Biology subjects

Pagan, A. J.

Publications and source records attributed to Pagan, A. J..

6 recordsLinked to original sources

bioRxiv2026

Macrophage adaptation to hypoxia in the tuberculous granuloma potentiates mycobacterium-induced mitochondrial damage and granuloma necrosis

Granulomas, organized macrophage-rich aggregates, are pathological hallmarks of tuberculosis. Characteristically, as Mycobacterium tuberculosis replicates within macrophages, the granuloma enlarges, and then the center undergoes necrosis, promoting mycobacterial growth in the debris (caseum). This sequence results in large, mycobacterium-rich granulomas that can rupture into airways, facilitating disease transmission. Here, using the zebrafish-Mycobacterium marinum tuberculosis model, we find that both stages - granuloma enlargement and necrosis - depend on the mycobacterial virulence factor, EsxA, that induces mitochondrial damage and apoptosis of infected macrophages. Initially, dying macrophages are engulfed by newly recruited macrophages. Then, the enlarging granuloma core becomes hypoxic and induces Hypoxia Inducible Factor-1 (HIF-1). HIF-1 suppresses mitochondrial respiration, sensitizing macrophages to EsxA mitotoxicity. These sensitized macrophages undergo accelerated apoptosis which outstrips clearance, causing mycobacterium-rich necrotic debris to accumulate in the granuloma cores. Thus, tuberculosis transmission depends on the dynamic interplay between a mycobacterial virulence factor and an adaptive host metabolic program.

microbiology↗

bioRxiv2022

mTOR-regulated Mitochondrial Metabolism Limits Mycobacterium-induced Cytotoxicity

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 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 humanitys most lethal pathogen, is successful in only a minority of infected individuals.

immunology↗

bioRxiv2022

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence

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- 6s 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-6s 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 StatementTuberculosis (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.

microbiology↗

bioRxiv2020

Elevated cerebrospinal fluid cytokine levels in tuberculous meningitis predict survival in response to dexamethasone

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 worlds 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.

immunology↗

bioRxiv2020

Mycobacterium marinum phthiocerol dimycocerosates enhance macrophage phagosomal permeabilization and membrane damage

Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an{Delta} mmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay-- a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.

microbiology↗

bioRxiv2020

Schistosoma mansoni eggs modulate the timing of granuloma formation to promote transmission

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. Egg-sized inert beads also induce granulomas rapidly, through a foreign body response. Strikingly, immature eggs evade macrophage recruitment altogether, revealing that the eggshell is immunological inert. These findings suggest that the parasite modulates the timing of granuloma formation to its advantage, inhibiting foreign body granuloma formation until it reaches the optimal maturation and location for extrusion. At this point, the parasite secretes specific antigens through the eggshell to trigger granulomas that might facilitate egg extrusion.

immunology↗