Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 577 records · Page 32Linked to original sources

bioRxiv2020

Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma

Retrotransposons are sequences which transpose within genomes using RNA as an intermediate. Long INterpersed Element-1 (LINE1 or L1) is the only active retrotransposon occupying around 17% of the human genome with an estimated 500,000 copies. An active L1 encodes two proteins (L1ORF1p and L1ORF2p); both of which are critical in the process of retrotransposition. In-order to propagate to the nextgeneration, L1s remain active in germ tissues and at an early stage of development. Surprisingly, by some unknown mechanism, L1 also shows activity in certain parts of the normal brain and many cancers. L1 activity is generally determined by assaying L1ORF1p because of its high expression and availability of the antibody. However, due to its lowerexpression and the unavailability of a robust antibody, detection of L1ORF2p has been limited. L1ORF2p is the crucial protein in the process of retrotransposition as it provides endonuclease and reverse transcriptase (RT) activity. Here, we report a novel human L1ORF2p antibody generated using an 80-amino-acid stretch from the RT domain, which is highly conserved among different species. The antibody detects significant L1ORF2p expression in murine germ tissues and human oral squamous cell carcinoma (OSCC) samples. This particular cancer is prevalent in India due to excessive use of tobacco. Here, using our in-house antibodies against L1 proteins, we show that more than fifty percent of samples are positive for L1 proteins. Overall, we reported a novel L1ORF2p antibody that detects L1 activity in germ tissues and OSCC

cancer biology↗

bioRxiv2020

Heterotrimeric kinesin-2 motor subunit, KLP68D, localises Drosophila odour receptor coreceptor in the distal domain of the olfactory cilia

Ciliary localisation of the odour receptor coreceptor (Orco) is essential for insect olfaction. Here, we show that in the Drosophila antenna Orco enters the bipartite cilia expressed on the olfactory sensory neurons in two discrete, one-hour intervals after the adult eclosion. Genetic analyses suggest that the heterotrimeric kinesin-2 is essential for Orco transfer from the base into the cilium. Using in vitro pulldown assay, we show that Orco binds to the C-terminal tail domain of KLP68D, the {beta}-subunit of kinesin-2. Reduced Orco enrichment decreases electrophysiological response to odours and loss of olfactory behaviour. Finally, we show that kinesin-2 function is necessary to compact Orco to an approximately four-micron stretch at the distal portion of the ciliary outer-segment bearing singlet microtubule filaments. Altogether, these results highlight an independent, tissue-specific regulation of Orco entry at specific developmental stages and its localisation to a ciliary subdomain by kinesin-2. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/395905v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@177d376org.highwire.dtl.DTLVardef@1dc14d6org.highwire.dtl.DTLVardef@1dc2d21org.highwire.dtl.DTLVardef@16e90c_HPS_FORMAT_FIGEXP M_FIG C_FIG Author SummaryJana, Jain, Dutta et al., show that the odour receptor coreceptor only enters the cilia expressed on olfactory sensory neurons at specified developmental stages requiring heterotrimeric kinesin-2. The motor also helps to localise the coreceptor in a compact, environment-exposed domain at the ciliary outer-segment. HighlightsO_LIOdorant receptor coreceptor (Orco) selectively enters the olfactory cilia. C_LIO_LIOrco localises in a specific domain at the distal segment of the olfactory cilium. C_LIO_LIOrco/ORx binds to the C-terminal tail domain of the kinesin-2{beta} motor subunit. C_LIO_LIOrco entry across the transition zone and its positioning require Kinesin-2. C_LI

cell biology↗

bioRxiv2020

A modified fluctuation test for elucidating drug resistance in microbial and cancer cells

