Maximiliano G. Gutiérrez

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases, but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here, we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines, were characterized by a metabolism typical of quiescent or memory T cells, and did not participate in inflammatory processes. In contrast, infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines, disseminated widely into the periphery, and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defences. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) lives within phagosomes and also disrupts these organelles to access the cytosol. The host pathways and mechanisms that contribute to maintaining Mtb phagosome integrity have not been investigated. Here, we examined the spatiotemporal dynamics of Mtb-containing phagosomes and identified an interferon-gamma-stimulated and Rab20-dependent membrane trafficking pathway in macrophages that maintains Mtb in spacious proteolytic phagolysosomes. This pathway functions to promote endosomal membrane influx in infected macrophages, and is required to preserve Mtb phagosome integrity and control Mtb replication. Rab20 is specifically and significantly upregulated in the sputum of human patients with active tuberculosis. Altogether, we uncover an immune-regulated cellular pathway of defense that promotes maintenance of Mtb within intact membrane-bound compartments for efficient elimination.

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.

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.

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.

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-kappaB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-kappaB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-kappaB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-kappaB during infection. Our results argue that NF-kappaB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-kappaB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-kappaB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs. Analysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden. PDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

The success of tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), relies on the ability to survive in host cells and escape to immune surveillance and activation. We recently demonstrated that Mtb manipulation of host lysosomal cathepsins in macrophages leads to decreased enzymatic activity and pathogen survival. In addition, while searching for microRNAs (miRNAs) involved in posttranscriptional gene regulation during mycobacteria infection of human macrophages, we found that selected miRNAs such as miR-106b-5p were specifically upregulated by pathogenic mycobacteria. Here, we show that miR-106b-5p is actively manipulated by Mtb to ensure its survival in macrophages. Using an in silico prediction approach, we identified miR-106b-5p with a potential binding to the 3'-untranslated region of cathepsin S (CtsS) mRNA. We demonstrated by luminescence-based methods that miR-106b-5p indeed targets CTSS mRNA resulting in protein translation silencing. Moreover, miR-106b-5p gain-of-function experiments lead to a decreased CtsS expression favoring Mtb intracellular survival. By contrast, miR-106b-5p loss-of-function in infected cells was concomitant with increased CtsS expression, with significant intracellular killing of Mtb and T-cell activation. Modulation of miR-106b-5p did not impact necrosis, apoptosis or autophagy arguing that miR-106b-5p directly targeted CtsS expression as a way for Mtb to avoid exposure to degradative enzymes in the endocytic pathway. Altogether, our data suggest that manipulation of miR-106b-5p as a potential target for host-directed therapy for Mtb infection.

The mechanisms that lead to phenotypic antibacterial tolerance in bacteria remain poorly understood.We investigate whether changes in NaCl concentration toward physiologically higher values affect antibacterial efficacy against Mycobacterium tuberculosis (Mtb), the causal agent of human tuberculosis.Indeed, multiclass phenotypic antibacterial tolerance is observed during Mtb growth in physiologic saline.This includes changes in sensitivity to ethionamide, ethambutol, D-cycloserine, several aminoglycosides, and quinolones.By employing organism-wide metabolomic and lipidomic approaches combined with phenotypic tests, we identified a time-dependent biphasic adaptive response after exposure of Mtb to physiological levels of NaCl.A first rapid, extensive, and reversible phase was associated with changes in core and amino acid metabolism.In a second phase, Mtb responded with a substantial remodelling of plasma membrane and outer lipid membrane composition.We demonstrate that phenotypic tolerance at physiological concentrations of NaCl is the result of changes in plasma and outer membrane lipid remodeling and not changes in core metabolism.Altogether, these results indicate that physiologic saline-induced antibacterial tolerance is kinetically coupled to cell envelope changes and demonstrate that metabolic changes and growth arrest are not the cause of phenotypic tolerance observed in Mtb exposed to physiologic concentrations of NaCl.Importantly, this work uncovers a role for bacterial cell envelope remodeling in antibacterial tolerance, alongside well-documented allterations in respiration, metabolism, and growth rate.

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.

Mycobacterium tuberculosis protein-tyrosine-phosphatase B (MptpB) is a secreted virulence factor that subverts antimicrobial activity in the host. We report here the structure-based design of selective MptpB inhibitors that reduce survival of multidrug-resistant tuberculosis strains in macrophages and enhance killing efficacy by first-line antibiotics. Monotherapy with an orally bioavailable MptpB inhibitor reduces infection burden in acute and chronic guinea pig models and improves the overall pathology. Our findings provide a new paradigm for tuberculosis treatment.

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1–deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte–associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

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.

In the world of pathogen-host cell interactions, the autophagic pathway has beenrecently described as a component of the innate immune response against intracellularmicroorganisms. Indeed, some bacterial survival mechanisms are hampered when thisprocess is activated. Mycobacterium tuberculosis infection of macrophages, for example,is impaired upon autophagy induction and the bacterial phagosomes are redirected toautophagosomes. On the other hand, pathogens like Coxiella burnetii are benefited bythis cellular response and subvert the autophagy process resulting in a more efficient replication.We study at the molecular level these two different faces of the autophagy processin pathogen life in order to elucidate the intricate routes modulated by the microorganismsas survival strategies.

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.

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.

Vibrio cholerae is the causative agent of cholera in humans. In addition to the criticalvirulence factors cholera toxin and toxin coregulated pilus, V. cholerae secretes V.cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensivevacuolation. We have shown that this vacuolation is related to the activation of autophagyin response to VCC action. Furthermore, we found that the autophagic pathway wasrequired to protect cells upon VCC intoxication. Based on additional data presented here,we propose a model aimed to explain the mechanism of cell protection. We postulatethat VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involvingfusion with lysosomes.

