Antonio J Pagán’s research while affiliated with University of Cambridge and other places
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Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity’s most lethal pathogen, is successful in only a minority of infected individuals.
Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations were required to prevent mitochondrial damage and death caused specifically by the mycobacterial ESX-1 secretion system. Our finding that the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism may help explain why Mycobacterium tuberculosis, albeit humanity’s most lethal pathogen, only causes disease in a minority of infected individuals.
This work was supported by a Wellcome Trust Principal Research Fellowship (103950/Z/14) and an NIH MERIT award (R37 AI054503) (L.R.).
Significance
Tuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis , and its close pathogenic relative Mycobacterium marinum , initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane–bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.
Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused specifically by ESAT-6, the principal secreted substrate of the specialized mycobacterial secretion system ESX-1, a key virulence mediator. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis , albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.
Mycobacterium tuberculosis and its close relative Mycobacterium marinum infect macrophages and induce the formation of granulomas, organized macrophage-rich immune aggregates. These mycobacterial pathogens can accelerate and co-opt granuloma formation for their benefit, using the specialized secretion system ESX-1, a key virulence determinant. ESX-1-mediated virulence is attributed to the damage it causes to the membranes of macrophage phagosomal compartments, within which the bacteria reside. This phagosomal damage, in turn, has been attributed to the membranolytic activity of ESAT-6, the major secreted substrate of ESX-1. However, mutations that perturb ESAT- 6’s membranolytic activity often result in global impairment of ESX-1 secretion. This has precluded an understanding of the causal and mechanistic relationships between ESAT-6 membranolysis and ESX-1-mediated virulence. Here, we identify two conserved residues in the unstructured C-terminal tail of ESAT-6 required for phagosomal damage, granuloma formation and virulence. Importantly, these ESAT-6 mutants have near- normal levels of secretion, far higher than the minimal threshold we establish is needed for ESX-1-mediated virulence early in infection. Unexpectedly, these loss-of-function ESAT-6 mutants retain the ability to lyse acidified liposomes. Thus, ESAT-6’s virulence functions in vivo can be uncoupled from this in vitro surrogate assay. These uncoupling mutants highlight an enigmatic functional domain of ESAT-6 and provide key tools to investigate the mechanism of phagosomal damage and virulence.
Significance Statement
Tuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis , and its close pathogenic relative Mycobacterium marinum , initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes.
Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C-terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.
Significance
Despite appropriate antibiotic treatment, tuberculous meningitis carries a high mortality ascribed to overexuberant inflammation. Genetic variations in the enzyme, LTA4H, alter inflammation, with individuals carrying the inflammation-associated LTA4H variant benefitting from antiinflammatory steroids administered alongside antibiotics. A prior study found poor correlation between LTA4H genotype and cerebrospinal fluid levels of cytokines, key mediators of inflammation. The study used “frequentist” statistical methods that can fail to detect true differences. Using Bayesian statistics, which can detect significant differences not found by frequentist methods, we found good correlation between LTA4H genotype and cytokine levels, and cytokine levels and outcome even independent of LTA4H genotype. These findings suggest that LTA4H and additional inflammation factors affect outcome and suggest tailoring steroid therapy to cytokine levels.
Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.
Adjunctive treatment with anti-inflammatory corticosteroids like dexamethasone increases survival in tuberculosis meningitis. Dexamethasone responsiveness associates with a C/T variant in Leukotriene A4 Hydrolase (LTA4H ), which regulates expression of the pro-inflammatory mediator leukotriene B4 (LTB4). TT homozygotes, with increased LTB4, have the highest survival when treated with dexamethasone and the lowest survival without. While the T allele is present in only a minority of the world’s population, corticosteroids confer modest survival benefit worldwide. Using Bayesian methods, we examined how pre-treatment levels of cerebrospinal fluid (CSF) pro-inflammatory cytokines affect survival in dexamethasone-treated tuberculous meningitis. LTA4H TT homozygosity was associated with global cytokine increases, including TNF. Association between higher cytokine levels and survival extended to non-TT patients, suggesting that other genetic variants may also induce dexamethasone-responsive pathological inflammation. These findings warrant studies that tailor dexamethasone therapy to pre-treatment CSF cytokine concentrations, while searching for additional genetic loci shaping the inflammatory milieu.