Clonal populations of microbial and cancer cells are often driven into a drug-tolerant persister state in response to drug therapy, and these persisters can subsequently adapt to the new drug environment via genetic and epigenetic mechanisms. Estimating the frequency with which drug-tolerance states arise, and its transition to drug-resistance, is critical for designing efficient treatment schedules. Here we study a stochastic model of cell proliferation where drug-tolerant persister cells transform into a drug-resistant state with a certain adaptation rate, and the resistant cells can then proliferate in the presence of the drug. Assuming a random number of persisters to begin with, we derive an exact analytical expression for the statistical moments and the distribution of the total cell count (i.e., colony size) over time. Interestingly, for Poisson initial conditions the noise in the colony size (as quantified by the Fano factor) becomes independent of the initial condition and only depends on the adaptation rate. Thus, experimentally quantifying the fluctuations in the colony sizes provides an estimate of the adaptation rate, which then can be used to infer the starting persister numbers from the mean colony size. Overall, our analysis introduces a modification of the classical Luria-Delbruck experiment, also called the "Fluctuation Test", providing a valuable tool to quantify the emergence of drug resistance in cell populations.

systems biology↗

bioRxiv2020

A benchmark dataset for individual tree crown delineation in co-registered airborne RGB, LiDAR and hyperspectral imagery from the National Ecological Observation Network

Broad scale remote sensing promises to build forest inventories at unprecedented scales. A crucial step in this process is designing individual tree segmentation algorithms to associate pixels into delineated tree crowns. While dozens of tree delineation algorithms have been proposed, their performance is typically not compared based on standard data or evaluation metrics, making it difficult to understand which algorithms perform best under what circumstances. There is a need for an open evaluation benchmark to minimize differences in reported results due to data quality, forest type and evaluation metrics, and to support evaluation of algorithms across a broad range of forest types. Combining RGB, LiDAR and hyperspectral sensor data from the National Ecological Observatory Networks Airborne Observation Platform with multiple types of evaluation data, we created a novel benchmark dataset to assess individual tree delineation methods. This benchmark dataset includes an R package to standardize evaluation metrics and simplify comparisons between methods. The benchmark dataset contains over 6,000 image-annotated crowns, 424 field-annotated crowns, and 3,777 overstory stem points from a wide range of forest types. In addition, we include over 10,000 training crowns for optional use. We discuss the different evaluation sources and assess the accuracy of the image-annotated crowns by comparing annotations among multiple annotators as well as to overlapping field-annotated crowns. We provide an example submission and score for an open-source baseline for future methods.

ecology↗

bioRxiv2020

Protective Effects of STI-2020 Antibody Delivered Post-Infection by the Intranasal or Intravenous Route in a Syrian Golden Hamster COVID-19 Model

We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.

microbiology↗

bioRxiv2020

Timing variability and midfrontal ~4 Hz rhythms correlate with cognition in Parkinson's disease

Patients with Parkinsons disease (PD) can have significant cognitive dysfunction; however, the mechanisms for these cognitive symptoms are unknown. Here, we used scalp electroencephalography (EEG) to investigate the cortical basis for PD-related cognitive impairments during interval timing, which requires participants to estimate temporal intervals of several seconds. Time estimation is an ideal task demand for investigating cognition in PD because it is simple, requires medial frontal cortical areas, and recruits basic executive processes such as working memory and attention. However, interval timing has never been systematically studied in PD patients with cognitive impairments. We report three main findings. First, 71 PD patients had increased temporal variability compared to 37 demographically-matched controls, and this variability correlated with cognitive dysfunction as measured by the Montreal Cognitive Assessment (MOCA). Second, PD patients had attenuated ~4 Hz EEG oscillatory activity at midfrontal electrodes in response to the interval-onset cue, which was also predictive of MOCA. Finally, trial-by-trial linear mixed-effects modeling demonstrated that cue-triggered ~4 Hz power predicted subsequent temporal estimates as a function of PD and MOCA. Our data suggest that impaired cue-evoked midfrontal ~4 Hz activity predicts increased timing variability that is indicative of cognitive dysfunction in PD. These findings link PD-related cognitive dysfunction with cortical mechanisms of cognitive control, which could advance novel biomarkers and neuromodulation for PD.

neuroscience↗

bioRxiv2020

Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking LAIR-collagen interaction