Summary In vitro studies are crucial for our understanding of the human macrophage immune functions. However, traditional in vitro culture media poorly reflect the metabolic composition of blood, potentially affecting the outcomes of these studies. Here, we analysed the impact of a physiological medium on human induced pluripotent stem cell (iPSC)-derived macrophages (iPSDM) function. Macrophages cultured in a human plasma-like medium (HPLM) were more permissive to Mycobacterium tuberculosis (Mtb) replication and showed decreased lipid metabolism with increased metabolic polarisation. Functionally, we discovered that HPLM-differentiated macrophages showed different metabolic organelle content and activity. Specifically, HPLM-differentiated macrophages displayed reduced lipid droplet and peroxisome content, increased lysosomal proteolytic activity, and increased mitochondrial activity and dynamics. Inhibiting or inducing lipid droplet formation revealed that lipid droplet content is a key factor influencing macrophage permissiveness to Mtb. These findings underscore the importance of using physiologically relevant media in vitro for accurately studying human macrophage function.

Phagocytosis and phagosome maturation are central 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 latex beads and M. tuberculosis as two phagocytic targets. We also describe two simple protocols for monitoring phagosome maturation: the use of the acidotropic probe LysoTracker and analyzing the recruitment of EGFP-tagged host proteins by phagosomes.

In this study, we characterized the role of Rv2617c in the virulence of Mycobacterium tuberculosis. Rv2617c is a protein of unknown function unique to M. tuberculosis complex (MTC) and Mycobacterium leprae. In vitro, this protein interacts with the virulence factor P36 (also named Erp) and KdpF, a protein linked to nitrosative stress. Here, we showed that knockout of the Rv2617c gene in M. tuberculosis CDC1551 reduced the replication of the pathogen in a mouse model of infection and favored the trafficking of mycobacteria to phagolysosomes. We also demonstrated that Rv2617c and P36 are required for resistance to in vitro hydrogen peroxide treatment in M. tuberculosis and Mycobacterium bovis, respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that infects a wide range of hosts including humans, causing Q fever, a disease characterized by high fever and flu-like symptoms. After its internalization the Coxiella-containing phagosomes interact with intracellular compartments and generate a large replicative vacuole that displays certain characteristics of a phagolysosome. We have shown that this bacterially-customized replicative vacuole also has the hallmarks of an autophagosomal compartment. Furthermore, in a recent publication we have reported that induction of autophagy is beneficial for the replication and survival of Coxiella. Different morphological forms of this bacterium have been described during its developmental cycle. Here we present additional data and discuss a model indicating that induction of autophagy favors the differentiation of the Coxiella small cell variants to the metabolically active large cells variants. We postulate that nutrient acquisition, likely by fusion with the nutrient-rich autophagic vacuoles, triggers the development of the large cell variants which actively multiply in the host cell.

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.

Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.

Noncytotoxic ferromagnetic FePt nanomotors possess hard magnetic properties that rival NdFeB micromagnets and enable active gene delivery. In article 2001114, Maximiliano G. Gutierrez, Peer Fischer, and co-workers describe their fabrication and material characterization, and show that FePt is promising for microrobotics and biomedical applications.

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside of infected cells through modulation of host antibacterial attack, i.e. avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In this interplay between host cell and Mtb, bacterial phosphatases play a central role. In this work, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic study, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis. Finally, mutation of Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosome containing mycobacteria in early endosome, indicating that Rv2577 plays a role in the virulence of Mtb.

A cellular membrane remodeling machinery repairs damaged organelles

Abstract Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro , in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms. One Sentence Summary We show that the lack of D-cycloserine resistance in Mycobacterium tuberculosis is due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

Mitochondrial dynamics and metabolism in immune cells are closely associated. However, if these interactions play a role in the human macrophage response to <i>Mycobacterium tuberculosis</i> (Mtb) remains largely unknown. We used human induced-pluripotent stem cell derived macrophages (iPSDM) together with high-content live-cell imaging, extracellular flux analysis and unbiased metabolomics to investigate mitochondrial dynamics, Mtb intracellular replication and mitochondrial metabolism. Mitochondrial tracking at the single-cell level revealed extensive mitochondrial morphology heterogeneity. Surprisingly, Mtb did not induce significant changes in the mitochondrial area, length or width during the first 48h of infection. We did not find a correlation between Mtb intracellular replication and changes in mitochondrial morphological parameters. However, Mtb induced a decrease in the mitochondrial membrane potential and protein turnover rate, as visualised by MitoTracker Red and the ratiometric reporter MitoTimer, respectively. The metabolic profile of infected macrophages showed an increase in oxygen consumption and extracellular acidification rate after 48h of infection. These changes were associated with decreased mitochondrial protein and transcripts levels. The inhibition of glycolysis with 2-Deoxy-d-glucose promoted Mtb replication, whereas oxamate (lactate dehydrogenase inhibitor) impaired Mtb intracellular replication. Collectively, our results show that macrophage metabolic reprogramming is required for the control of Mtb replication. Unlike other intracellular pathogens, changes in host cell metabolism induced by Mtb might correlate with disruption of mitochondrial function rather than morphology.

