Biology subjects

Dhar, N.

Publications and source records attributed to Dhar, N..

12 recordsLinked to original sources

bioRxiv2026

A genetically encoded biosensor reveals heterogenous cAMP dynamics coordinating growth and resuscitation in Mycobacterium tuberculosis

3',5'- Cyclic adenosine monophosphate (cAMP) serves as a global regulatory hub in Mycobacterium tuberculosis (Mtb), which dedicates approximately 1% of its genome to the synthesis, degradation and sensing of this secondary messenger. Despite cAMPs central role in Mtb virulence and metabolic adaptation, we currently do not have any tools to monitor cAMP dynamics in real time at single-cell resolution. Here, we report the development and validation of rmgCarvi, a genetically encoded biosensor adapted from eukaryotic systems for use in mycobacteria. By utilizing cAMP-responsive cpGFP and constitutive mCherry fluorescence, rmgCarvi provides a high-fidelity ratiometric readout of intracellular cAMP levels, revealing that carbon source and growth state act as primary determinants of cAMP dynamics. Real-time single-cell imaging using rmgCarvi demonstrated robust correlations between cAMP and growth rate, with pronounced cAMP spikes coinciding with cell division. In starved Mtb populations, a bimodal distribution in cAMP levels dictates a distinct hysteresis during regrowth upon nutrient supplementation. Low-cAMP subpopulations are associated with strong induction in cAMP levels and a longer lag before initiation of growth, while high cAMP cells do not exhibit significant induction in cAMP and have recovery times uncorrelated with prior cAMP levels. Finally, use of rmgCarvi in infection studies captured host species-specific responses, including a strong cAMP induction upon internalization followed by a decline in murine, but not human, macrophages. By resolving cAMP dynamics with unprecedented cellular and temporal resolution, rmgCarvi provides a framework for understanding how Mtb tunes this crucial metabolite to navigate the complex microenvironments in the host. Significance StatementWe report the evaluation of a ratiometric genetic biosensor, rmgCarvi, for real-time single-cell monitoring of cAMP dynamics in mycobacteria. This biosensor reveals how Mycobacterium tuberculosis precisely tunes cAMP within a narrow range ("Goldilocks zone") to coordinate metabolic adaptation, and growth across complex nutrient environments. In nutrient-starved populations, we identify a bimodal cAMP distribution that functions as a phenotypic memory, dictating resuscitation kinetics upon nutrient restoration. As the first cAMP biosensor validated in any mycobacterial species, rmgCarvi facilitates the discovery of host-specific signaling dynamics during infection and provides a platform for screening of compounds that perturb cAMP homeostasis. This study provides a framework to explore how Mtb utilizes this secondary messenger molecule to survive and adapt within complex host environments.

microbiology↗

bioRxiv2026

Human prostaglandin reductases dearomatize and inactivate benzothiazinone antitubercular drugs

Macozinone (MCZ, PBTZ169) is a potent clinical stage benzothiazinone antitubercular agent that covalently inhibits the essential mycobacterial flavoenzyme DprE1. In some mammals, MCZ undergoes reductive dearomatization to H2MCZ, a Hydride Meisenheimer Complex, identified as the major circulating metabolite in humans. We demonstrate for the first time that the NADPH-dependent human prostaglandin reductases PTGR1 and PTGR2 catalyze MCZ dearomatization into H2MCZ, resulting in loss of antimycobacterial activity. This reaction represents a heretofore undescribed host-mediated metabolic inactivation pathway for a therapeutic agent. Although H2MCZ may constitute a transient reactive intermediate, ex vivo and cellular data indicate that it does not contribute to DprE1 inhibition in vivo. Pharmacological inhibition of PTGR1 and PTGR2 using diclofenac, indomethacin, dicumarol, or the selective inhibitor PTGR2-IN-1 suppresses H2MCZ formation and partially restores MCZ antimycobacterial activity in vitro. Together, our findings uncover a previously unrecognized noncanonical enzymatic mechanism of drug metabolism involving dearomatization in humans. Targeting prostaglandin reductases may represent a strategy to enhance benzothiazinone exposure and efficacy.

