Autophagy is rapidly developing into a new immunological paradigm. The latest links now include overlaps between autophagy and innate immune signaling via TBK-1 and IKKα/β, and the role of autophagy in inflammation directed by the inflammasome. Autophagy's innate immunity connections include responses to pathogen and damage associated molecular patterns including alarming such as HMGB1 and IL-1β, Toll-like receptors, Nod-like receptors including NLRC4, NLRP3 and NLRP4, and RIG-I-like receptors. Autophagic adaptors referred to as SLRs (sequestosome 1/p62-like receptors) are themselves a category of pattern recognition receptors. SLRs empower autophagy to eliminate intracellular microbes by direct capture and by facilitating generation and delivery of antimicrobial peptides, and also serve as inflammatory signaling platforms. SLRs contribute to autophagic control of intracellular microbes, including Mycobacterium tuberculosis, Salmonella, Listeria, Shigella, HIV-1 and Sindbis viruses, but act as double edged sword and contribute to inflammation and cell death. Autophagy roles in innate immunity continue to expand vertically and laterally, and now include antimicrobial function downstream of vitamin D3 action in tuberculosis and AIDS. Recent data expand the connections between immunity related GTPases and autophagy to include not only IRGM but also several members of the Gbp (guanlyate-binding proteins) family. The efficacy with which autophagy handles microbes, microbial products and sterile endogenous irritants governs whether the outcome will be with suppression of or with excess inflammation, the latter reflected in human diseases that have strong inflammatory components including tuberculosis and Crohn's disease.
The in vivo kinetics of antigen-presenting cells (APCs) in patients with advanced and convalescent tuberculosis (TB) is not well characterized. In order to target Mycobacterium tuberculosis (MTB) peptides- and HLA-DR-holding monocytes and macrophages, 2 MTB peptide-specific CD4+ T-cell receptor (TCR) tetramers eu and hu were successfully constructed. Peripheral blood (PBL) samples from inpatients with advanced pulmonary TB (PTB) were analyzed using flow cytometry, and the percentages of tetramer-bound CD14+ monocytes ranged from 0.26–1.44% and 0.21–0.95%, respectively; significantly higher than those measured in PBL samples obtained from non-TB patients, healthy donors, and umbilical cords. These tetramers were also able to specifically detect macrophages in situ via immunofluorescent staining. The results of the continuous time-point tracking of the tetramer-positive rates in PBL samples from active PTB outpatients undergoing treatment show that the median percentages were at first low before treatment, increased to their highest levels during the first month, and then began to decrease during the second month until finally reaching and maintaining a relatively low level after 3–6 months. These results suggest that there is a relatively low level of MTB-specific monocytes in advanced and untreated patients. Further experiments show that MTB induces apoptosis in CD14+ cells, and the percentage of apoptotic monocytes dramatically decreases after treatment. Therefore, the relatively low level of MTB-specific monocytes is probably related to the apoptosis or necrosis of APCs due to live bacteria and their growth. The bactericidal effects of anti-TB drugs, as well as other unknown factors, would induce a peak value during the first month of treatment, and a relatively low level would be subsequently reached and maintained until all of the involved factors reached equilibrium. These tetramers have diagnostic potential and can provide valuable insights into the mechanisms of antigen presentation and its relationship with TB infection and latent TB infection.
Mycobacterium tuberculosis (MTB) is one of the most dangerous pathogens in the world. It is estimated that one-third of the world population contracts the bacteria during their lives. Approximately 5–10% of infected individuals will eventually develop an active form of the disease. Cellular immunity plays an important role in immunity against tuberculosis (TB); however, the host's defense mechanisms are not completely understood. Here, we developed a novel tool: MTB antigen-specific tetrameric CD4+ T-cell receptor (TCR) complexes that can detect MTB peptide-specific antigen presenting cells (APCs) in blood and local tissues. We found that a relatively low level of antigen-specific monocytes (i.e., APCs) was detected in peripheral blood (PBL) samples from untreated TB patients, and then increased to their peak levels during the first month after treatment, which probably had something to do with the decrease in APC apoptosis. Our research provides a new method for tracking dynamic changes in APCs that are associated with TB infection and latent TB infection, and an additional tool for the studies of TB immunity and its pathogenesis.