Summary Mycobacterium tuberculosis (Mtb) infection can becleared by the innate immune system before theinitiation of an adaptive immune response. Thisinnate protection requires a variety of robust cellautonomous responses from many different hostimmune cell types. However, Mtb has evolvedstrategies to circumvent some of these defences.In this mini-review, we discuss these host–pathogen interactions with a focus on studies per-formed in human cells and/or supported by humangenetics studies (such as genome-wide associa-tion studies).Introduction Mycobacterium tuberculosis (Mtb) is particularly effectiveat subverting many of the host immune defences, and thisis one reason why it is such a successful human pathogenthat has been particularly hard to eradicate. The outcomeof infection by Mtb and therefore the clinical manifestationof tuberculosis (TB) depend on many combined factors,such as host genetics, bacterial genetics (virulencefactors), the health and nutritional status of the host andwhether there has been any prior exposure/immunity andvaccination history. Around half of individuals exposed toMtb do not exhibit a positive tuberculin skin test (Morrison

Summary 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 lysosomal leakage of Cathepsin B and Cathepsin L and was independent of proteasome degradation and mitophagy. In a mouse model of endomembrane damage, metabolic analysis confirmed that in vivo, live lung macrophages that internalised crystals displayed impaired mitochondrial function and increased glycolytic and lipid metabolism. Single-cell RNA-sequencing analysis of bronchoalveolar lavage revealed that lysosomal damage skewed metabolic and immune responses primarily in CD36 + /LIPA + and Krt79 + /Car4 + subsets of alveolar macrophages. Importantly, modulation of macrophage metabolism with 2-Deoxy- d- glucose and oxamate impacted the host response to Mycobacterium tuberculosis (Mtb) infection in an endomembrane damage dependent manner. This work uncovers a new inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