pharmacology and toxicology↗

bioRxiv2026

Menaquinone depletion resensitises bedaquiline-resistant tuberculosis

Tuberculosis remains a leading cause of global mortality, and rising bedaquiline resistance threatens the effectiveness of current drug-resistant treatment regimens. Bedaquiline resistance typically arises through mutations in Rv0678 that upregulate drug efflux and confer cross-resistance to multiple drug classes. Here, we identify and optimise a chemical series targeting MenG, a central enzyme in the menaquinone biosynthesis pathway, yielding potent bactericidal inhibitors with in vivo efficacy. Strikingly, MenG inhibition restored bedaquiline susceptibility in efflux-mediated resistant strains, an effect confirmed in vivo where combination therapy achieved a 99.8% reduction in bacterial burden compared with bedaquiline alone. Potentiation also extended to pretomanid and other key agents. Disruption of upstream menaquinone and shikimate pathway enzymes produced similar resensitisation, establishing these pathways as tractable targets for restoring drug susceptibility in Mycobacterium tuberculosis. These findings provide a novel strategy to overcome bedaquiline resistance and strengthen future regimens for efflux-mediated drug-resistant tuberculosis.

microbiology↗

bioRxiv2025

Impact of cell culture model systems on SARS-CoV-2 and MERS-CoV infection dynamics and antiviral responses

Cell cultures are widely used to study infectious respiratory diseases and to test therapeutics; however, they do not faithfully recapitulate the architecture and complexity of the human respiratory tract. Lung organoids have emerged as an alternative model that partially overcomes this key disadvantage. Lung organoids can be cultured in various formats that offer potential for studying highly pathogenic viruses. However, the effects of these different formats on virus infection remain unexplored, leaving their relative value unclear. In this study, we generated primary lung organoids from human donor cells and used them to derive monolayers and air-liquid interface (ALI) cultures with the goal of comparing the replication kinetics of two circulating highly pathogenic coronaviruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV). Infection studies revealed that organoid-derived monolayers displayed limited infection and the innate immune response was impaired against bacterial lipopolysaccharide (LPS) but not against virus-like double-stranded dsRNA or poly(I:C). Meanwhile, organoids and organoid-derived ALI cultures retained viral permissivity, with ALI cultures displaying diverse antiviral immune responses against both coronaviruses. SARS-CoV-2 and MERS-CoV demonstrated differential replication kinetics in organoid and organoid-derived ALI cultures. Therefore, primary organoid-derived cells in two-dimensional monolayer or three-dimensional ALI formats influence virus infection and host antiviral responses. Our study informs the selection of culture conditions for organoid-based respiratory disease research and therapeutic testing. ImportanceThe COVID-19 pandemic heralded the upsurge in human-derived lung organoid based studies due to their cellular heterogeneity that emulates the cellular complexity of the respiratory tract. A major disadvantage of organoid models resides in their apical-in conformation that "hides" cells and proteins that are typically exposed to the air-liquid interface (ALI) in the airways and are targets of viruses. Here, we generated monolayers and ALI cultures to facilitate cell exposure to highly relevant pathogens and compared them to parental organoids. Organoids at the ALI captured infection and immune responses better than organoids and organoid-derived monolayer cultures. Organoids at the ALI are a viable approach to improve identification and characterization of virus infection, host responses, and therapeutic testing.