Schistosome eggs provoke the formation of granulomas, organized immune aggregates, around them. For the host, the granulomatous response can be both protective and pathological. Granulomas are also postulated to facilitate egg extrusion through the gut lumen, a necessary step for parasite transmission. We used zebrafish larvae to visualize the granulomatous response to Schistosoma mansoni eggs and inert egg-sized beads. Mature eggs rapidly recruit macrophages, which form granulomas within days. Beads also induce granulomas rapidly, through a foreign body response. Strikingly, immature eggs do not recruit macrophages, revealing that the eggshell is immunologically inert. Our findings suggest that the eggshell inhibits foreign body granuloma formation long enough for the miracidium to mature. Then parasite antigens secreted through the eggshell trigger granulomas that facilitate egg extrusion into the environment. In support of this model, we find that only mature S. mansoni eggs are shed into the feces of mice and humans.
Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.
... In addition, how metabolism benefits host immune cell defense against invading pathogens has aroused attention. mTOR has been demonstrated to protect host macrophage from mycobacterium-induced cell death through increasing glycolysis-fueled mitochondrial energy [50,51]. Therefore, it could be that the different responses to wounding and infection in the tlr2 mutant are caused by mitochondrial dysfunction due to the aberrant expression of genes involved in metabolism. ...
... New studies have suggested a possible anti-pathogen effect for this drug in different doses and conditions, for example against lymphocytic choriomeningitis virus (LCMV), vaccinia viruses or as a host cell mediator against tuberculosis (Araki et al., 2009;Singh and Subbian, 2018). Other studies on zebrafish model of Mycobacterium marinum infection indicated that mTOR can effectively boost host resistance to infection (Pagan et al., 2016). Information on the antileishmanial effect of this drug is still limited, although our previous study on L. major infection showed a strong anti-parasitic effect for rapamycin (Khadir et al., 2018). ...
... Mtb control in other studies [23,30,68] and is a defining feature of Mtb-infected MDMs in WT mice [28,29]. GSEA was performed as an additional approach to test if MDM expression of MHCII molecules, and presumably receipt of signals Table. ...
... Similarly, ESAT-6 from M. marinum was also found to play a direct role in producing pores in the membranes of the Mycobacterium-containing vacuole, facilitating its escape from the vacuole and cell-to-cell spread [33]. The C-terminal end of ESAT-6 is found to be essential for phagosomal damage, granuloma formation, and mycobacterial virulence [70]. Also, ESAT-6 activates the NLRP3 inflammasome to secrete IL-18 and IL-1b that recruit neutrophils [71]. ...
... Further elucidation of this mechanism may illuminate whether the association of SLIT3 genetic effects on TBM survival modulates the effects of steroids and/or LTA4H-dependent pathways. For example, an LTA4H genetic variant was associated with cerebrospinal fluid cytokines and inflammatory cell recruitment, a mechanism which could also be impacted by SLIT3 and contribute additional effects on survival (78,82). ...
... Blocking SjE16.7 in vivo could significantly alleviate egg-induced liver immunopathological damage (77,78). Takaki et al. used zebrafish larvae as a model to study egg-induced macrophage recruitment and granuloma formation and found that the Schistosoma japonicum-derived protein omega-1 could mediate the initial stage of macrophage recruitment, which Chemotactic activity depends on its RNase activity (79). When lentivirus was used to interfere with omega-1 in eggs, the tail vein injection of eggs into mice could significantly inhibit the immunopathological changes of egg granulomas in the lungs (80). ...
... For the immunolocalization of the most highly expressed SmVAL9 and SmVAL29 in eggs and miracidia, antisera were raised against proteins produced recombinantly in the bacterial system. Primarily mature eggs were selected for immunolocalization, as these are the eggs that should be the most active in terms of secretion [3,43]. Our experiments revealed that the tissue localization of both proteins in eggs and miracidia is identical. ...