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1). Leukocyte associated immunoglobulin-like receptor-2 (LAIR-2) is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 and that LAIR-2 could release LAIR-1 mediated immune suppression. Analysis of public datasets shows high LAIR-2 expression being associated with a favorable outcome in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 reduces tumor growth and increases T cell expansion and effector function in humanized tumor models. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+ immune cells are localized. Our findings show that NC410 might be a powerful new strategy for cancer immunotherapy for immune-excluded tumors.

cancer biology↗

bioRxiv2020

Signature of selection in composite Vrindavani cattle of India

Vrindavani is an Indian composite cattle breed developed by crossbreeding taurine dairy breeds with native indicine cattle in the 1960s. About 190,000 semen doses of Vrindavani bulls have been distributed to the farmers till date. The animals are under artificial and natural selection for higher milk production and adaptation to the tropical climate, respectively. However, the selection response for production and adaptation traits in the Vrindavani genome is not explored. In this study, we provide the first overview of the selection signatures in the Vrindavani genome. 96 Vrindavani cattle were genotyped using the BovineSNP50 BeadChip and the SNP genotype data of its constituent breeds were collected from a public database. Within-breed selection signatures in Vrindavani were investigated using the integrated haplotype score (iHS). Vrindavani was also compared to each of its parental breeds to discover between-population signatures of selection using two approaches, cross-population extended haplotype homozygosity (XP-EHH) and fixation index (FST). Selection of signature identifies 11 common region identified by more than one harbouring genes such as LRP1B, TNNI3K, APOB, CACNA2D1, FAM110B and SPATA17 associated with production and adaptation. Overall, our results suggested stronger selective pressure on regions responsible for adaptation compared to milk yield.

genetics↗

bioRxiv2020

Meta-GWAS for quantitative trait loci identification in soybean

We report a meta-Genome Wide Association Study involving 73 published studies in soybean (Glycine max L. [Merr.]) covering 17,556 unique accessions, with improved statistical power for robust detection of loci associated with a broad range of traits. De novo GWAS and meta-analysis were conducted for composition traits including fatty acid and amino acid composition traits, disease resistance traits, and agronomic traits including seed yield, plant height, stem lodging, seed weight, seed mottling, seed quality, flowering timing, and pod shattering. To examine differences in detectability and test statistical power between single- and multi-environment GWAS, comparison of meta-GWAS results to those from the constituent experiments were performed. Using meta-GWAS analysis and the analysis of individual studies, we report 483 quantitative trait loci (QTL) at 393 unique loci. Using stringent criteria to detect significant marker trait associations, 66 candidate genes were identified, including 17 candidate genes for agronomic traits, 19 for seed related traits, and 33 for disease reaction traits. This study identified potentially valuable candidate genes that affect multiple traits. The success in narrowing down the genomic region for some loci through overlapping mapping results of multiple studies is a promising avenue for community-based studies and plant breeding applications.

genetics↗

bioRxiv2020

Naturally secreted bacterial outer membrane vesicles: potential platform for a vaccine against Campylobacter jejuni