Abstract Neurodegenerative diseases such as Parkinson’s (PD) have a complex aetiology consisting of an interplay of genetic and environmental factors. Inflammation and infection are proposed external factors that trigger disease progression. Tuberculous and cryptococcal meningitis frequently lead to long-term neurological sequelae but their association with the development of PD are unexplored. In this study, we protein profiled the CSF from 76 patients with or without infectious meningitis and found that proteins commonly associated with PD (LRRK2, tau and alpha-synuclein) were significantly elevated, establishing a link between neuroinflammation and infection. Importantly, these findings suggest that LRRK2, tau and alpha-synuclein could represent biomarkers of neuroinflammation.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the host’s immune system. Here, we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed cells into potent bactericidal phagocytes. We found that 579 and 1,495 genes were respectively differentially expressed in naive- and M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of lysosome-associated genes. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB, involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections. eLife digest The discovery of antibiotic drugs, which treat diseases caused by bacteria, has been a hugely valuable advance in modern medicine. They work by targeting specific cellular processes in bacteria, ultimately stopping them from multiplying or killing them outright. Antibiotics sometimes also affect their human hosts and can cause side-effects, such as gut problems or skin reactions. Recent evidence suggests that antibiotics also have an impact on the human immune system. This may happen either indirectly, by affecting ‘friendly’ bacteria normally present in the body, or through direct effects on immune cells. In turn, this could change the effectiveness of drug treatments. For example, if an antibiotic weakens immune cells, the body could have difficulty fighting off the existing infection – or become more vulnerable to new ones. However, even though new drugs are being introduced to combat the worldwide rise of antibiotic-resistant bacteria, their effects on immunity are still not well understood. For example, bedaquiline is an antibiotic recently developed to treat tuberculosis infections that are resistant to several drugs. Giraud-Gatineau et al. wanted to determine if bedaquiline altered the human immune response to bacterial infection independently from its direct anti-microbial effects. Macrophages engulf foreign particles like bacteria and break them down using enzymes stored within small internal compartments, or ‘lysosomes’. Initial experiments using human macrophages, grown both with and without bedaquiline, showed that the drug did not harm the cells and that they grew normally. A combination of microscope imaging and genetic analysis revealed that exposure to bedaquiline not only increased the number of lysosomes within macrophage cells, but also the activity of genes and proteins that increase lysosomes’ ability to break down foreign particles. These results suggested that bedaquiline treatment might make macrophages better at fighting infection, even if the drug itself had no direct effect on bacterial cells. Further studies, where macrophages were first treated with bedaquiline and then exposed to different types of bacteria known to be resistant to the drug, confirmed this hypothesis: in every case, the treated macrophages became efficient bacterial killers. In contrast, older anti-tuberculosis drugs did not have any such potentiating effect on the macrophages. This work sheds new light on our how antibiotic drugs can interact with the cells of the human immune system, and can sometimes even boost our innate defences. Such immune-boosting effects could one day be exploited to make more effective treatments against bacterial infections. Introduction Antibiotics are commonly used in the treatment of bacterial infections, and, in effectively combating such diseases, have substantially increased human life expectancy. As with most drugs, antibiotic treatment can also alter host metabolism, leading to adverse side-effects, including nausea, hepatotoxicity, skin reactions, and gastrointestinal and neurological disorders. Such side-effects can become critical when antibiotic treatment is long and involves several drugs, as in the treatment of tuberculosis (TB), where 2–28% of patients develop mild liver injury during treatment with first-line drugs (Agal et al., 2005). Antibiotics can interfere with the immune system, indirectly through the disturbance of the body’s microbiota (Ubeda and Pamer, 2012), or directly by modulating the functions of immune cells. Such interactions may impact treatment efficacy or the susceptibility of the host to concomitant infection. For example, after treatment completion, TB patients are more vulnerable to reactivation and reinfection of the disease, suggesting therapy-related immune impairment (Cox et al., 2008). Drug-sensitive TB can be cured by combining up to four antibiotics in a 6-month treatment; specifically, isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and pyrazinamide (PZA) for 2 months, and INH and RIF for additional 4 months. INH induces apoptosis of activated CD4+ T cells in Mycobacterium tuberculosis (MTB)-infected mice (Tousif et al., 2014) and leads to a decrease in Th1 cytokine production in household contacts with latent TB under preventive INH therapy (Biraro et al., 2015). RIF has immunomodulatory properties and acts as a mild immunosuppressive agent in psoriasis (Tsankov and Grozdev, 2011). RIF reduces inflammation by inhibiting IκBα degradation, mitogen-activated protein kinase (MAPK) phosphorylation (Bi et al., 2011), and Toll-like receptor 4 signaling (Wang et al., 2013). PZA treatment of MTB-infected human monocytes and mice significantly reduces the release of pro-inflammatory cytokines and chemokines (Manca et al., 2013). Recently, Puyskens et al. showed that several anti-TB drugs bind to the aryl hydrocarbon receptor and may impact host defense (Puyskens et al., 2020). It is therefore necessary to understand how antibiotic treatment modulates macrophage functions, and more generally, how it impacts the host immune response. The worldwide rise in antibiotic resistance is a major threat to global health care. A growing number of bacterial infections, such as pneumonia, salmonellosis, and TB, are becoming harder to treat as the antibiotics used to treat them become less effective. While new antibiotics are being developed and brought to the clinic, their effects on the human immune system are not being studied in-depth. Here, we have investigated the impact of a recently approved anti-TB drug, bedaquiline (BDQ), on the transcriptional responses of human macrophages infected with MTB. Macrophages are the primary cell target of MTB, which has evolved several strategies to survive and multiply inside the macrophage phagosome, including prevention of phagosome acidification (Sturgill-Koszycki et al., 1994), inhibition of phagolysosomal fusion (Armstrong and Hart, 1975) and phagosomal rupture (Simeone et al., 2012; van der Wel et al., 2007). They play a central role in the host response to TB pathogenesis, by orchestrating the formation of granulomas, presenting mycobacterial antigens to T cells, and killing the bacillus upon IFN-γ activation (Cambier et al., 2014). BDQ is a diarylquinoline that specifically inhibits a subunit of the bacterial adenosine triphosphate (ATP) synthase, decreasing intracellular ATP levels (Andries et al., 2005; Koul et al., 2007). It has 20,000 times less affinity for human ATP synthase (Haagsma et al., 2009). The most common side effects of BDQ are nausea, joint and chest pain, headache, and arrhythmias (Diacon et al., 2012; TMC207-C208 Study Group et al., 2014). However, possible interactions between BDQ and the host immune response have not been studied in detail. Understanding the impact of BDQ on the host immune response may help to develop strategies aiming at improving drug efficacy and limiting side-effects, including cytotoxicity, alteration of cell metabolism, and immunomodulation. Results BDQ modulates the response of naïve and MTB-infected macrophages We treated human monocyte-derived macrophages from four healthy donors with BDQ at 5 µg/mL, which corresponds to the concentration detected in the plasma of TB patients treated with BDQ (Andries et al., 2005). This concentration did not affect cell viability over an incubation period of 7 days (Figure 1—figure supplement 1). After 18 hr of treatment, we characterized the genome-wide gene expression profiles of BDQ-treated macrophages by RNAseq, with DMSO-treated cells serving as a control. The expression of 579 genes was affected by BDQ (FDR < 0.05, Figure 1—source datas 1 and 2), with 186 being upregulated and 393 being downregulated. We classified all 579 genes by performing gene-set enrichment analysis using ClueGO cluster analysis (Bindea et al., 2009). The gene set upregulated by BDQ was significantly enriched for genes associated with lysosome, phagocytic vesicle membrane, vacuolar lumen, hydrolase activity and lipid homeostasis (Figure 1A). Figure 1 with 2 supplements see all Download asset Open asset BDQ modulates the response of human macrophages. Cells from four individual donors were treated with BDQ (5 μg/mL) for 18 hr. Differentially expressed genes were identified by mRNAseq. (A) Gene ontology enrichment analysis of genes whose expression is upregulated by BDQ treatment, using the Cytoscape app ClueGO (FDR < 0.05; LogFC >0.5). (B) Cells were infected with BDQ-resistant MTB for 