Biology subjects

Ghosh, A.

Publications and source records attributed to Ghosh, A..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by directly interacting with either a protein effector or with the 5-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis. Thus, our findings of c-di-AMP messenger controlling drug resistance phenotypes of mycobacteria against four different classes of antibiotics is a unique observation that will contribute to scientific advancement in the field. One Sentence SummaryHere we describe how second messenger c-di-AMP modulates the antibiotic sensitivity and resistance profile of M. smegmatis by diverse mechanisms.

microbiology↗

bioRxiv2023

Peripheral vascular strand development in nodules is controlled by a bHLH/HLH heterodimer

Only the Leguminosae family can develop root nodules with peripheral vasculature, an adaptation that grants them an advantage in optimizing nitrogen fixation efficiency. Medicago truncatula develops indeterminate nodules that possess peripheral vascular-strands encircling the central infection zone. How vascular-strands shifted from the nodule central part to the periphery remains unresolved. Here we show, MtbHLH1 (renamed as Nodule Vascular bundle Development 1) is required for the proper organization of vascular strands. In nvd1 nodules, vascular strands pass through the infection zone. NVD2, an HLH transcription factor that lacks a DNA-binding domain, is activated by NVD1. Mutant nvd2 nodules display a similar partially central vasculature. NVD2 is expressed along the nodule vascular bundle and NVD2:GFP fusion protein localizes to the nodule vascular endodermis. The formation of the peripheral vasculature is dependent on the proper stoichiometry of NVD1 and NVD2 heterodimers, as NVD2 controls NVD1-mediated transcriptional activation by sequestering NVD1. Transcription of NVD1 is activated by auxin and Auxin Responsive transcription Factor (MtARF5). Transcriptome sequencing of nvd1 and nvd2 nodules and visualization of in situ auxin and cytokinin signal outputs indicated aberrant auxin/cytokinin balance in these nodules. Our findings showed that the NVD1-NVD2 heterodimer plays a key role in the formation of an orderly peripheral vascular bundle around Medicago nodules.

plant biology↗

bioRxiv2023

Learning better with Dale's Law: A Spectral Perspective

Most recurrent neural networks (RNNs) do not include a fundamental constraint of real neural circuits: Dales Law, which implies that neurons must be excitatory (E) or inhibitory (I). Dales Law is generally absent from RNNs because simply partitioning a standard networks units into E and I populations impairs learning. However, here we extend a recent feedforward bio-inspired EI network architecture, named Dales ANNs, to recurrent networks, and demonstrate that good performance is possible while respecting Dales Law. This begs the question: What makes some forms of EI network learn poorly and others learn well? And, why does the simple approach of incorporating Dales Law impair learning? Historically the answer was thought to be the sign constraints on EI network parameters, and this was a motivation behind Dales ANNs. However, here we show the spectral properties of the recurrent weight matrix at initialisation are more impactful on network performance than sign constraints. We find that simple EI partitioning results in a singular value distribution that is multimodal and dispersed, whereas standard RNNs have an unimodal, more clustered singular value distribution, as do recurrent Dales ANNs. We also show that the spectral properties and performance of partitioned EI networks are worse for small networks with fewer I units, and we present normalised SVD entropy as a measure of spectrum pathology that correlates with performance. Overall, this work sheds light on a long-standing mystery in neuroscience-inspired AI and computational neuroscience, paving the way for greater alignment between neural networks and biology.

neuroscience↗

bioRxiv2023

Sse1, Hsp110 chaperone of yeast, controls the cellular fate during Endoplasmic Reticulum-stress