Acute diarrheal illness and gastroenteritis caused by Campylobacter jejuni (C. jejuni) infection remain significant public health risks in developing countries with substantial mortality and morbidity in humans, particularly in children under the age of five. Despite improved global awareness in sanitation and hygiene practices, including food safety measures, C. jejuni infections continue to rise even across the developed nations and no vaccine is currently available for humans. Genetic diversities among C. jejuni strains as well as limited understanding of immunological correlates of host protection remain primary impediments for developing an effective vaccine against C. jejuni. Given the role of bacterial outer membrane-associated proteins in intestinal adherence and invasion as well as modulating dynamic interplay between host and pathogens, bacterial outer membrane vesicles (OMVs) have emerged as potential vaccine platforms against a number of enteric pathogens, including C. jejuni. In the present study, we describe a mucosal vaccine strategy using chitosan (CS) coated OMVs (CS-OMVs) to induce specific immune responses against C. jejuni in mice. However, considering the challenges of mucosal delivery of OMVs in terms of exposure to variable pH, risk of enzymatic degradation, rapid gut transit, and low permeability across the intestinal epithelium, we preferentially used CS as a non-toxic, mucoadhesive polymer to coat OMVs. Mucosal administration of CS-OMVs induced high titre of systemic (IgG) and local (secretory IgA) antibodies in mice. The neutralizing ability of secretory IgA (sIgA) produced in the intestine was confirmed by in vitro inhibition of cell adherence and invasion of C. jejuni while in vivo challenge study in OMVs immunized mice showed a significant reduction in cecal colonization of C. jejuni. Moreover, to investigate the immunological correlates of the observed protection, present data suggest OMVs driven T cell proliferation with an increased population of CD4+ and CD8+ T cells. In addition to antibody isotype profile, significant upregulation of IFN-{gamma} and IL-6 gene expression in mesenteric lymph nodes collected from OMVs immunized mice further suggests that mucosal delivery of OMVs promotes a Th1/Th2 mixed type immune responses. Together, we provide strong experimental evidence that as an acellular and non-replicating canonical end product of bacterial secretion, mucosal delivery of OMVs may represent a promising platform for developing an effective vaccine against C. jejuni. Author SummaryDespite the loss of 7.5 million disability-adjusted life years, which is over and above any other globally prevalent enteric or enterotoxigenic pathogens, C. jejuni remains a neglected foodborne pathogen, particularly in tropical countries. Even with the improved global awareness in sanitation and hygiene practices, including food safety measures C. jejuni infections continue to rise globally and no vaccine is currently available for humans. In light of the importance of the diverse cargo selection by bacterial OMVs, the present study describes a mucosal vaccine strategy using chitosan-coated OMVs to induce specific immune responses against C. jejuni in mice. We provide here strong experimental evidence that as a non-replicating canonical end product of bacterial secretion, mucosal delivery of OMVs represents an attractive vaccine platform against C. jejuni.

immunology↗

bioRxiv2020

Using Machine Learning To Develop A Fully Automated Soybean Nodule Acquisition Pipeline (SNAP)

Nodules form on plant roots through the symbiotic relationship between soybean (Glycine max L. Merr.) roots and bacteria (Bradyrhizobium japonicum), and are an important structure where atmospheric nitrogen (N2) is fixed into bio-available ammonia (NH3) for plant growth and developmental. Nodule quantification on soybean roots is a laborious and tedious task; therefore, assessment is done on a less informative qualitative scale. We report the Soybean Nodule Acquisition Pipeline (SNAP) for nodule quantification that combines RetinaNet and UNet deep learning architectures for object (i.e., nodule) detection and segmentation. SNAP was built using data from 691 unique roots from diverse soybean genotypes, vegetative growth stages, and field locations; and has a prediction accuracy of 99%. SNAP reduces the human labor and inconsistencies of counting nodules, while acquiring quantifiable traits related to nodule growth, location and distribution on roots. The ability of SNAP to phenotype nodules on soybean roots at a higher throughput enables researchers to assess the genetic and environmental factors, and their interactions on nodulation from an early development stage. The application of SNAP in research and breeding pipelines may lead to more nitrogen use efficient soybean and other legume species cultivars, as well as enhanced insight into the plant-Bradyrhizobium relationship.

plant biology↗

bioRxiv2020

Vascular features, sucrose transport and Sucrose Synthase activity contribute to the differential source-sink relationship between a cultivated and a wild rice

The source-sink relationship is key to overall crop performance. Detailed understanding of the factors that determine source-sink dynamics is imperative for the balance of biomass and grain yield in crop plants. We investigated the differences in the source-sink relationship between a cultivated rice Oryza sativa cv. Nipponbare and a wild rice Oryza australiensis that show striking differences in biomass and grain yield. Oryza australiensis, accumulating higher biomass, not only showed higher photosynthesis per unit leaf area but also exported more sucrose from leaves than Nipponbare. However, grain features and sugar levels suggested limited sucrose mobilization to the grains in the wild rice due to vasculature and sucrose transporter functions. Low cell wall invertase activity and high sucrose synthase cleavage activity followed by higher expression of cellulose synthase genes in Oryza australiensis stem utilized photosynthates preferentially for the synthesis of structural carbohydrates, resulting in high biomass. In contrast, the source-sink relationship favored high grain yield in Nipponbare via accumulation of transitory starch in the stem, due to higher expression of starch biosynthetic genes, which is mobilized to panicles at the grain filling stage. Thus, vascular features, sucrose transport, and functions of sugar metabolic enzymes explained the differences in the source-sink relationship between Nipponbare and Oryza australiensis. HighlightVascular features, sucrose transport, and sugar metabolic enzyme activity contribute to the differential source-sink relationship between the selected cultivated and wild rice that differ in biomass and grain yield.