The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers—even those who work in the field—to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.
autophagy; lysosome; mitophagy; pexophagy; stress; vacuole
One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.
Identifying coordinately regulated genes and their control by environmentally-initiated signal transduction pathways is important for understanding bacterial virulence mechanisms. The work reported here provides a comprehensive, high resolution, transcriptome map of the opportunistic pathogen Pseudomonas aeruginosa using RNA-seq. The results suggest that P. aeruginosa senses the temperature during the transition from its natural environment to a mammalian host, and this plays a key role in regulating the coordinated expression of several virulence factors. A large number of antisense transcripts and non-coding RNAs were identified, with preferential clustering in the regions acquired through horizontal gene transfer, suggesting that a part of the non-coding genome has a distinct evolutionary origin. We created an online data viewer, the Pseudomonas transcriptome browser, to facilitate access to the transcriptome data from this study as well as the subsequent results of work deposited by other investigators. The resources generated through our analyses provide a valuable tool to the P. aeruginosa research community and set the foundation for a systems biology approach towards understanding the complexity of the regulatory networks controlling the multiple lifestyles of this highly versatile organism.
Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus–secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus–containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.
Staphylococcus aureus is a microorganism that causes serious infectious diseases such as pneumonia, endocarditis, osteomyelitis, and wound infections. This pathogen can infect various types of non-professional phagocytic cells and after internalization is able to escape the phagolysosomal compartment towards the cytoplasm, where it actively replicates. Subsequently, the eukaryotic host cell is killed to spread the infection. Besides the clinical importance of this microorganism, the molecular mechanisms of S. aureus infection are not completely understood. S. aureus induces an autophagic response in infected cells, which is beneficial for bacterial replication and cell killing. We have previously shown that Hla is responsible for this autophagy activation. We found that the Hla-induced autophagic response occurs by a “non-canonical" pathway independent of PI3K/Beclin1 complex but dependent on Atg5. Here we show that cAMP has a key role in the regulation of Hla-induced autophagic response. cAMP, through EPAC/Rap2b and via calpain activation, inhibits S. aureus–induced autophagy. Additionally, we show that EPAC and Rap2b are recruited to the S. aureus–containing phagosome. Our study contributes to the understanding of the molecular mechanisms used by S. aureus to survive, a key step in Staphylococcal pathogenicity.
Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.
Tuberculosis is responsible for approximately 2 million deaths worldwide each year. Current treatment regimens require administration of multiple drugs over several months and resistance to these drugs is on the rise. Mycobacterium tuberculosis, the causative agent of the disease, can proliferate within host cells. It has been recently observed that autophagy (cellular self-eating) can kill intracellular M. tuberculosis. We report that the antiprotozoal drug nitazoxanide and its metabolite tizoxanide induce autophagy, inhibit signaling by mTORC1, a major negative regulator of autophagy, and prevent M. tuberculosis proliferation in infected macrophages. We show that nitazoxanide exerts at least some of its pharmacological effects by targeting the quinone reductase NQO1. Our results uncover a novel mechanism of action for the drug nitazoxanide, and show that pharmacological modulation of autophagy can suppress intracellular M. tuberculosis proliferation.
Low vitamin D levels in human immunodeficiency virus type-1 (HIV) infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3), the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP) and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A1, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.
Macroautophagy (autophagy - ‘self-eating’, lysosome-dependent degradation and recycling of the intracellular components in response to stress) is an important host defense mechanism against viral and mycobacterial infections. Recent studies have described that activation of autophagy in macrophages reduces the viability of Mycobacterium tuberculosis and HIV due to an intimate autophagy-phagocytosis interaction. Low serum levels of the 25-hydroxycholecalciferol form of vitamin D have been associated with an increased risk for active tuberculosis and HIV disease progression as well as M. tuberculosis susceptibility. In this study, we report that the active form of vitamin D, 1α,25-dihydroxycholecalciferol inhibits the replication of HIV and M. tuberculosis in a concentration dependent manner. Moreover, by inhibiting key stages in the autophagy pathway, we demonstrate that the inhibition of HIV and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Furthermore, through the use of RNA interference for the human cathelicidin microbial peptide we demonstrate that cathelicidin is essential for the 1α,25-dihydroxycholecalciferol induced autophagic flux and inhibition of HIV replication and mycobacterial growth. These findings suggest that the induction of autophagy has the potential to be useful in the treatment of persons co-infected with HIV and M. tuberculosis.
Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.
The major cause of AIDS deaths globally has been tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (MTb). Co-infection with MTb exacerbates human immunodeficiency virus type1 (HIV-1) replication and disease progression via both innate and adaptive host immune responses to MTb infection. In this report, we present evidence that the transcription factor NFAT5 plays a crucial role in MTb-induced HIV-1 replication in human peripheral blood cells and monocytes. We also show that MTb infection itself stimulates NFAT5 gene expression in human monocytes and that its expression involves the TLR signalling pathway and requires the downstream adaptor proteins MyD88, IRAK1, and TRAF6. This identification of a novel role for NFAT5 in TB/HIV-1 co-infection reveals that NFAT5 is a major mediator of TLR-dependent gene expression and thus provides a potential new therapeutic target for treatment of HIV-1 and possibly other diseases.
Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.
Tuberculosis is one of the world's most pernicious diseases. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has a lipid-rich cell wall that contains immunostimulatory properties. One of the lipid cell wall components, trehalose 6,6′-dimycolate (TDM), is a Mincle ligand and an immunogenic factor of Mtb that induces inflammatory responses leading to granuloma formation. Defining the major target and cellular functions of TDM may be requisite for delaying or preventing mycobacterial TDM-induced inflammation. Here, we demonstrated that neutrophils are important for the early phase of TDM-induced lung inflammation. Neutrophils are recruited during the initial stage of TDM-induced lung inflammation and Mincle is required for neutrophil access to TDM-challenged sites by enhancing neutrophil integrin expression, cytoskeleton remodeling, and cell adhesion. Furthermore, neutrophils aggravate TDM-induced lung inflammation by producing proinflammatory cytokines/chemokines. These findings open new perspectives for the role of Mincle signaling on neutrophils during TDM-induced inflammatory responses.
Autophagy was viewed until very recently primarily as a metabolic and intracellular biomass and organelle quality and quantity control pathway. It has now been recognized that autophagy represents a bona fide immunological process with a wide array of roles in immunity. The immunological functions of autophagy, as we understand them now, span both innate and adaptive immunity. They range from unique and sometimes highly specialized immunological effectors and regulatory functions (referred to here as type I immunophagy) to generic homeostatic influence on immune cells (type II immunophagy), akin to the effects on survival and homeostasis of other cell types in the body. As a concept-building tool for understanding why and how autophagy is intertwined with immunity, it is useful to consider that the presently complex picture has emerged in increments, starting in part from the realization that autophagy acts as an evolutionarily ancient microbial clearance mechanism defending eukaryotic cells against intracellular pathogens. In this review, we build a step-wise model of how the core axis of autophagy as a cell-autonomous immune defense against microbes evolved into a complex but orderly web of intersections with innate and adaptive immunity processes. The connections between autophagy and conventional immunity systems include Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-I-like receptors (RLRs), damage-associated molecular patterns (DAMPs) such as HMGB1, other known innate and adaptive immunity receptors and cytokines, sequestasome (p62)-like receptors (SLR) that act as autophagy adapters, immunity related GTPase IRGM, innate and adaptive functions of macrophages and dendritic cells, and differential effects on development and homeostasis of T and B-lymphocyte subsets. The disease contexts covered here include tuberculosis, infections with human immunodeficiency virus and other viruses, Salmonella, Listeria, Shigella, Toxoplasma, and inflammatory disorders such as Crohn's disease and multiple sclerosis.
autophagy; dendritic cells; T cells; AIDS; bacterial; inflammatory bowel disease
adaptors; autophagy; cargo; mitophagy; stress; xenophagy
In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.
autophagy; tuberculosis; ribosome; ubiquitin; antimicrobial peptides
Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a virulence factor involved in extrapulmonary dissemination and a strong diagnostic antigen against tuberculosis, is both surface-associated and secreted. The role of HBHA in macrophages during M. tuberculosis infection, however, is less well known. Here, we show that recombinant HBHA produced by Mycobacterium smegmatis effectively induces apoptosis in murine macrophages. DNA fragmentation, nuclear condensation, caspase activation, and poly (ADP-ribose) polymerase cleavage were observed in apoptotic macrophages treated with HBHA. Enhanced reactive oxygen species (ROS) production and Bax activation were essential for HBHA-induced apoptosis, as evidenced by a restoration of the viability of macrophages pretreated with N-acetylcysteine, a potent ROS scavenger, or transfected with Bax siRNA. HBHA is targeted to the mitochondrial compartment of HBHA-treated and M. tuberculosis-infected macrophages. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in both macrophages and isolated mitochondria treated with HBHA. Disruption of HBHA gene led to the restoration of ΔΨm impairment in infected macrophages, resulting in reduced apoptosis. Taken together, our data suggest that HBHA may act as a strong pathogenic factor to cause apoptosis of professional phagocytes infected with M. tuberculosis.
Cell death is a common outcome during infection with a number of pathogenic microorganisms. Therefore, defining the factors responsible for killing of host cells is important to uncovering mechanisms of pathogenesis. World-wide, two billon people are latently infected with Mycobacterium tuberculosis, which is still killing 2–3 million people each year. Heparin-binding hemagglutinin (HBHA) protein of M. tuberculosis is known to interact specifically with non-phagocytic cells and to be involved in dissemination from lungs to other tissues. Nevertheless, the role of HBHA in phagocytic cells such as macrophages, which are the first cells of the immune system to encounter inhaled pathogens, has been unknown. In the present study, we suggest HBHA as a critical bacterial protein for macrophage cell death. After M. tuberculosis infection or HBHA treatment of macrophages, HBHA targeted to mitochondria and then caused mitochondrial damage and oxidative stress, which eventually lead to apoptosis. A mutant of M. tuberculosis lacking HBHA induced less apoptosis with moderated mitochondrial damage. These experiments provide a candidate virulence factor which may be a novel target for tuberculosis treatment.
Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocompatibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of Mycobacterium bovis BCG that is unable to escape lysosomal transfer and show that presentation of mycobacterial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metalloprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. The zmp1 deletion mutant was more immunogenic in vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-γ)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.
Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes host macrophages where it persists in immature phagosomes by avoiding their maturation into phagolysosomes. The mechanisms of how M. tuberculosis inhibits phagolysosome biogenesis have been researched in detail and the maturation block at least partially depends on the manipulation of host phosphoinositide interconversions, with phosphatidylinositol 3-phosphate (PI3P) being a central target since it has been shown to be required for phagolysosome biogenesis. PI3P earmarks intracellular organelles for binding and assembly of effector molecules that interact with PI3P or its derivatives, including Class E Vps proteins such as Hrs and ESCRT components, early endosome antigen 1, which are required for sequential protein and membrane sorting within the endosomal and, by extension, phagosomal systems. In a search of a cellular mechanism that can bypass the tubercule bacillus-imposed PI3P block, researchers have uncovered a new general bactericidal process, autophagy, which can eliminate intracellular pathogens. This is a new, rapidly growing field replete with possibilities for novel, previously untried immunologic and pharmacologic interventions applicable not only to TB but to other stubborn bacterial, parasitic and viral diseases.
autophagy; macrophage; phagosome; phosphoinositide; Rab; tuberculosis
Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.
Tuberculosis in its latent form infects one-third of the total human population, hiding in structures called granulomas in the lung. The dense tissue formed by the granuloma severely limits the amount of oxygen available and yet somehow the bacteria manage to survive for many years. In this study we have examined TB bacteria artificially locked at specific oxygen tensions to understand how they maintain basic metabolic functions in the absence of oxygen. As oxygen is lowered intracellular ATP levels fall and reduced cofactors such as NADH accumulate, unable to close the respiratory cycle using electron transport to donate their extra electrons to molecular oxygen. As they begin to suffocate, the bacteria flip the direction of their tricarboxylic acid (TCA) cycle enzymes from an oxidative direction to a reductive direction and begin to actively secrete succinic acid. Similar to lactate secretion in bacteria capable of growing anaerobically, this process is fermentative in nature, sufficient to enable the TB bacteria to continue basic physiologic functions like maintaining a proton gradient across the membrane, but not sufficient to allow them to grow. Understanding these processes may pinpoint vulnerabilities that could help develop interventions to treat latent infections before people develop destructive lung disease.
Mammalian target of rapamycin (mTOR) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles 1, 2 intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates 3-8. Here we report that lysosomal positioning coordinates anabolic and catabolic responses to changes in nutrient availability by orchestrating early plasma membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, while starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH (pHi). Lysosomal positioning regulates mTORC1 signalling, which, in turn, influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting both at the initiation and termination stages of the process. Our findings provide a fundamental physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
Autophagy is a key cytoplasmic biomass and organellar quality and quantity control pathway of the eukaryotic cell. It is particularly suited to capture and degrade large, multi-macromolecular cytosplasmic targets earmarked for degradation or turnover. Typical autophagic cargos represent large swaths of cytosol as a source of energy and anabolic precursors at times of growth restrictions imposed by the absence of growth factors, nutrient limitation or hypoxia. Autophagy is the only effective mechanism for removal of whole organelles such as leaky or surplus mitochondria, disposal of potentially toxic protein aggregates too large for proteasomal removal, and elimination of intracellular microbes including bacteria, protozoa and viruses. Recent studies have shown that human immunodeficiency virus (HIV) is targeted for eliminated by autophagy but that this is countered by the viral protein Nef. Here we review these relationships and underscore the untapped potential of autophagy as a druggable antiviral process.
Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.
Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Cure and possible eradication of tuberculosis are limited by the lack of availability of any drug that can kill dormant Mtb. Understanding of the processes critical for dormancy and a reliable dormancy model suitable for high throughput screening of chemicals will help to discover drugs that can kill dormant Mtb. Storage of lipids for utilization as energy source is critically needed for dormancy. In the human lung, Mtb-infected macrophages are sequestered inside the hypoxic environments of the physical enclosure called granuloma in which Mtb becomes dormant. None of the currently used cell culture models of Mtb infection mimic this situation. We developed a model that mimics the environment inside the human granuloma by incubating Mtb-infected macrophages under hypoxia. We found that, under these conditions, macrophages accumulate lipid droplets and Mtb within these macrophages acquire a dormancy phenotype. We report how the pathogen inside the macrophages utilizes the host lipids to store lipids within the pathogen and acquire the hallmark traits of dormant Mtb. Thus, our novel model of Mtb dormancy may enable better understanding of the metabolic processes vital for the dormant pathogen and help to discover drugs that can kill latent pathogens.
IRGM, a human immunity related GTPase, confers autophagic defense against intracellular pathogens by an unknown mechanism. Here we report the unexpected mode of IRGM action. IRGM showed differential affinity for mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd but not IRGMb splice isoforms caused mitochondrial depolarization and autophagy-independent but Bax/Bak-dependent cell death. By acting on mitochondria IRGM confers autophagic protection or cell death, explaining IRGM action both in defense against tuberculosis and in damaging inflammation in Crohn's disease.
Dendritic cells (DCs) in mucosal surfaces are early targets for human immunodeficiency virus-1 (HIV-1). DCs mount rapid and robust immune responses upon pathogen encounter. However, immune response in the early events of HIV-1 transmission appears limited, suggesting that HIV-1 evade early immune control by DCs. We report that HIV-1 induces a rapid shutdown of autophagy and immunoamphisomes in DCs. HIV-1 envelope activated the mammalian target of rapamycin pathway in DCs, leading to autophagy exhaustion. HIV-1-induced inhibition of autophagy in DC increased cell-associated HIV-1 and transfer of HIV-1 infection to CD4+ T cells. HIV-1-mediated downregulation of autophagy in DCs impaired innate and adaptive immune responses. Immunoamphisomes in DCs engulf incoming pathogens and appear to amplify pathogen degradation as well as Toll-like receptor responses and antigen presentation. The findings that HIV-1 downregulates autophagy and impedes immune functions of DCs represent a pathogenesis mechanism that can be pharmacologically countered with therapeutic and prophylactic implications.
Autophagy initiation is strictly dependent on phosphatidylinositol 3-phosphate (PI3P) synthesis. PI3P production is under tight control of PI3Kinase, hVps34, in complex with Beclin-1. Mammalian cells express several PI3P phosphatases that belong to the myotubularin family. Even though some of them have been linked to serious human diseases, their cellular function is largely unknown. Two recent studies indicate that PI3P metabolism involved in autophagy initiation is further regulated by the PI3P phosphatases Jumpy and MTMR3. Additional pools of PI3P, upstream of mTOR and on the endocytic pathway, may modulate autophagy indirectly, suggesting that other PI3P phosphatases might be involved in this process. This review sums up our knowledge on PI3P phosphatases and discusses the recent progress on their role in autophagy.
autophagy; myotubularin; PI3P; phosphatase; Jumpy; MTMR14
Autophagy is a ubiquitous eukaryotic cytoplasmic quality and quantity control pathway. The role of autophagy in cytoplasmic homeostasis seamlessly extends to cell-autonomous defense against intracellular microbes. Recent studies also point to fully integrated, multitiered regulatory and effector connections between autophagy and nearly all facets of innate and adaptive immunity. Autophagy in the immune system as a whole confers measured immune responses; on the flip side, alterations in autophagy can lead to inflammation and tissue damage, as evidenced by Crohn's disease predisposition polymorphisms in autophagy basal apparatus (Atg16L) and regulatory (IRGM) genes. Polymorphisms in the IRGM gene in human populations have also been linked to predisposition to tuberculosis. There are several areas of most recent growth: (i) links between autophagy regulators and infectious disease predisposition in human populations; (iii) demonstration of autophagy role in infection control in vivo in animal models; (ii) the definition of specific anti-autophagic defenses in highly evolved pathogens; and (iii) recognition of connections between the ubiquitin system and autophagy of bacteria (and interestingly mitochondria, which are incidentally organelles of bacterial evolutionary origin) via a growing list of modifier and adapter proteins including p62/SQSTM1, NDP52, Atg32, Parkin and Nix/BNIP3L.
Autophagy allows cells to self-digest portions of their own cytoplasm for a multitude of physiological purposes including innate and adaptive immunity functions. In one of its innate immunity manifestations, autophagy is known to contribute to the killing of intracellular microbes including Mycobacterium tuberculosis, although the molecular mechanisms have been unclear. Here, we delineated sequential steps of the autophagic pathway necessary to control intracellular M. tuberculosis and found that in addition to autophagy initiation and maturation, an accessory autophagy-targeting molecule p62 (A170 or SQSTM1) was required for mycobactericidal activity. The p62 adapter protein delivered specific ribosomal and bulk ubiquitinated cytosolic proteins to autolysosomes where they were proteolytically converted into products capable of killing M. tuberculosis. Thus, p62 brings cytosolic proteins to autolysosomes where they are processed from innocuous precursors into neo-antimicrobial peptides, explaining in part the unique bactericidal properties of autophagic organelles.
autophagy; p62; NBR1; ribosome; tuberculosis
The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D−/− hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.
Tissue macrophages frequently undergo a program of cell death, termed apoptosis, following sustained ingestion and killing of bacteria. In macrophages, induction of apoptosis enhances bacterial killing when macrophages' initial killing capacity is exhausted. We have investigated the mechanism of apoptosis in macrophages exposed to pneumococci, the commonest cause of bacterial pneumonia. We show that the cell structure containing ingested bacteria, the phagolysosome, becomes permeabilized early in the death process. Pneumococcal exposure activates a phagolysosomal enzyme, cathepsin D, which induces apoptosis. Cathepsin D activation is required for permeabilization of mitochondria, an organelle implicated in apoptosis induction. Cathepsin D reduces levels of a negative regulator of apoptosis in macrophages, Mcl-1, by enhancing its association with an enzyme, which mediates its degradation. The importance of these findings was confirmed in a bone marrow transplant model in which mice either received bone marrow from mice containing or lacking the cathepsin D gene. This model showed that reduced apoptosis of alveolar macrophages occurred when cathepsin D was lacking, and that this impaired clearance of pneumococci in the mouse lung. We conclude that during bacterial challenge, lysosomal permeabilization and cathepsin D activation triggers a novel death pathway, in a timely fashion, linking bacterial killing to apoptosis induction.