Sse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as Nucleotide Exchange Factor (NEF), as protein-disaggregase and as a Chaperone linked to Protein Synthesis (CLIPS), are well documented. In the currently study, we show that SSE1 genetically interacts with IRE1 and HAC1, the Endoplasmic Reticulum-Unfolded Protein Response (ER-UPR) sensors implicating its role in ER protein homeostasis. Interestingly, absence of this chaperone imparts unusual resistance to tunicamycin-induced ER stress which depends on the intact Ire1-Hac1 mediated ER-UPR signalling. Furthermore, cells lacking SSE1 show ER-stress-responsive inefficient reorganization of translating ribosomes from polysomes to monosomes and increased monosome content that drive uninterrupted protein translation. In consequence, the kinetics of ER-UPR is starkly different in sse1{Delta} strain where we show that stress response induction and restoration of homeostasis is prominently faster in contrast to the wildtype (WT) cells. Importantly, Sse1 plays a critical role in controlling the ER-stress mediated cell division arrest which is escaped in sse1{Delta} strain during chronic tunicamycin stress. Consequently, sse1{Delta} strain shows significantly higher cell viability in comparison to WT yeast, following short-term as well as long-term tunicamycin stress. In summary, we demonstrate a new role of Sse1 in ER protein homeostasis where the chaperone genetically interacts with ER-UPR pathway, controls the protein translation during ER stress and the kinetics of ER-UPR. More importantly, we show the crtical role of Sse1 in regulating the ER-stress-induced cell division arrest and cell death during global ER stress by tunicamycin. Author SummarySse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. It performs various functions as Nucleotide Exchange Factor (NEF), as a protein-disaggregase and as a Chaperone linked to Protein Synthesis (CLIPS) to maintain a healthy proteome in the cytosol. In the present study, we show that SSE1 is a critical player to maintain Endoplasmic Reticulum (ER) protein homeostasis. We report that SSE1 genetically interacts with the ER-Unfolded Protein Response (ER-UPR) sensors, IRE1 and HAC1. In absence of SSE1, yeast cells gain unusual fitness to tunicamycin (Tm)-induced ER stress which depends on the functional Ire1-Hac1 mediated ER-UPR signalling. Furthermore, Sse1 controls the ribosomal organization during tunicamycin-induced ER-stress and in absence of the chaperone the amount of polysomes and monosmes are high leading to uninterrupted protein translation during ER stress. Consequently, sse1{Delta} strain exhibits starkly different ER-UPR kinetics compared to wildtype (WT) cells. Importantly, our data reveal that cell division arrest and cell death due to Tm-induced ER-stress is critically controlled by Sse1.

cell biology↗

bioRxiv2023

GATA-regulated transcriptional program dictate cell fate equilibrium to establish the maternal-fetal exchange interface and fetal development

The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of GATA2 and GATA3 in labyrinth trophoblast progenitors have underdeveloped placenta and die [~] embryonic day 9.5 (E9.5). Single cell RNA Seq (scRNA-Seq) analysis revealed excessive accumulation of multipotent labyrinth trophoblast progenitors upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate to matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast (CTB)-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promote trophoblast progenitor-to-SynT commitment ensuring establishment of the maternal-fetal exchange interface.

developmental biology↗

bioRxiv2023

Computational Framework for Statistical Epistasis Supports XOR Penetrance Function in a Living System

Statistical epistasis has been studied extensively because of its potential to provide evidence for genetic interactions for phenotypes, but there have been methodological limitations to its exhaustive, widespread application. We present new algorithms for the interaction coefficients for standard regression models for epistasis that permit many varied encodings for the interaction terms for loci and efficient memory usage. The algorithms are given for two-way and three-way epistasis and may be generalized to higher order epistasis. Statistical tests for the interaction coefficients are also provided. We also present an efficient matrix based algorithm for permutation testing for two-way epistasis. We offer a proof and experimental evidence that methods that look for epistasis only at loci that have main effects may not be justified. Given the computational efficiency of the algorithm, we applied the method to a rat data set and mouse data set, with at least 10000 loci and 1000 samples each, using the standard Cartesian encoding and the XOR penetrance function for the interactions, to test for evidence of statistical epistasis for the phenotype of body mass index. This study revealed that the XOR penetrance function found greater evidence for statistical epistasis in many more pairs of loci in both data sets and in the rat data set, those pairs of loci found using the XOR penetrance function are enriched for biologically relevant pathways. Author summaryEpistasis, the interaction between two or more genes, is likely integral to the study of genetics and present throughout nature. Yet, it is seldom fully explored as most approaches primarily focus on single-locus effects (such as GWAS), partly because analyzing all pairwise and higher-order interactions requires significant computational resources. Many current methods for epistasis detection only consider a Cartesian encoding for interaction terms. This is likely limiting as epistatic interactions can evolve to produce varied relationships between genes, some non-linear. In this work we describe computationally efficient algorithms for the detection of statistical epistasis that allow for varied interaction encodings for modeling epistasis. Our methodology efficiently detects pairwise and three-way epistatic interactions in two closely related species (rat and mouse) under both Cartesian and XOR interaction encodings. Our results in both species show that many biologically relevant epistatic relationships would have been undetected if only one interaction encoding was applied providing evidence that more varied models for interaction may need to be applied to describe epistasis that occurs in living systems.

genetics↗

bioRxiv2023

Intermediate antiparallel beta structure in amyloid plaques revealed by infrared spectroscopic imaging