plant biology↗

bioRxiv2020

Role of intercellular coupling and delay on the synchronization of genetic oscillators

Living cells encode diverse biological clocks for circadian timekeeping and formation of rhythmic structures during embryonic development. A key open question is how these clocks synchronize across cells through intercellular coupling mechanisms. To address this question, we leverage the classical motif for genetic clocks the Goodwin oscillator where a gene product inhibits its own synthesis via time-delayed negative feedback. More specifically, we consider an interconnected system of two identical Goodwin oscillators (each operating in a single cell), where state information is conveyed between cells via a signaling pathway whose dynamics is modeled as a first-order system. In essence, the interaction between oscillators is characterized by an intercellular coupling strength and an intercellular time delay that represents the signaling response time. Systematic stability analysis characterizes the parameter regimes that lead to oscillatory dynamics, with high coupling strength found to destroy sustained oscillations. Within the oscillatory parameter regime we find both in-phase and anti-phase oscillations with the former more likely to occur for small intercellular time delays. Finally, we consider the stochastic formulation of the model with low-copy number fluctuations in biomolecular components. Interestingly, stochasticity leads to qualitatively different behaviors where in-phase oscillations are susceptible to the inherent fluctuations but not the anti-phase oscillations. In the context of the segmentation clock, such synchronized in-phase oscillations between cells are critical for the proper generation of repetitive segments during embryo development that eventually leads to the formation of the vertebral column.

systems biology↗

bioRxiv2020

Division stochasticity can be transmitted to protein expression through chromosome replication

Stochastic fluctuations (noise) are a fundamental characteristic of protein production. Some sources of this stochasticity are still under debate. In this work, we explore how these fluctuations can originate from the stochasticity on division events. To do that, we consider the classical gene expression model with chromosome replication following the known Helmstetter & Cooper model. This model predicts intervals of the cell cycle where bacteria can have more than one copy of a particular gene. Considering the transcription rate as proportional to the number of chromosomes and division based on a continuous rate model, we explore how stochasticity in division or equivalently in cell size, could be transmitted to gene expression. Our simulations suggest that division can be an important source of such fluctuations only if chromosomes are replicating, otherwise, this noise is not well transmitted. This effect happens even if replication is deterministic. This work can be helpful for understanding cell cycle dynamics and their interplay with phenotypic variability.

systems biology↗

bioRxiv2020

Genome-wide SNP data unravel the ancestry and signatures of divergent selection in Ghurrah pigs of India

The evolution and domestication of pigs is a complex and ongoing process. Despite its rich biodiversity and proximity to the geographical origins of Sus scrofa domesticus, the place of Indian pigs in the global phylogeny is unclear. Using microarray-derived (porcine 60K SNP chip) genotypes of 11 Ghurrah pigs from North-Western India and a public dataset comprising 2113 pigs of 146 breeds, we determined the genomic ancestry of Ghurrah pigs and compared their genetic constitution to European and Asian breeds to ascertain signatures of divergent selection. Results showed that Ghurrah pigs contain genes of Asian and European ancestry with signs of inter-species introgression. Using Admixture LD - decay statistics, the European admixture event was dated to the recent past, coinciding with the start of cross-breeding efforts in India. The complex Asian ancestry pattern of the breed resembled that of wild boars of South - Central China and Thailand, possibly suggesting introgression from an Indian wild boar relative. FST and XP - EHH comparisons with Asian breeds highlighted divergent selection in genomic regions associated with odontogenesis and skeletal muscle development. Comparisons with European commercial breeds revealed that genomic regions governing olfaction and response to sensory stimulation were under selection in Ghurrah pigs. QTL for meat and carcass traits also showed divergent selection between European breeds and Ghurrah pigs. Our results present the first genomic characterization of an Indian pig breed using dense microarray-derived genotypes and highlight the importance of further genomic characterization of Indian domestic and wild pigs.