24 hr and then treated with BDQ (5 μg/mL) for an additional 18 hr. Gene ontology enrichment analysis of genes whose expression is up-regulated by BDQ treatment in BDQr-MTB-infected cells, using the Cytoscape app ClueGO (FDR < 0.05; LogFC >0.5). (C) Venn diagram showing the number of genes regulated by BDQ treatment in naive and BDQr-MTB-infected macrophages, relative to untreated controls. (D) Heatmap showing differential expression of genes differentially expressed by BDQ in naive and BDQr-MTB-infected cells. Each column corresponds to one donor. Data were normalized to determine the log ratio with respect to the median expression of each gene. Figure 1—source data 1 Genes whose expression is upregulated in naive macrophages upon BDQ treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data1-v1.xlsx Download elife-55692-fig1-data1-v1.xlsx Figure 1—source data 2 Genes whose expression is downregulated in naive macrophages upon BDQ treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data2-v1.xlsx Download elife-55692-fig1-data2-v1.xlsx Figure 1—source data 3 Genes whose expression is upregulated in BDQr-MTB-infected macrophages upon BDQ treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data3-v1.xlsx Download elife-55692-fig1-data3-v1.xlsx Figure 1—source data 4 Genes whose expression is downregulated in BDQr-MTB-infected macrophages upon BDQ treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data4-v1.xlsx Download elife-55692-fig1-data4-v1.xlsx Figure 1—source data 5 Genes differentially expressed in BDQr-MTB infected macrophages by BDQ. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data5-v1.xlsx Download elife-55692-fig1-data5-v1.xlsx Figure 1—source data 6 Genes differentially expressed in naive macrophages by BDQ. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data6-v1.xlsx Download elife-55692-fig1-data6-v1.xlsx Figure 1—source data 7 Differentially expressed genes both in naive and in BDQr-MTB infected macrophages upon BDQ treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data7-v1.xlsx Download elife-55692-fig1-data7-v1.xlsx Table 1 Gene Ontology (GO) functional annotation of genes differentially expressed by BDQ only in naïve- and BDQr-MTB-infected macrophages. Specific NAIVE BDQ genesGO categoryavg. LogFCp-valueCell division-0.518.34E-05Sphingolipid metabolic process0.331.42E-04Angiogenesis0.675.16E-04Spindle-0.635.46E-04Lysosomal lumen0.351.21E-04Glycosphingolipid metabolic process0.351.21E-03Response to oxidative stress0.491.26E-03Mitotic cell cycle-0.581.29E-03Specific INFECTED BDQ genesGO categoryavg. LogFCp-valueEndoplasmatic reticulum-Golgi intermediate compartment-0.382.90E-07Membrane raft-0.344.93E-05Cellular protein metabolic process-0.353.32E-04Lipid binding-0.364.88E-04Ribonucleoprotein complex binding-0.375.17E-04Protein dephosphorylation-0.345.43E-04Lysosomal membrane-0.346.31E-04Ubiquitin-dependent protein catabolic process0.746.48E-04 We next evaluated if BDQ could modulate gene expression in MTB-infected cells. In order to exclude potential differences due to the MTB bacillary load between treated and untreated cells, we generated a virulent BDQ-resistant strain of M. tuberculosis (BDQr-MTB). The selected clone, which carried a Ala63→Pro mutation in subunit c of the ATP synthase (Andries et al., 2005; Figure 1—figure supplement 2A), had a similar generation time to wild-type bacteria when cultured in 7H9 liquid medium, although we observed a slower growth after 7 days of treatment. (Figure 1—figure supplement 2B). We also noted no difference in virulence or in intracellular growth of both wild-type- and BDQ-resistant MTB (Figure 1—figure supplement 2C-E). As expected, the MIC99 (defined as the concentration required to prevent 99% growth) for susceptible MTB was 0.07 µg/mL, a value similar to previously published study (Andries et al., 2005), while the MIC99 of the BDQr-MTB was 36 µg/mL. We infected macrophages with BDQr-MTB. After 24 hr of infection, cells were incubated for an additional 18 hr with BDQ (5 µg/mL). The bacillary load of resistant MTB inside macrophages was the same in untreated cells as in cells after 18 hr of BDQ treatment. In contrast, in the same experiment using BDQ-susceptible MTB, there was a 70% decrease in the bacillary load (Figure 1—figure supplement 2C). Following treatment, we characterized the genome-wide gene expression profiles of MTB-infected macrophages, as described above. The expression of 1,495 genes was affected by BDQ (FDR < 0.05, Figure 1A, Figure 1—source datas 3 and 4), with 499 being upregulated and 996 being downregulated. More genes were thus affected by BDQ treatment in MTB-infected cells than in naive macrophages. This probably reflects the fact that MTB infection induces an extensive remodeling of the transcriptome (Barreiro et al., 2012; Tailleux et al., 2008). The genes differentially expressed by BDQ only in MTB-infected macrophages are enriched in genes related to assembly of the endoplasmic reticulum-Golgi intermediate compartment, membrane raft and cellular protein metabolic process (Table 1, Figure 1—source datas 5 and 6). This probably reflects the cell adaptation to infection. Functional annotation of the gene set upregulated by BDQ also revealed that similar pathways were affected by BDQ in naive and BDQr-MTB-infected macrophages, with an enrichment for genes associated with glucose/phospholipid metabolism and lysosome (Figure 1B, Figure 1—source datas 3 and 4). 452 genes were differentially expressed in both naive and MTB-infected cells upon BDQ treatment with an over-representation of lysosome-associated genes (Figure 1C–D, Figure 1—source data 7). BDQ affects host metabolism As metabolic pathways were over-represented in our RNAseq analysis, we investigated if glycolysis is affected by BDQ treatment using the Seahorse Extracellular Flux analyzer. This assay measures the rate of proton accumulation in the extracellular medium during glycolysis (glycoPER) and can discriminate between basal glycolysis, induced glycolytic capacity (by addition of rotenone/antimycin A (Rot/AA), an inhibitor of the mitochondrial electron transport chain), and non-glycolytic acidification (by addition of the glycolytic inhibitor 2-deoxy-D-glycose (2-DG)). After incubation with BDQ, we observed a 30% decrease in basal glycolysis and glycolytic capacity compared to untreated cells (Figure 2A–B, Figure 2—figure supplement 1A–B). Figure 2 with 1 supplement see all Download asset Open asset Modulation of host metabolism by BDQ. (A–B) The Glycolytic Rate Assay was performed in heat killed-MTB stimulated macrophages treated with BDQ, in the presence of rotenone/antimycin A (Rot/AA) and 2-deoxy-D-glycose (2-DG), inhibitors of the mitochondrial electron transport chain and glycolysis, respectively (one-way ANOVA test). One representative experiment (of two) is shown. (C) Lipid profile of BDQr-MTB infected cells treated with BDQ, by MALDI-TOF (unpaired two tailed Student’s t test). PI: Phosphotidylinositol; CL: Cardiolipids; PE: Phosphatidylethanolamine; PG: Phosphatidylglycerol. Numbers correspond to mass-to-charge ratio (m/z). Cells derived from three donors were analyzed. Error bars represent the mean ± SD and significant differences between treatments are indicated by an asterisk, in which *p<0.05, **p<0.01, ***p<0.001. We assessed phospholipid metabolism, a pathway also identified in our ClueGO cluster analysis (Figure 1B). Like glycolysis, lipid metabolism affects macrophage phenotype and function (Remmerie and Scott, 2018). We analyzed the lipid profile of BDQ-treated cells using MALDI-TOF mass spectrometry. We observed an increase of phosphatidylinositols upon incubation with BDQ (Figure 2C, Figure 2—figure supplement 1C). No significant changes were observed in the levels of phosphatidylethanolamines, phosphatidylglycerols, or cardiolipins. Taken together, these data show that BDQ induced a significant metabolic reprogramming of both MTB-infected and resting macrophages. BDQ increases macrophage lysosomal activity Macrophages are involved in innate immunity and tissue homeostasis through their detection and elimination of microbes, debris, and dead cells, which occurs in lysosomes (Wynn et al., 2013). Lysosomes are acidic and hydrolytic organelles responsible for the digestion of macromolecules. Recent work has shown that they are also signaling platforms, which respond to nutrient and cellular stress (Lawrence and Zoncu, 2019). Functional annotations based on the GO database of the differentially expressed genes suggested a substantial impact of BDQ treatment on lysosome function (Figure 1A–B). We identified 38 and 54 differentially expressed genes by BDQ, respectively in naïve- and BDQr-MTB infected cells (FDR < 0.05, Figure 3A). These genes are involved in lysosome biogenesis, transport and degradation of small molecules, and lysosomal acidification. They included genes coding for components of vacuolar ATPase (V-ATPase), hydrolases, and SLC11A1 (NRAMP1), a divalent transition metal transporter involved in host resistance to pathogens, including MTB (Meilang et al., 2012). Figure 3 with 2 supplements see all Download asset Open asset BDQ activates the lysosomal pathway in human MTB-infected macrophages. (A) Heatmap showing differential expression of genes included in the Lysosome KEGG category (p-value<0.05). Each column corresponds to one donor. Data were normalized to determine the log ratio with respect to the median expression of each gene. (B) Macrophages were infected with BDQr-MTB expressing the GFP protein and incubated with BDQ (5 μg/mL) for 3 hr, 18 hr and 48 hr. Acid organelles were then labeled with 100 nM LysoTracker DND-99 for 1 hr. The fluorescence intensity was quantified by flow cytometry. (C–E) Cells