready for future outbreaks.

Human stem cell-based models for studying host-pathogen interactions.

The use of human cell lines and primary cells as in vitro models represents a valuable approach to study cellular responses to infection. However, with the advent of new molecular technologies and tools available, there is a growing need to develop more physiologically relevant systems to overcome cell line model limitations and better mimic human disease. Since the discovery of human stem cells, its use has revolutionised the development of in vitro models. This is because after differentiation, these cells have the potential to reflect in vivo cell phenotypes and allow for probing questions in numerous fields of the biological sciences. Moreover, the possibility to combine the advantages of stem cell-derived cell types with genome editing technologies and engineered 3D microenvironments, provides enormous potential for producing in vitro systems to investigate cellular responses to infection that are both relevant and predictive. Here, we discuss recent advances in the use of human stem cells to model host-pathogen interactions, highlighting emerging technologies in the field of stem cell biology that can be exploited to investigate the fundamental biology of infection. TAKE AWAYS: hPSC overcome current limitations to study host-pathogen interactions in vitro. Genome editing can be used in hPSC to study cellular responses to infection. hPSC, 3D models and genome editing can recreate physiological in vitro systems.

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.