microbiology↗

bioRxiv2024

Wag31, a membrane tether, is crucial for lipid homeostasis in mycobacteria

The mycobacterial cytoskeletal protein Wag31 is necessary for maintaining cell shape and directing cellular growth and elongation. Wag31 has a characteristic N-terminal DivIVA-domain and a C-terminal coiled-coil domain. While the role of Wag31 in polar elongation is known, there is limited mechanistic insight on how it orchestrates growth and elongation. In this report, we delineate roles of the N- and C-terminal domains of Wag31 using genetics, state-of-the-art multi-omics, biochemical, and imaging approaches. We show that Wag31 predominantly interacts with several membrane-associated proteins involved in lipid metabolism, cell wall synthesis and division. Native levels of Wag31 are critical for the maintenance and distribution of membrane lipids. Both depletion and overexpression of Wag31 perturbs lipid homeostasis, leading to the formation of intracellular lipid inclusions (ILIs). Protein-lipid crosslinking and imaging studies reveal that purified Wag31 can bind and effectively tether Cardiolipin (CL)-containing liposomes. We further show that the tethering activity lies in the DivIVA-domain containing N-terminal of Wag31 while the C-terminal mediates protein-protein interactions of Wag31. Despite retaining its ability to interact with partner proteins, the DivIVA domain-deleted Wag31 mutant shows defects in liposome tethering in vitro and non-polar localization of CL in vivo, which eventually causes lethality. Our study suggests that membrane tethering licenses Wag31 to form scaffolds that help orchestrate protein-lipid and protein-protein interactions necessary for mycobacterial growth and survival.

microbiology↗

bioRxiv2023

Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of polysaccharide-specific B cell immunity

Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25+ cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this assay to simultaneously analyse 14 Streptococcus pneumoniae or 5 Streptococcus agalactiae serotype-specific B cell populations. The phenotype of polysaccharide-specific B cells was associated with serotype specificity, vaccination history and donor population. For example, we observed a link between non-class switched (IgM+) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3. Moreover, B cells had increased activation in donors from South Africa, which has high-incidence of S. agalactiae invasive disease, compared to European donors. This assay allows for the characterization of heterogeneity in B cell immunity that may underlie immunization efficacy.

immunology↗

bioRxiv2023

Uptake-independent killing of macrophages by extracellular aggregates of Mycobacterium tuberculosis is ESX-1 and PDIM-dependent

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of small numbers of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular replication of Mtb leads to death of the infected macrophages, release of bacterial aggregates, and rapid growth of the extracellular aggregates on host-cell debris. Here, we show that extracellular Mtb aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use single-cell time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytoplasmic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb type VII secretion system ESX-1, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface-exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that ESX-1-mediated secretion of the EsxA/EsxB virulence factors is not required for uptake-independent killing of macrophages after contact with extracellular Mtb aggregates. In the absence of EsxA/EsxB secretion, killing is mediated by the 50-kDa isoform of the ESX-1-secreted protein EspB, while blocking secretion of both EsxA/EsxB and processed EspB reduces killing to background levels. Treatment with a small-molecule ESX-1 inhibitor reduces uptake-independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti-phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung. Significance statementMycobacterium tuberculosis (Mtb) can survive inside the lung macrophages that normally provide the first line of defense against bacterial infections. Intracellular replication of Mtb ultimately results in the death and lysis of infected macrophages, allowing the bacteria to spread to other cells and propagate the infection. Our study shows that extracellular Mtb aggregates that form on the debris of dead host cells can induce macrophage death in a contact-dependent but uptake-independent manner, allowing the bacteria to evade the host defenses associated with uptake by macrophages. Killing of macrophages by extracellular Mtb aggregates is driven by the Mtb ESX-1 secretion system and the surface-exposed lipid phthiocerol dimycocerosate. Our results suggest that novel drugs targeting Mtb factors required for host-cell killing by extracellular Mtb aggregates may reduce bacterial spreading and expansion of necrotic tuberculosis lesions, which are known to be poorly penetrated by conventional antibiotics.

microbiology↗

bioRxiv2021

The mycobacterial mutasome: composition and recruitment in live cells

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded "mycobacterial mutasome" - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III {beta} subunit ({beta} clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted {beta} clamp-binding motif abolishes ImuB-{beta} clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this {beta} clamp-binding antibiotic collapses pre-formed ImuB-{beta} clamp complexes. These observations establish the essentiality of the ImuB-{beta} clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

microbiology↗

bioRxiv2021

Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbours of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

microbiology↗

bioRxiv2020

Invasion into the bladder wall generates solitary subpopulations of uropathogenic Escherichia coli that are protected from killing by antibiotics and neutrophil swarms in an organoid model