were infected with GFP expressing BDQr-MTB (green) and treated with BDQ (5 μg/mL). After 18 hr and 48 hr of treatment, cells were labelled with LysoTracker (red) and fluorescence was analyzed by confocal microscopy. DAPI (blue) was used to visualize nuclei (scale bar: 10 μm). The quantification of LysoTracker staining and the percentage of LysoTracker-positive MTB phagosomes were performed using Icy software. (F) Macrophages were activated with heat-killed MTB and treated with BDQ for 18 hr and 48 hr. Cells were then incubated with DQ-Green BSA. Fluorescence was quantified by flow cytometry. Significant differences between BDQ treatment and control (DMSO) are indicated by an asterisk. One representative experiment (of at least three) is shown. Error bars represent the mean ± SD. *p<0.05, **p<0.01, ***p<0.001. To validate our transcriptomic data, we incubated BDQ-treated, BDQr-MTB-infected cells with LysoTracker Red DND-99, a red fluorescent probe that labels acidic organelles, and analyzed them using flow cytometry. No differences were observed between control and treatment after 3 hr of BDQ treatment (Figure 3B). However, at 18 hr and 48 hr post-treatment, fluorescence intensity was substantially increased in macrophages incubated with BDQ compared to DMSO-treated cells (1.7 and 5.4 times more, respectively). These results were supported by confocal microscopy, which revealed the appearance of numerous acidic compartments upon treatment (Figure 3C), up to five times more in BDQ-treated macrophages than untreated cells at 48 hr post-treatment (p<0.001, Figure 3D). We also observed a large number of MTB phagosomes co-localized with LysoTracker-positive compartments (Figure 3E). As the expression of many genes coding for hydrolases was upregulated upon BDQ treatment (Figure 3A), we tested the effect of the drug on late endosomal/lysosomal proteolytic activity. BDQ-treated macrophages were incubated with DQ-Green BSA, a self-quenched non-fluorescent probe that produces brightly fluorescent peptides following hydrolysis by lysosomal proteases. At 18 hr and 48 hr post-treatment, we observed a dose-dependent increase in fluorescence intensity upon treatment with BDQ (up to 5.5 times more than untreated cells, p<0.01, Figure 3F). Similar results were obtained when we incubated naive macrophages with BDQ (Figure 3—figure supplement 1). Together, these data demonstrate that BDQ induces biogenesis of competent lysosomes. We performed additional experiments to confirm that the main effects of BDQ on lysosome were independent of infection with live bacteria. Briefly, cells were untreated or stimulated with LPS (TLR4 agonist), Pam3CSK4 (TLR1/2 agonist), heat-killed bacteria (hk-MTB), drug-susceptible MTB or BDQr-MTB, and treated with BDQ. After 18 hr, RNA was collected and we performed RT-qPCR on a panel of lysosomal genes. We also analyzed the intensity of the LysoTracker staining using flow cytometry (Figure 3—figure supplement 2). Our results clearly show that the main effects on lysosome biogenesis/activation occurred with BDQ treatment and were not exclusively seen after infection with live MTB. BDQ potentiates PZA antimycobacterial activity The capacity of BDQ to induce acidic compartments may potentiate the efficacy of other drugs, whose activity is pH dependent. In vivo studies have suggested a synergistic interaction between BDQ and PZA (Ibrahim et al., 2007), and it is commonly assumed that a low pH is required for PZA activity against MTB (Zhang and Mitchison, 2003). We thus infected macrophages with BDQr-MTB and treated them with BDQ and PZA. After 7 days of treatment, cells were lysed and bacteria counted. PZA showed moderate bactericidal activity, with 50 µg/mL PZA resulting in a 36% decrease in bacterial numbers compared to untreated cells (Figure 4A). We confirmed that the combination of PZA with BDQ was highly bactericidal on MTB, leading to a 83% decrease in colony forming units using 50 µg/mL PZA. This decrease was not a result of an additive effect between the two drugs, as BDQ alone at 1 µg/mL had no antibacterial activity. We also found no synergy between BDQ and PZA on the BDQ-resistant mutant cultivated in Middlebrook 7H9 liquid medium (Figure 4B). Thus, the potentiation of PZA activity by BDQ is most likely due to the effect of BDQ on the host cell, and in particular on the increase of lysosomal acidification. Figure 4 with 1 supplement see all Download asset Open asset BDQ potentiates PZA antimycobacterial activity. (A) Macrophages were infected with BDQr-MTB and treated with BDQ (1 μg/mL) and PZA. After 7 of days treatment, cells were lysed and bacteria were enumerated by CFU (counted in triplicate). (B) Optical density (OD) measurements of bacterial growth of BDQr-MTB in the presence of BDQ (1 μg/mL) and different concentrations of PZA. Bacteria were cultured in 7H9 medium supplemented with 10% OADC enrichment with/without the drugs. One representative experiment (of at least three) is shown. Error bars represent the mean ± SD. *p<0.05, **p<0.01, ***p<0.001. We next tested whether BDQ would synergize with the other first-line anti-TB drugs in liquid culture or in BDQr-MTB-infected macrophages. We found that BDQ did not potentiate the activity of ethambutol (EMB), isoniazid (INH) and rifampicin (RIF) in either case. While we cannot exclude the possibility that BDQ may have additive effects with other anti-TB drugs, as has been described (Ibrahim et al., 2007), we saw no evidence of synergism with any of the first-line agents (Figure 4—figure supplement 1). Classical anti-TB drugs did not activate the lysosomal pathway in human macrophages Bacterial hydroxyl radical released after treatment with INH or PZA can directly induce host cell autophagy (Kim et al., 2012). We thus tested whether other antibiotics might have similar effects to BDQ. We characterize the genome-wide gene expression profiles of naïve macrophages and macrophages stimulated with hk-MTB, and treated with amikacin (AMK), EMB, INH, PZA or RIF for 18 hr. We chose drug concentrations based on the concentrations detected in the plasma of treated TB patients. Following treatment, only RIF and PZA significantly modulate gene expression in macrophages. 556 and 752 genes were differentially expressed in cells stimulated with heat-killed bacteria and exposed to RIF and PZA, respectively (Figure 5A, Figure 5—figure supplement 1, Figure 5—source datas 1–5). We classified these genes by performing gene-set enrichment analysis and confirmed that the lysosomal pathway was not induced upon RIF or PZA treatment (Table 2). The expression of only two genes belonging to this pathway was upregulated by RIF, and only one by PZA, compared to 46 whose expression was modulated by BDQ (Figure 5B–C). Consistent with these results, none of these antibiotics were able to increase LysoTracker staining (Figure 5D). Figure 5 with 1 supplement see all Download asset Open asset Classical anti-TB drugs did not activate the lysosomal pathway in human macrophages. (A) Number of differentially-expressed genes upon treatment with commonly used anti-TB drugs relative to untreated control. Briefly, naïve- and hk-MTB-stimulated macrophages were treated with AMK (20 µg/mL), EMB (20 µg/mL), INH (10 µg/mL), PZA (200 µg/mL) or RIF (20 µg/mL). After 18 hr, differentially expressed (DE) genes were identified by mRNAseq. (B–C) Venn diagram showing the number of genes regulated by PZA and RIF in naive and hk-MTB-stimulated macrophages, in comparison with the number of lysosomal genes differentially expressed by BDQ (FDR < 0.05, absLogFC >0.1). (D) Macrophages were incubated for 48 hr with AMK, BDQ, EMB, INH, PZA or RIF, and then stained with Lysotracker. Fluorescence intensity was analyzed by flow cytometry. One representative experiment (of at least three) is shown. Error bars represent the mean ± SD. *p<0.05. Figure 5—source data 1 Differentially expressed genes in hk-MTB stimulated macrophages upon EMB treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data1-v1.xlsx Download elife-55692-fig5-data1-v1.xlsx Figure 5—source data 2 Differentially expressed genes in hk-MTB stimulated macrophages upon RIF treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data2-v1.xlsx Download elife-55692-fig5-data2-v1.xlsx Figure 5—source data 3 Differentially expressed genes in naive macrophages upon RIF treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data3-v1.xlsx Download elife-55692-fig5-data3-v1.xlsx Figure 5—source data 4 Differentially expressed genes in hk-MTB stimulated macrophages upon PZA treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data4-v1.xlsx Download elife-55692-fig5-data4-v1.xlsx Figure 5—source data 5 Regulated genes naive macrophages upon PZA treatment. FDR < 0.05. https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data5-v1.xlsx Download elife-55692-fig5-data5-v1.xlsx Table 2 Gene Ontology (GO) functional annotation of differentially expressed genes in naïve- or hk-MTB-stimulated cells treated with PZA or RIF. hk-MTB + PZAGO categoryavg. LogFCp-valueIntegral to lumenal side of endoplasmic reticulum membrane0.162.79E-04Cytokine-mediated signaling pathway0.273.28E-04Interferon-gamma-mediated signaling pathway0.213.56E-04MHC class I receptor activity0.167.82E-04Cytosolic small ribosomal subunit0.267.98E-03MHC class I protein complex0.161.04E-03Regulation of immune response0.221.33E-03Negative regulation of MAPK cascade0.201.34E-03Naïve + RIFGO categoryavg. LogFCp-valueMitotic cell cycle-0.371.53E-17DNA replication-0.342.59E-13Cell cycle checkpoint-0.364.98E-10S phase of mitotic cell cycle-0.326.29E-10DNA strand elongation involved in DNA replication-0.331.88E-09G1/S transition of mitotic cell cycle-0.362.02E-09Cell division-0.343