genomics↗

bioRxiv2020

Aging associated altered response to intracellular bacterial infections and its implication on the host

The effects of senescence and aging on geriatric diseases has been well explored but how these influence infections in the elderly have been scarcely addressed. Here, we show that several innate immune responses are elevated in senescent cells and old mice, allowing them to promptly respond to bacterial infections. We have identified higher levels of iNOS as a crucial host response and show that p38 MAPK in senescent cells acts as a negative regulator of iNOS transcription. In old mice, however the ability to impede bacterial proliferation does not correlate with increased survival as elevated immune responses persist unabated eventually affecting the host. The use of anti-inflammatory drugs that could consequently be recommended also decreases iNOS disarming the host of a critical innate immune response. Overall, our study highlights that infection associated mortality in the elderly is not merely an outcome of pathogen load but is also influenced by the hosts ability to resolve inflammation induced damage. Summary statementUsing cellular models and old mice we demonstrate the effect of aging on host response to bacterial infections. Aged systems mount a more effective anti-bacterial innate immune response but its persistence results in mortality of the host.

microbiology↗

bioRxiv2020

NEON Crowns: a remote sensing derived dataset of 100 million individual tree crowns

Forests provide essential biodiversity, ecosystem and economic services. Information on individual trees is important for understanding the state of forest ecosystems but obtaining individual-level data at broad scales is challenging due to the costs and logistics of data collection. While advances in remote sensing techniques allow surveys of individual trees at unprecedented extents, there remain significant technical and computational challenges in turning sensor data into tangible information. Using deep learning methods, we produced an open-source dataset of individual-level crown estimates for 100 million trees at 37 sites across the United States surveyed by the National Ecological Observatory Networks Airborne Observation Platform. Each canopy tree crown is represented by a rectangular bounding box and includes information on the height, crown area, and spatial location of the tree. Tree crowns identified using this technique correspond well with hand-labeled crowns, exhibiting both high levels of overlap and good correspondence in height estimates. These data have the potential to drive significant expansion of individual-level research on trees by facilitating both regional analyses at scales of ~10,000 ha and cross-region comparisons encompassing forest types from most of the United States.

ecology↗

bioRxiv2020

Disulfiram inhibits M. tuberculosis growth by altering methionine pool, redox status and host-immune response

Methionine biosynthetic pathway, essential for the growth of Mycobacterium tuberculosis (Mtb) in the host, represents an attractive target for the development of novel anti-tuberculars. Here, we have biochemically characterized homoserine acetyl transferase (HSAT viz. MetA) of Mtb, which catalyses the first committed step of methionine and S-adenosylmethionine (SAM) biosynthesis. High-throughput screening of a 2300 compound library resulted in identification of thiram, an anti-fungal organosulfur compound, as the most potent MetA inhibitor. Further analysis of thiram analogs led to the identification of orally bioavailable disulfiram (DIS, an anti-alcoholism FDA approved drug) as a novel inhibitor of MetA. Both thiram and DIS restricted the growth of drug-sensitive and drug-resistant Mtb strains in a bactericidal manner. ThermoFlour assay demonstrated direct binding of DIS with MetA. Metabolomic and transcriptomic studies showed DIS mediated perturbation of methionine and redox homeostasis, respectively, in Mtb. In concordance, the effect of DIS on Mtb growth was partially rescued by supplementation with either L-methionine as well as N-acetyl cysteine, suggesting a multi-target killing mechanism. In Mtb-infected mice, DIS administration restricted bacterial growth, increased efficacy of isoniazid, ameliorated lung pathology, modulated lung immune cell landscape and protective immune response. Taken together, our results demonstrate that DIS can be repurposed for designing an effective anti-tubercular therapy.

microbiology↗