Aggregation of amyloid beta (A{beta}) peptides into extracellular plaques is a hallmark of the molecular pathology of Alzheimers disease (AD). Amyloid aggregates have been extensively studied in-vitro, and it is well known that mature amyloid fibrils contain an ordered parallel {beta} structure. The structural evolution from unaggregated peptide to fibrils can be mediated through intermediate structures that deviate significantly from mature fibrils, such as antiparallel {beta}-sheets. However, it is currently unknown if these intermediate structures exist in plaques, which limits the translation of findings from in-vitro structural characterizations of amyloid aggregates to AD. This arises from the inability to extend common structural biology techniques to ex-vivo tissue measurements. Here we report the use of infrared (IR) imaging, wherein we can spatially localize plaques and probe their protein structural distributions with the molecular sensitivity of IR spectroscopy. Analyzing individual plaques in AD tissues, we demonstrate that fibrillar amyloid plaques exhibit antiparallel {beta}-sheet signatures, thus providing a direct connection between in-vitro structures and amyloid aggregates in AD brain. We further validate results with IR imaging of in-vitro aggregates and show that antiparallel {beta}-sheet structure is a distinct structural facet of amyloid fibrils.

biophysics↗

bioRxiv2023

Interspecies generation of functional muscle stem cells

Satellite cells, the stem cells of skeletal muscle tissue, hold a prodigious regeneration capacity. However, low satellite cell yield from autologous or donor-derived muscles precludes adoption of satellite cell transplantation for the treatment of muscle diseases including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether sufficient quantity of satellite cells can be produced in allogeneic or xenogeneic animal hosts. First, we report on exclusive satellite cell production in intraspecies mouse chimeras by injection of CRISPR/Cas9-corrected DMD-induced pluripotent stem cells (iPSCs) into blastocysts carrying an ablation system of host Pax7+ satellite cells. Additionally, injection of genetically-corrected DMD-iPSCs into rat blastocysts produced interspecies rat-mouse chimeras harboring mouse muscle stem cells that efficiently restored dystrophin expression in DMD mice. This study thus provides a proof-of-principle for the generation of therapeutically-competent stem cells between divergent species, raising the possibility of procuring human stem cells in large animals for regenerative medicine purposes.

cell biology↗

bioRxiv2023

A self-renewing biomimetic skeletal muscle construct engineered using induced myogenic progenitor cells

Skeletal muscle is a highly organized and regenerative tissue that maintains its homeostasis primarily by activation and differentiation of muscle stem cells. Mimicking an in vitro skeletal muscle differentiation program that contains self-renewing adult muscle stem cells and aligned myotubes has been challenging. Here, we set out to engineer a biomimetic skeletal muscle construct that can self-regenerate and produce aligned myotubes using induced myogenic progenitor cells (iMPCs), a heterogeneous culture consisting of skeletal muscle stem, progenitor and differentiated cells. Utilizing electrospinning, we fabricated polycaprolactone (PCL) substrates that enabled iMPC-differentiation into aligned myotubes by controlling PCL fiber orientation. Newly-conceived constructs contained highly organized multinucleated myotubes in conjunction with self-renewing muscle stem cells, whose differentiation capacity was augmented by Matrigel supplementation. Additionally, we demonstrate using single cell RNA-sequencing (scRNA-seq) that iMPC-derived constructs faithfully recapitulate a step-wise myogenic differentiation program. Notably, when the constructs were subjected to a damaging myonecrotic agent, self-renewing muscle stem cells rapidly differentiated into aligned myotubes, akin to skeletal muscle repair in vivo. Taken together, we report on a novel in vitro system that mirrors myogenic regeneration and muscle fiber alignment, and can serve as a platform to study myogenesis, model muscular dystrophies or perform drug screens.

bioengineering↗

bioRxiv2023

Intermediate antiparallel fibrils in Aβ40 Dutch mutant aggregation: nanoscale insights from AFM-IR

Cerebral Amyloid Angiopathy (CAA), which involves amyloid deposition in blood vessels leading to fatal cerebral hemorrhage and recurring strokes, is present in the majority Alzheimers disease cases. Familial mutations in the amyloid {beta} peptide is correlated to higher risks of CAA, and are mostly comprised of mutations at residues 22 and 23. While the structure of the wild type A{beta} peptide has been investigated in great detail, less is known about the structure of mutants involved in CAA and evolutions thereof. This is particularly true for mutations at residue 22, for which detailed molecular structures, as typically determined from Nuclear Magnetic Resonance (NMR) spectroscopy or electron microscopy, do not exist. In this report, we have used nanoscale infrared (IR) spectroscopy augmented with Atomic Force Microscopy (AFM-IR) to investigate structural evolution of the A{beta} Dutch mutant (E22Q) at the single aggregate level. We show that that in the oligomeric stage, the structural ensemble is distinctly bimodal, with the two subtypes differing with respect to population of parallel {beta}-sheets. Fibrils on the other hand are structurally homogeneous, with early-stage fibrils distinctly anti parallel in character, which develop parallel {beta}-sheets upon maturation. Furthermore, the antiparallel structure is found to be a persistent feature across different stages of aggregation.

biophysics↗

bioRxiv2023

Joint testing of rare variant burden scores using non-negative least squares

Gene-based burden tests are a popular and powerful approach for analysis of exome-wide association studies. These approaches combine sets of variants within a gene into a single burden score that is then tested for association. Typically, a range of burden scores are calculated and tested across a range of annotation classes and frequency bins. Correlation between these tests can complicate the multiple testing correction and hamper interpretation of the results. We introduce a new method called the Sparse Burden Association Test (SBAT) that tests the joint set of burden scores under the assumption that causal burden scores act in the same effect direction. The method simultaneously assesses the significance of the model fit and selects the set of burden scores that best explain the association at the same time. Using simulated data, we show that the method is well calibrated and highlight some scenarios where the test outperforms existing gene-based tests. We apply the method to 73 quantitative traits from the UK Biobank which further illustrates the power of the method. This test is implemented in the REGENIE software.

genetics↗

bioRxiv2023

Multiple forms of neural processing when repeating voluntary thumb flexions

There is considerable trial-to-trial variability in single cortical neurons when performing the same action repeatedly. One possibility is that neural populations are more stable in representing actions; alternatively, they too may be distinctly engaged from trial-to-trial. To address the nature of variability in large neural populations, we captured the EEG signals time-locked to repeated voluntary thumb flexion movements ([~]500 repetitions, 23 subjects). By using non-negative matrix factorization on the low-frequency sensorimotor cortical signals, we quantified the trial-to-trial motor-related potentials (MRPs) in terms of prototypical signals (meta-MRPs) and their corresponding prominence at each trial (meta-trials). Clustering the meta-MRPs across the sampled population revealed 5 distinct signal patterns. There were brain-wide correlates of these meta-MRP clusters. Cortical hemispheres were distinctly recruited from trial-to-trial as certain clusters were accompanied by bilateral motor negativity while others were characterized by ipsilateral motor negativity. The sensory feedback too was distinctly processed from trial-to-trial as the central post-movement positivity was present only for certain clusters. A poorly understood pre-motor positivity accompanied all clusters albeit varying in their timing from trial-to-trial. These patterns - including the time-varying positivity preceding the movement - were rendered invisible by the traditional averaging of the signals. We suggest that the variability in EEG signals is not just noise but a consequence of distinct activation patterns deployed by the cortex. We support the idea that the cortex flexibly switches between distinct forms of neural processing to achieve the same behavioral goals.

neuroscience↗

bioRxiv2023

Description of three new species of predatory Genus Hexacentrus (Orthoptera: Tettigoniidae) from India, with bioacoustic and morphological characterization.

Hexacentrus is a genus of predatory katydids. In India, the genus Hexacentrus is represented by 7 species of which 6 are morphologically characterized while one is only acoustically characterized. In this study, we describe three new species under the genus from India based on detailed morphological, stridulatory, and acoustic features.

zoology↗

bioRxiv2023

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

Bacterial RNP bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We found 111 BR-body enriched proteins, including several RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, showing BR-bodies are more complex than previously assumed. While most BR-body enriched proteins that were tested cannot phase separate, we identified five that undergo RNA-dependent phase separation in vitro, showing other RNP condensates interface with BR-bodies. RNA degradosome protein clients are recruited more strongly to RNase E droplets than droplets of other RNP condensates, implying that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble with preferred directionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing. HighlightsO_LIBR-body proteomics identified 111 proteins enriched in BR-bodies. C_LIO_LIBR-bodies associate with an interconnected network of RNP condensates. C_LIO_LIBR-body condensation is modulated by its interaction network. C_LIO_LIRNA is required for rapid BR-body condensation. C_LI Graphical AbstractSummary of the BR-body protein interactome. Lines between two protein circles represent a direct interaction.

microbiology↗

bioRxiv2023

Automated quantitative trait locus analysis (AutoQTL)

