Biology subjects

Glasgow, J. N.

Publications and source records attributed to Glasgow, J. N..

6 recordsLinked to original sources

bioRxiv2026

Glucose selectively drives a rapid oxidative burst and immunometabolic reprogramming in human neutrophils during Mycobacterium tuberculosis infection

Neutrophil functions have been linked to tuberculosis (TB)-associated tissue damage; however, the mechanisms driving immunopathology in the human TB lung remain poorly understood, due partly to the scarcity of human tissue for study. Here, we examine the metabolic and bioenergetic reprogramming of human neutrophils in response to Mycobacterium tuberculosis (Mtb) infection. In human necrotic TB granulomas, levels of NETosis-associated proteins are increased and co-localize with GLUT3, linking nutrient uptake to tissue damage. In vitro, Mtb elicits an immediate, contact-dependent oxidative burst in human neutrophils, and the magnitude of this response is carbon source-dependent. Glucose enables the most robust responses, indicating that glucose metabolism is a key driver of neutrophil-mediated inflammatory damage during TB. Mtb-induced responses are distinct from those induced by PMA, non-tuberculous mycobacteria, or other pathogenic intracellular bacteria, and are mediated through multiple neutrophil surface receptors. Notably, our data show that while the oxidative burst is carbon source-dependent, cytokine production is not. Further, Mtb infection reprograms neutrophil metabolism from glycolysis to the pentose phosphate pathway (PPP), generating NADPH required for the oxidative burst. Inhibiting G6PD, NADPH oxidase, or PAD4 significantly reduces this response, highlighting the PPP as a promising host target for mitigating TB immunopathology.

microbiology↗

bioRxiv2023

Detection of Mycobacterium tuberculosis in human tissue via RNA in situ hybridization

RationaleAccurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identify Mycobacterium tuberculosis (Mtb) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection of Mtb mRNA. ObjectivesTo employ RNAscope to determine the spatial distribution of Mtb mRNA within tuberculous human tissue, to appraise ZN-negative tissue from confirmed TB patients, and to provide proof-of-concept of RNAscope as a platform to inform TB diagnosis and Mtb biology. MethodsWe examined ante- and postmortem human TB tissue using RNAscope to detect Mtb mRNA and a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). Measurements and main resultsWe adapted RNAscope for Mtb and identified intact and disintegrated Mtb bacilli and intra- and extracellular Mtb mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. ConclusionsRNAscope has diagnostic potential and can guide therapeutic intervention as it detects Mtb mRNA and morphology in ZN-negative tissues from TB patients, and Mtb mRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations of Mtb bacilli exist in vivo.

microbiology↗

bioRxiv2022

NAD(H) homeostasis is essential for host protection mediated by glycolytic myeloid cells in tuberculosis

Mycobacterium tuberculosis (Mtb) disrupts glycolytic flux in infected myeloid cells through an unclear mechanism. Flux through the glycolytic pathway in myeloid cells is inextricably linked to the availability of NAD+, which is maintained by NAD+ salvage and lactate metabolism. Using lung tissue from tuberculosis (TB) patients and myeloid deficient LDHA (LdhaLysM-/-) mice, we demonstrate that glycolysis in myeloid cells is essential for protective immunity in TB. Glycolytic myeloid cells are essential for the early recruitment of multiple classes of immune cells and the protective effects of IFN{gamma}. We identified NAD+ depletion as central to the glycolytic inhibition caused by Mtb. Lastly, we show that the NAD+ precursor nicotinamide exerts a host-directed, antimycobacterial effect, and that nicotinamide prophylaxis and treatment reduces Mtb lung burden in vivo. These findings provide new insight into how Mtb alters host metabolism through perturbation of NAD(H) homeostasis and reprogramming of glycolysis, highlighting this pathway as a potential therapeutic target.

microbiology↗

bioRxiv2021

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the Dos dormancy regulon

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 {micro}M) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase. Significance StatementHydrogen sulfide (H2S) is an important signaling molecule in eukaryotes and bacteria, and along with CO and NO, is an important part of host defense against Mycobacterium tuberculosis (Mtb). However, the mechanism(s) by which Mtb senses and responds to H2S is unknown. Here, we report that the Mtb heme sensor kinase DosS, a known sensor of CO and NO, is also a sensor of H2S. We found that H2S binds DosS in its ferric (Fe3+) state, which is considered as its inactive state, to induce the Dos dormancy regulon during infection. These data highlight the unusual capacity of Mtb to sense multiple gasotransmitters through a single sensing protein.

biochemistry↗

bioRxiv2021

Mycobacterium tuberculosis H2S functions as a sink to modulate central metabolism, bioenergetics, and drug susceptibility

H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.

microbiology↗

bioRxiv2020

3D microarchitecture of the human tuberculous granuloma

Our current understanding of the pathophysiology of human pulmonary TB is limited by the paucity of human TB lung tissue for study and reliance on 2D analytical methods. Here, to overcome the limitations of conventional 2D histopathology, we used high-resolution 3D X-ray imaging ({micro}CT/nCT) to characterize necrotic lesions within human tuberculous lung tissues in relation to the airways and vasculature. We observed marked heterogeneity in the 3D structure and volume of lesions. Also, 3D imaging of large human TB lung sections provides unanticipated new insight into the spatial organization of TB lesions in relation to airways and the vascular system. Contrary to the current dogma depicting granulomas as simple spherical structures, we show that TB lesions exhibit complex, cylindrical, branched-type morphologies, which are connected to, and shaped by, the small airways. Our results highlight the likelihood that a single structurally complex lesion could be wrongly viewed as multiple independent lesions when evaluated in 2D. These findings have strong implications for understanding the pathophysiology and evolution of TB disease and suggest that aerosolized drug delivery strategies for TB should be reconsidered.

microbiology↗