... The rescue was apparent in macrophages that were not treated with IFN-γ, which we used for subsequent experiments. We were surprised that ATG5 and ATG7 restricted ΔppsD Mtb growth, because PDIM contributes to phagosomal damage, which is generally thought to be a prerequisite for xenophagy 41,[47][48][49]53,54 . The bacterial ESX-1 type VII secretion system (T7SS) is also required for phagosomal damage, and it was not identified in the screen. ...
... The fadD26-papA5 operon, starting from the fadD26 gene, encodes proteins involved in PDIM/PGL synthesis and translocation, including FadD26, the PpsA-E and DrrA-C proteins [16]. Mycobacterial mutants with disruptions in the PDIM/PGL biosynthetic or transport pathway exhibit significantly impaired virulence [17,18], demonstrating that PDIMs/PGLs are important virulence factors. PDIMs not only promote mycobacterial macrophage entry, but also prevent phagosomal acidification, permeabilize phagosomal membranes in infected macrophages, and prompt bacillary dissemination by facilitating macrophage necrosis [14,[18][19][20]. ...
... Furthermore, our cases raise the question of why a granulomatous response was observed. Granuloma formation is typically associated with chronic infections that resist eradication, such as Mycobacterium tuberculosis or fungal pathogens, and is driven by a Th1-type immune response involving IFN-γ mediated macrophage activation and T-cell recruitment [21]. The presence of granulomas in these cases suggests that hAdV-B may have evaded clearance and triggered a similar immunologic cascade. ...
![Figure 1. mTORC1-deficient zebrafish are hypersusceptible to Mm infection (A) Hindbrain ventricle (hbv) and caudal vein (cv) injection routes used in this study. Larvae were infected with $150 Mm expressing tdTomato (B), (C), and (E-J) or tdKatushka2 (D) fluorescent proteins via the caudal vein 2 days post-fertilization (dpf). (B) Overlaid micrographs of widefield mycobacterial fluorescence (Mm, red) and bright field in mtor fh178/fh178 or WT siblings (mtor +/+ ) 4 days post-infection (dpi). (C) Quantification of bacterial fluorescence (fluorescent pixel counts [FPCs]) in animals from mtor fh178/+ incross 4 dpi. Symbols represent individual animals. Horizontal lines indicate mean values. (D) Confocal micrograph optical sections of mtor fh178/fh178 and a WT sibling expressing Tg(mpeg1:YFP) 4 dpi, showing a granuloma in the WT animal and mycobacterial cording in the mtor fh178/fh178 animal. Mm (magenta) and macrophages (green) are shown. Arrowheads indicate intracellular Mm.](https://www.researchgate.net/publication/363532388/figure/fig1/AS:11431281084310434@1663131927601/mTORC1-deficient-zebrafish-are-hypersusceptible-to-Mm-infection-A-Hindbrain-ventricle_Q320.jpg)
![TNF affects late-stage granuloma formation
Comparison of macrophage recruitment and granuloma formation in WT and TNFR1 mutant zebrafish larvae with fluorescent macrophages following implantation with a single schistosome egg into their hindbrain ventricle. (A) Zebrafish larva at 36 hours post-fertilization with the hindbrain ventricle (HBV) site of injection and implantation outlined. Scale bar, 300 μm. (B-D) Granuloma formation at 5 days post-implantation. (B) Percent of animals with; granuloma formation (confluent epithelioid macrophage aggregates), macrophages recruited (>6 macrophages in contact with the egg), or minimal recruitment (0–6 macrophages in contact with egg) [4]. (C) Granuloma size and (D) images, with each image from top to bottom corresponding with each red data point, top to bottom, respectively. Scale bar, 50 μm. Horizontal bars in (C), means. Statistics, Student’s t-test. (E) Mean macrophage recruitment kinetics during the first 6 hours post-implantation. Error bars, SEM. Sample size, n = 5 animals per group.](https://www.researchgate.net/publication/348607181/figure/fig1/AS:11431281274127931@1724862463819/TNF-affects-late-stage-granuloma-formation-Comparison-of-macrophage-recruitment-and_Q320.jpg)