Mitochondrial dynamics and metabolism are closely associated, however, if these interactions play a role in the human macrophage response to <i>Mycobacterium tuberculosis</i> (Mtb) remain largely unknown. Here, human induced-pluripotent stem cell derived macrophages (iPSDM) were used to define whether mitochondrial dynamics and metabolism are modulated by Mtb infection. Macrophage bioenergetic response to infection was analysed by extracellular flux analysis (Seahorse) and unbiased metabolomics. In a complementary approach, high-content live cell imaging and single cell analysis was used to investigate changes in mitochondrial dynamics and Mtb intracellular replication. The metabolic profile of infected human macrophages showed an increase in the oxygen consumption and extracellular acidification rate after 48h of infection. Moreover, the lipidomics analysis showed a significant upregulation in the total levels of sphingomyelin and ceramide. Interestingly, these changes in metabolism were not associated with a disruption of the mitochondrial network. However, infection decreased mitochondrial protein levels, which correlated with RNA-Seq analysis showing downregulation of mitochondrial transcripts. The inhibition of glycolysis with 2-Deoxy-d-glucose promoted Mtb replication, whereas oxamate (a lactate dehydrogenase inhibitor) impaired Mtb growth. Our results show that macrophage metabolic reprogramming is required for the control of Mtb replication. Unlike other intracellular pathogens, Mtb did not induce fragmentation of the mitochondrial network, suggesting that manipulation of host cell metabolism might correlate with disruption of mitochondrial function rather than morphology.