BackgroundQuantitative Trait Locus (QTL) analysis and Genome-Wide Association Studies (GWAS) have the power to identify variants that capture significant levels of phenotypic variance in complex traits. However, effort and time are required to select the best methods and optimize parameters and pre-processing steps. Although machine learning approaches have been shown to greatly assist in optimization and data processing, applying them to QTL analysis and GWAS is challenging due to the complexity of large, heterogenous datasets. Here, we describe proof-of-concept for an automated machine learning approach, AutoQTL, with the ability to automate many complex decisions related to analysis of complex traits and generate diverse solutions to describe relationships that exist in genetic data. ResultsUsing a dataset of 18 putative QTL from a large-scale GWAS of body mass index in the laboratory rat, Rattus norvegicus, AutoQTL captures the phenotypic variance explained under a standard additive model while also providing evidence of non-additive effects including deviations from additivity and 2-way epistatic interactions from simulated data via multiple optimal solutions. Additionally, feature importance metrics provide different insights into the inheritance models and predictive power of multiple GWAS-derived putative QTL. ConclusionsThis proof-of-concept illustrates that automated machine learning techniques can be applied to genetic data and has the potential to detect both additive and non-additive effects via various optimal solutions and feature importance metrics. In the future, we aim to expand AutoQTL to accommodate omics-level datasets with intelligent feature selection strategies.

genetics↗

bioRxiv2022

Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins

Metal-Induced Energy Transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and sub-cellular components across multiple timescales, from a few milliseconds to hours, with negligible phototoxic effects.

biophysics↗

bioRxiv2022

Establishment and characterisation of oviductal organoids from farm and companion animals

Organoid technology has provided us with a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of human diseases. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. Here, we have developed a platform to grow organoids from animal tissue samples and characterized oviductal organoids from five domestic animal species. Organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected equine, bovine, feline, canine, and porcine oviducts. The addition of WNT, TGFB, BMP, Rock, and Notch signalling pathway activators or inhibitors in the culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were observed and described. Well-defined and synchronised motile ciliated cells were observed in differentiated organoids in mature populations. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have developed a detailed cross-species comparison of oviductal organoid models, which will be valuable for advancing assisted reproductive technologies and fertility studies in these animal species in the future. Summary sentenceOrganoids can be derived from the oviductal epithelium of cow, cat, dog, horse, and pig to advance assisted reproductive technologies in animals.

developmental biology↗

bioRxiv2022

C-4-Modified Isotetrones Prevent Biofilm Growth and Persister Cell Resuscitation in Mycobacterium smegmatis

Hyperphosphorylated guanosine nucleotide (p)ppGpp, synthesized by Rel proteins, regulates the stringent response pathway responsible for biofilm growth and persister cell formation in the stationary phase of mycobacteria. The discovery of vitamin C as a potent inhibitor of Rel protein activities raises the prospect of such a tetrone lactone to prevent biofilm growth and persister cell formation. The closely related isotetrone lactone derivatives are identified in the present study as potent inhibitors of the above processes in a mycobacterium. Isotetrone lactone derivatives are synthesized from appropriate -ketocarboxylic acids, derived from the a-amino acids. Aldol condensation with formaldehyde, followed by the lactone formation, completes synthesis of isotetrone derivatives, possessing varied substituents at C-4 carbon, in good yields. A series of biochemical evaluations of biofilm growth and persister cell formation in M. smegmatis is conducted. Among the derivatives, isotetrone possessing phenyl substituent at C-4 carbon completely inhibit the biofilm formation at 400 g mL-1 concentration, 84 h of post-exposure, followed by a moderate inhibition by the isotetrone possessing p-hydroxyphenyl substituent. Whereas, the latter isotetrone inhibits the growth of cells at 400 g mL-1 f.c. when monitored for 2 weeks, under PBS starvation condition. Isotetrones also potentiate the inhibition of antibiotic tolerant regrowth of cells by ciprofloxacin antibiotic (0.75 g mL-1) and thus act as bio-enhancers. The combination is shown to significantly arrest the emergence of ciprofloxacin-resistant genetic mutants. The observations suggest that isotetrones in combination with ciprofloxacin are therapeutically superior when administered together. Systematic molecular dynamics studies show that isotetrone derivative binds to Rel protein more efficiently than vitamin C and the binding is aided by hydrogen bonding, van der Waals and electrostatic interactions at a binding site possessing serine, threonine, lysine and arginine residues. The present study establishes that the identified isotetrone derivatives (i) act as inhibitors of M. smegmatis biofilm growth and (ii) arrest the re-emergence of recalcitrant persister cells when administered together with ciprofloxacin antibiotic. Results of this study establish that isotetrones as new chemical entities that interfere with stringent response pathways in a mycobacterium under stress and permit overcoming the multidrug-resistant persister cell emergence in the bacterium.

microbiology↗