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs) requiring antibiotic therapy. Recurrent infections, which occur in a quarter of treated individuals, may arise from "quiescent intracellular reservoirs" of bacteria that invade deeper layers of the bladder wall following infection and exfoliation of superficial umbrella cells. Here, we present a novel bladder organoid model of UPEC infection that recapitulates the stratified bladder architecture within a small volume suitable for live-cell imaging of host-pathogen dynamics with high spatiotemporal resolution. We confirm that bacteria injected into the organoid lumen rapidly enter superficial cells that resemble umbrella cells and proliferate to generate tightly packed colonies that resemble intracellular bacterial communities (IBCs), a hallmark of UPEC pathogenesis. Unexpectedly, at early stages of infection we detect individual "solitary" bacteria that penetrate deeper layers of the organoid wall, where they evade killing by antibiotics and neutrophils. Volumetric serial block face scanning electron microscopy of infected organoids reveals that solitary bacteria can be found throughout the bladder wall and may be intracellular or pericellular (sandwiched between uroepithelial cells). Unlike bacteria within IBCs, which are coccoid-shaped and non-flagellated, solitary bacteria within the bladder wall are rod-shaped and flagellated. We conclude that early invasion of deeper layers of the bladder wall, independent of IBC formation, results in the establishment of reservoirs of solitary bacteria that resist elimination by antibiotics and the host innate immune response.

microbiology↗

bioRxiv2020

Rapid endothelialitis and vascular inflammation characterise SARS-CoV-2 infection in a human lung-on-chip model

Severe cases of COVID-19 present with hypercoagulopathies and systemic endothelialitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of immune cell mediated cytokine storms is unknown. This is in part because in vitro models are typically epithelial cell monocultures or fail to recapitulate vascular physiology. We use a vascularised lung-on-chip model where, consistent with monoculture reports, low numbers of SARS-CoV-2 virions are released apically from alveolar epithelial cells. However, rapid infection of the underlying endothelial layer leads to the generation of clusters of endothelial cells with low or no CD31 expression, a progressive loss of endothelial barrier integrity, and a pro-coagulatory microenvironment. These morphological changes do not occur if these cells are exposed to the virus apically. Viral RNA persists in individual cells, which generates a response that is skewed towards NF-KB mediated inflammation, is typified by IL-6 secretion even in the absence of immune cells, and is transient in epithelial cells but persistent in endothelial cells. Perfusion with Tocilizumab, an inhibitor of trans IL-6 signalling slows the loss of barrier integrity but does not prevent the formation of endothelial cell clusters with reduced CD31 expression. SARS-CoV-2 mediated endothelial cell damage occurs despite a lack of rapid viral replication, in a cell-type specific manner and independently of immune-cell mediated cytokine storms, whose effect would only exacerbate the damage.

microbiology↗

bioRxiv2020

A lung-on-chip infection model reveals protective and permissive roles of alveolar epithelial cells in tuberculosis

Mycobacterium tuberculosis (Mtb) makes first contact with a host in the alveolar space, an interaction largely inaccessible to experimental observation. We establish a lung-on-chip model for early tuberculosis and use time-lapse imaging to reveal the dynamics of host-Mtb interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models. By reconstituting host physiology in a modular manner, we probe the role of pulmonary surfactant secreted by alveolar epithelial cells (AECs) in early infection. This is difficult to study directly in animal models, as surfactant-deficient animals are either non-viable or develop acute lung pathologies. We demonstrate that surfactant deficiency results in rapid and uncontrolled Mtb growth in both macrophages and AECs. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins 1,2 from the bacterial cell surface and consistent with this mechanism of action, we show that attenuation of bacteria lacking the virulence-associated ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why individuals with compromised surfactant function, such as smokers and elderly persons, are at increased risk of developing active tuberculosis.

microbiology↗