Abstract 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. However, the host and pathogen determinants driving 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 both 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 cells (MNCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multi-nucleation. Importantly, we extend our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNCs. Our findings implicate MNC 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.

In 2019 we started a new annual meeting, aimed at bringing together researchers from across the United Kingdom studying cellular microbiology and the cell biology of host-pathogen interactions. In contrast to large glamourous meetings, featuring the great and the good from across the world, we wanted to create a forum for early career researchers to present their work and enjoy lively discussion. In particular, we hope that focussing on making the meeting accessible, affordable, and informal would help integrate and build the U.K. community working on this exciting topic.

Background: Anxiety and stress among college students have sharply increased over the past two decades.COVID-19 exacerbated the mental health and academic challenges of United States (US) college students, particularly students of color.Objective: To investigate the impact of COVID-19 on anxiety, stress and coping of Hispanic/Latine, Asian, and non-Hispanic White (NHW) US college students.Explore students' academic perceptions as predictors of anxiety and stress.Methods: In this descriptive, cross-sectional study, 855 participants were students attending California State University, Fullerton (CSUF), a large public university in metropolitan Orange County, California, USA.From April to May 2020, participants answered an online survey of anxiety, stress, and coping.Demographic information and academic experiences were also collected online using a researcher-designed questionnaire.Frequencies and percentages summarized categorical data, and associations were analyzed using crosstabs, correlations, and multiple regression.Results: Participants identifying as Hispanic/Latine (n = 449), Asian (n = 169), and non-Hispanic White (NHW; n = 237) indicated difficulty transitioning to remote instruction and low academic engagement post-COVID-19.Over 70% of students reported moderate to high anxiety, while 80+% reported significant stress; these rates were typically higher for Asian and Hispanic/Latine participants than NHW participants.The leading healthy coping activity was exercise; the leading unhealthy coping behavior was alcohol abuse.Academic perceptions negatively predicted anxiety and stress levels.Conclusion: COVID-19 adversely affected the psychological well-being of participants of color.Administrators should address students' mental health needs and provide culturally sensitive services.Additional remote instruction resources should be provided to these vulnerable students.

Mitochondrial dynamics and metabolism in immune cells are closely associated. However, if these interactions play a role in the human macrophage response to <i>Mycobacterium tuberculosis</i> (Mtb) remains largely unknown. We used human induced-pluripotent stem cell-derived macrophages together with high-content live-cell imaging, extracellular flux analysis and labelled metabolomics to investigate mitochondrial dynamics, Mtb intracellular replication and mitochondrial metabolism. Mitochondrial tracking at the single-cell level revealed extensive mitochondrial morphology heterogeneity. Mtb did not induce significant changes in mitochondrial area, length or width during the first 48h of infection. However, infection with Mtb WT but not with an attenuated strain lacking the region of difference 1 (Mtb ΔRD1) induced a decrease in the mitochondrial membrane potential and protein turnover rate, as visualised by MitoTracker Red and the ratiometric reporter MitoTimer, respectively. Notably, these changes correlated with a selective degradation of mitochondrial proteins and downregulated mitochondrial transcripts. The metabolic profile of infected macrophages showed an increase in oxygen consumption and extracellular acidification rate after 48h of infection. The inhibition of glycolysis with 2-Deoxy-d-glucose promoted Mtb replication, whereas oxamate (lactate dehydrogenase inhibitor) impaired Mtb intracellular replication. Collectively, our results show that macrophage metabolic reprogramming is required for the control of Mtb replication. Unlike other intracellular pathogens, changes in host cell metabolism induced by Mtb might correlate with disruption of mitochondrial function rather than morphology.

Summary The intracellular population of Mycobacterium tuberculosis (Mtb) is dynamically segregated 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 dual-imaging approach that allows quantitative monitoring of host subcellular acidification and Mtb intrabacterial pH profiles by live-fluorescence microscopy in a biosafety level 3 laboratory. By combining this live imaging approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. Importantly, we show that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo . By using Mtb mutants with different intra-macrophage localisation, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo . We anticipate this dual imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. Highlights Mtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophages Pyrazinamide, but not other frontline antibiotics, acts as a protonophore in cellulo Pyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidification Cytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistance Pyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo