Biology subjects

Kumari, S.

Publications and source records attributed to Kumari, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Integrative plasma lipidomics and proteomics profiling to decipher potential biomarkers of dilated cardiomyopathy

BackgroundDilated cardiomyopathy (DCM), primarily characterised by left ventricular dilatation and systolic dysfunction, is one of the leading causes of heart failure and requires a critical clinical investigative strategy. However, conventional imaging techniques such as echocardiography and MRI, along with some classical CVD markers (NTproBNP, cTnT), fall short in diagnosing DCM-specific phenotypes. Thus, the need for biochemical markers with enhanced accuracy to DCM is of enormous importance. Lipids and proteins play essential roles in maintaining myocardial function. The homeostatic disruption of such biomolecules might contribute to DCM pathogenesis, thus offering to serve as potential biomarkers for DCM. Moreover, the lack of global lipidomics studies and specific protein markers in DCM patients prompted us to explore the disease pathophysiology through an integrative omics-based analysis coupled with an ML-derived approach. ObjectiveTo identify accurate, precise and specific circulatory lipidomic and proteomic biomarkers of DCM using high-resolution mass spectrometry and machine learning (ML)-based approaches. MethodsHigh-resolution-mass-spectrometry-based lipidomics and proteomics were applied to identify lipid and protein biomarkers in a cohort (n=360) of healthy and DCM patients. Top protein classifiers were further evaluated using single-cell transcriptomics on publicly available datasets from DCM myocardium and validated using ELISA. A biomarker panel was built by the integration of lipidomics and validated proteomics data using machine-learning-based approaches. ResultsA total of 125 lipids and 10 proteins have been primarily discovered with significant alterations in DCM (0.8 [≥] FC [≥] 1.2; padj < 0.05). Using a Boruta-based ML approach, we identified 39 lipids and 10 proteins as primary discriminators between DCM and controls. ELISA validation confirmed the potential of B2M (6.85 {+/-} 2.86 g/ml vs. 4.26 {+/-} 1.25 g/ml; p < 0.0001) and Tetranectin (CLEC3B) (1.99 {+/-} 0.88 g/ml vs. 2.49 {+/-} 0.90 g/ml; p = 0.0006) to emerge as protein biomarkers of DCM. In line with that, the single-cell transcriptomic analysis showed a similar trend of alteration of tetranectin (CLEC3B) in cardiomyocytes and {beta}2microglobulin (B2M) in varied cell types of the myocardium. Further, Integrated ROC analysis combining the top 8 lipid discriminators with B2M and CLEC3B achieved an AUC of 0.995, demonstrating enhanced diagnostic precision compared to the classical CVD marker NTproBNP (0.965). ConclusionThis study offers a system-omics-based perspective on first-global lipidomic and proteomic changes associated with DCM pathophysiology, with a high potential for diagnostic application.

systems biology↗

bioRxiv2025

Transcription factor LHX2 suppresses astrocyte proliferation in the postnatal mammalian cerebral cortex

In the developing cerebral cortex astrocytes arise from progenitors in the ventricular and subventricular zones (V-SVZ), and also from local proliferation within the parenchyma. In the mouse neocortex, astrocytes that occupy upper versus deep layers (UL/DL) are known to be distinct populations in terms of molecular and morphological features. Transcription factor LHX2 is expressed both in V-SVZ gliogenic progenitors and in differentiated astrocytes throughout development and into adulthood. Here we show that loss of Lhx2 at birth results in an increased astrocyte proliferation in UL but not the DL of the cortex in the first postnatal week. Consistent with this, transcriptomic signatures of UL astrocytes increase. By 3 months, Lhx2 mutant astrocytes display upregulation of GFAP, and transcriptomic signatures associated with "reactive" astrocytes, in the absence of injury. These results demonstrate a novel role for Lhx2 in regulating proliferation and molecular features of cortical astrocytes. Summary StatementLoss of Lhx2 causes increased astrocyte proliferation in the upper but not deep layers of the mammalian cortex, and upregulation of reactive gliosis-like signatures in the absence of injury.

neuroscience↗

bioRxiv2024

GRAS Family Transcription Factor Binding Behaviors in Sorghum bicolor, Oyrza, and Maize

Identifying non-coding regions that control gene expression has become an essential aspect of understanding gene regulatory networks that can play a role in crop improvements such as crop manipulation, stress response, and plant evolution. Transcription Factor (TF)-binding approaches can provide additional valuable insights and targets for reverse genetic approaches such as EMS-induced or natural SNP variant screens or CRISPR editing techniques (e.g. promoter bashing). Here, we present the first ever DAP-seq profiles of three GRAS family TFs (SHR, SCL23, and SCL3) in the crop Sorghum bicolor, Oryza sativa japonica, and Zea mays. The binding behaviors of the three GRAS TFs display unique and shared gene targets and categories of previously characterized DNA-binding motifs as well as novel sequences that could potentially be GRAS family-specific recognition motifs. Additional transcriptomic and chromatin accessibility data further facilitates the identification of root-specific GRAS regulatory targets corresponding to previous studies. These results provide unique insights into the GRAS family of TFs and novel regulatory targets for further molecular characterization.

plant biology↗

bioRxiv2024

RCoxNet: deep learning framework for enhanced cancer survival prediction integrating random walk with restart with mutation and clinical data

Cancer poses a significant global health challenge, characterized by a complex disease progression and disrupted growth regulation. A thorough understanding of cellular and molecular biological mechanisms is essential for developing novel treatments and improving the accuracy of patient survival predictions. While prior studies have leveraged gene expression and clinical data to forecast survival outcomes through current machine learning and deep learning approaches, gene mutation data--despite being a widely recognized metric--has rarely been incorporated due to its limited information, inadequate representation of gene relationships, and data sparsity, which negatively affects the robustness, effectiveness, and interpretability of current survival analysis approaches. To overcome the challenges of mutation data sparsity, we propose RCoxNet, a novel deep learning neural network framework that integrates the Random Walk with Restart (RWR) algorithm with a deep learning Cox Proportional Hazards model. By applying this framework to mutation data from cBioportal, our model achieved an average concordance index of 0.62 {+/-} 0.05 across four cancer types, outperforming existing deep neural network models. Additionally, we identified clinical features critical for differentiating between predicted high- and low-risk patients, with the relevance of these features being partially supported by previous studies.

bioinformatics↗

bioRxiv2024

Molecular basis of the urate transporter URAT1 inhibition by gout drugs

Hyperuricemia is a condition when uric acid, a waste product of purine metabolism, accumulates in the blood1. Untreated hyperuricemia can lead to crystal formation of monosodium urate in the joints, causing a painful inflammatory disease known as gout. These conditions are associated with many other diseases and affect a significant and increasing proportion of the population2-4. The human urate transporter 1 (URAT1) is responsible for the reabsorption of [~]90% of uric acid in the kidneys back into the blood, making it a primary target for treating hyperuricemia and gout5. Despite decades of research and development, clinically available URAT1 inhibitors have limitations because the molecular basis of URAT1 inhibition by gout drugs remains unknown5. Here we present cryo-electron microscopy structures of URAT1 alone and in complex with three clinically relevant inhibitors: benzbromarone, lesinurad, and the novel compound TD-3. Together with functional experiments and molecular dynamics simulations, we reveal that these inhibitors bind selectively to URAT1 in inward-open states. Furthermore, we discover differences in the inhibitor dependent URAT1 conformations as well as interaction networks, which contribute to drug specificity. Our findings illuminate a general theme for URAT1 inhibition, paving the way for the design of next-generation URAT1 inhibitors in the treatment of gout and hyperuricemia.

biophysics↗

bioRxiv2024

(p)ppGpp and DksA play crucial role in reducing the efficacy of b-lactam antibiotics by modulating bacterial membrane permeability

The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0{Delta}dksA V. cholerae mutant to different -lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to -lactam antibiotics. These findings may help in the development of adjuvants for -lactam antibiotics by inhibiting the functions of stringent response modulators. ImportanceThe (p)ppGpp biosynthetic pathway is widely conserved in bacteria. Intracellular levels of (p)ppGpp and the transcription factor DksA play crucial roles in bacterial multiplication and viability in the presence of antibiotics and/or other xenobiotics. The present findings have shown that (p)ppGpp and DksA significantly reduces the efficacy of -lactam and other antibiotics by modulating the availability of peptidoglycan and cell membrane-associated metabolites by reducing membrane permeability. Nevertheless, the whole-cell proteome analysis of (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA strains identified the biosynthetic pathways and associated enzymes that are directly modulated by the stringent response effector molecules. Thus, the (p)ppGpp metabolic pathways and DksA could be a potential target for increasing the efficacy of antibiotics and developing antibiotic adjuvants.

microbiology↗

bioRxiv2024

Binding of SARS-CoV-2 nucleocapsid protein to uninfected epithelial cells induces antibody-mediated complement deposition

SARS-CoV-2 infection triggers strong antibody response toward Nucleocapsid-Protein (NP), suggesting extracellular presence beyond its intra-virion RNA binding. Interestingly, NP was found to decorate infected and proximal uninfected cell-surfaces. Here, we propose a new mechanism through which extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated linear-glycosaminoglycans by spatial rearrangement of its RNA-binding sites facilitated by the flexible, positively charged, linker. Coating of uninfected lung-derived cells with purified NP attracted anti-NP-IgG from lung fluids and sera collected from COVID-19 patients. The magnitude of this immune recognition was significantly elevated in moderate compared to mild COVID-19 cases. Importantly, binding of anti-NP-IgG present in sera generated clusters that triggered C3b deposition by the classical complement pathway. Heparin analog enoxaparin outcompeted NP-binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings unveil how extracellular NP may exacerbate COVID-19 tissue damage, and suggest leads for preventative therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/585388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1084e20org.highwire.dtl.DTLVardef@4126caorg.highwire.dtl.DTLVardef@159c610org.highwire.dtl.DTLVardef@da7b7f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIgG from patients sera target NP-bound cells resulting in complement activation C_LIO_LIThe flexible linker allows NP to both bind linear sulfated GAGs and wrap around RNA C_LIO_LIHeparin analogs prevent NP surface binding and alleviate complement activation C_LIO_LICell-ELISA anti-NP IgG levels differ between mild and moderate COVID-19 C_LI

immunology↗

bioRxiv2024

Ion-channel degeneracy and heterogeneities in the emergence of signature physiological characteristics of dentate gyrus granule cells

Complex systems are neither fully determined nor completely random. Biological complex systems, including single neurons, manifest intermediate regimes of randomness that recruit integration of specific combinations of functionally segregated subsystems. Such emergence of biological function provides the substrate for the expression of degeneracy, the ability of disparate combinations of subsystems to yield similar function. Here, we present evidence for the expression of degeneracy in morphologically realistic models of dentate gyrus granule cells (GC) through functional integration of disparate ion-channel combinations. We performed a 45-parameter randomized search spanning 16 active and passive ion channels, each biophysically constrained by their gating kinetics and localization profiles, to search for valid GC models. Valid models were those that satisfied 17 sub- and supra-threshold cellular-scale electrophysiological measurements from rat GCs. A vast majority (>99%) of the 15,000 random models were not electrophysiologically valid, demonstrating that arbitrarily random ion-channel combinations wouldnt yield GC functions. The 141 valid models (0.94% of 15,000) manifested heterogeneities in and cross-dependencies across local and propagating electrophysiological measurements, which matched with their respective biological counterparts. Importantly, these valid models were widespread throughout the parametric space and manifested weak cross-dependencies across different parameters. These observations together showed that GC physiology could neither be obtained by entirely random ion-channel combinations nor is there an entirely determined single parametric combination that satisfied all constraints. The complexity, the heterogeneities in measurement and parametric spaces, and degeneracy associated with GC physiology should be rigorously accounted for, while assessing GCs and their robustness under physiological and pathological conditions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/580453v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1fe6e90org.highwire.dtl.DTLVardef@10cc473org.highwire.dtl.DTLVardef@135732dorg.highwire.dtl.DTLVardef@1caec0_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

LHX2 regulates dendritic morphogenesis in layer II/III of the neocortex via distinct pathways in progenitors and postmitotic neurons.

In the mammalian neocortex, excitatory neurons that send projections via the corpus callosum are critical to integrating information across the two brain hemispheres. The molecular mechanisms governing the development of the dendritic arbours and spines of these callosal neurons are poorly understood, yet these features are critical to their physiological properties. LIM Homeodomain 2 (Lhx2), a regulator of fundamental processes in cortical development, is expressed in postmitotic callosal neurons occupying layer II/III of the neocortex and also in their progenitors residing in the embryonic day (E) 15.5 ventricular zone of the mouse neocortex. We tested whether this factor is essential for dendritic arbour configuration and spine morphogenesis of layer II/III neurons. Here, we report loss of Lhx2, either in postmitotic layer II/III neurons or their progenitors, resulted in shrunken dendritic arbours and perturbed spine morphology. The defects were more pronounced upon Lhx2 disruption in progenitors, and were recapitulated when this was driven exclusively in basal progenitors. In postmitotic neurons, LHX2 regulates dendritic and spine morphogenesis via the canonical Wnt /{beta} Catenin signalling pathway. Constitutive activation of this pathway in postmitotic neurons mimics the Lhx2 loss-of-function phenotype. In E15.5 progenitors, LHX2 acts in part via bHLH transcription factor NEUROG2 to regulate dendritic morphogenesis. We demonstrate that loss of Lhx2 causes a massive increase in Neurog2 expression, and that Neurog2 knockdown partially rescues the loss of Lhx2 phenotype. Our study uncovers novel LHX2 functions consistent with its temporally dynamic and diverse roles in development. TeaserThe mature architecture of a neuron is shaped by distinct genetic mechanisms that act in its mother cell and after it is born.

neuroscience↗

bioRxiv2024

P66Shc Mediates SUMO2-induced Endothelial Dysfunction

BackgroundSumoylation is a post-translational modification that can regulate different physiological functions. Increased sumoylation, specifically conjugation of SUMO2/3 (small ubiquitin-like modifier 2/3), is detrimental to vascular health. However, the molecular mechanism mediating this effect is poorly understood. MethodsWe used cell-based assays and mass spectrometry to show that p66Shc is a direct target of SUMO2 and SUMO2 regulates p66Shc function via lysine-81 modification. To determine the effects of SUMO2-p66ShcK81 on vascular function, we generated p66ShcK81R knockin mice and crossbred to LDLr-/- mice to induce hyperlipidemia. Next, to determine p66ShcK81-SUMO2ylation-induced changes in endothelial cell signaling, we performed mass spectrometry followed by Ingenuity Pathway Analysis. ResultsOur data reveal that p66Shc mediates the effects of SUMO2 on endothelial cells. Mass spectrometry identified that SUMO2 modified lysine-81 in the unique collagen homology-2 domain of p66Shc. SUMO2ylation of p66Shc increased phosphorylation at serine-36, causing it to translocate to the mitochondria, a step critical for oxidative function of p66Shc. Notably, sumoylation-deficient p66Shc (p66ShcK81R) was resistant to SUMO2-induced p66ShcS36 phosphorylation and mitochondrial translocation. P66ShcK81R knockin mice were resistant to endothelial dysfunction induced by SUMO2ylation and hyperlipidemia. Ingenuity Pathway Analysis revealed multiple signaling pathways regulated by p66ShcK81-SUMO2ylation in endothelial cells, highlighting JAK-STAT as a major pathway affected by SUMO2-p66ShcK81. ConclusionsCollectively, our work reveals SUMO2-p66Shc signaling as a fundamental regulator of vascular endothelial function. We discovered that p66ShcK81 is an upstream modification regulating p66Shc signaling and mediates hyperlipidemia-induced endothelial dysfunction and oxidative stress.

cell biology↗

bioRxiv2024

Forced mouth opening induces post-traumatic hyperalgesia and associated peripheral sensitization after temporomandibular joints injury in mice

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. The pathophysiology of TMD is not fully understood, and it is necessary to understand pathophysiology underlying painful TMD conditions to develop more effective treatment methods. Recent studies suggested that external or intrinsic trauma to TMJ is associated with chronic TMD in patients. Here, we investigated the effects of the TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors. FMO increased mechanical hyperalgesia assessed by von Frey test, spontaneous pain-like behaviors assessed by mouse grimace scale, and anxiety-like behaviors assessed by open-field test. In vivo GCaMP Ca2+ imaging of intact trigeminal ganglia (TG) showed increased spontaneous Ca2+ activity and mechanical hypersensitivity of TG neurons in the FMO compared to the sham group. Ca2+ responses evoked by cold, heat, and capsaicin stimuli were also increased. FMO-induced hyperalgesia and neuronal hyperactivities were not sex dependent. TG neurons sensitized following FMO were primarily small to medium-sized nociceptive afferents. Consistently, most TMJ afferents in the TG were small-sized peptidergic neurons expressing calcitonin gene-related peptides, whereas nonpeptidergic TMJ afferents were relatively low. FMO-induced intraneural inflammation in the surrounding tissues of the TMJ indicates potentially novel mechanisms of peripheral sensitization following TMJ injury. These results suggest that the TMJ injury leads to persistent post-traumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors.

neuroscience↗

bioRxiv2023

Hybrid de novo and haplotype resolved genome assembly of Vechur cattle - elucidating genetic varaiation

Cattle contribute to the nutritional needs and economy of a place. Performance and fitness depends on the response and adaptation to local climatic conditions. Genomic and genetic studies are important for advancing cattle breeding and availability of relevant reference genomes are essential. In the present study, the genome of a Vechur calf was sequenced on both short-read illumina and long-read nanopore platforms. Hybrid de novo assembly approach was deployed to obtain an average contigs length of 1.97 Mbp and N50 of 4.94 Mbp. Using a short-read genome sequence of the corresponding sire and dam, a haplotype-resolved genome was also assembled. In comparison to the taurine reference genome, we found 28982 autosomal structural variants, 16926990 SNVs with 883544 SNVs homozygous in the trio samples suggesting high prevalence of these SNPs in the population. Many of these SNPs have been reported to be associated with various QTLs including growth, milk yield and milk fat content which are crucial determinants of cattle production. Further, population genotype data analysis indicated that the present sample belongs to an Indian cattle breed forming a unique cluster of Bos indicus. Subsequent FST analysis revealed differentiation of Vechur cattle genome at multiple loci, especially those regions related to whole body growth and cell division especially IGF1, HMGA2, RRM2 and CD68 loci suggesting a possible role of these genes in its small stature and better disease resistance capabilities in comparison with the local crossbreeds. This provides an opportunity to select and engineer cattle breeds optimized for local conditions.

genomics↗

bioRxiv2023

WASP facilitates tumor mechanosensitivity in T lymphocytes

Cytotoxic T lymphocytes (CTLs) carry out immunosurveillance by scanning target cells of diverse physical properties for the presence of antigens. While the recognition of cognate antigen by the T cell receptor is the primary signal for CTL activation, it has become increasingly clear that the mechanical stiffness of target cells plays an important role in antigen-triggered T cell responses. However, the molecular machinery within CTLs that transduces the mechanical information of tumor cells remains unclear. We find that CTLs mechanosensitive ability requires the activity of the actin-organizing protein Wiskott-Aldrich Syndrome Protein (WASP). WASP activation is modulated by the mechanical properties of antigen-presenting contexts across a wide range of target cell stiffnesses and activated WASP then mediates mechanosensitive activation of early TCR signaling markers in the CTL. Our results provide a molecular link between antigen mechanosensing and CTL immune response and suggest that CTL-intrinsic cytoskeletal organizing principles enable the processing of mechanical information from diverse target cells.

immunology↗

bioRxiv2023

A single-dose intramuscular immunization of pigs with lipid-nanoparticle DNA vaccines based on the hemagglutinin antigen confers complete protection against challenge infection with the homologous influenza virus strain

Influenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as a nanocarrier to deliver DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. Intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only one out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines. ImportanceInfluenza A virus of swine (IAV-S) is a significant pathogen of swine. The virus also poses a great public health concern due to its zoonotic potential. Although whole-inactivated virus (WIV) vaccines are available to control IAV-S, their heterologous efficacy is limited due to the substantial genetic/antigenic variation of the viral genome. This study provides compelling evidence demonstrating that lipid nanoparticle encapsulated DNA (LNP-DNA) vaccines induce robust systemic and mucosal immunity and complete protection of pigs against challenge infection with the homologous IAV-S strain. Importantly, the LNP-DNA vaccine approach meets the regulatory requirements to serve as an adaptable platform that can be frequently updated to match the emergence of new IAV-S variants. Thus, this LNP-DNA vaccine strategy exhibits great potential in effectively mitigating the economic impact of IAV-S on the swine industry and the associated public health threat.

microbiology↗

bioRxiv2023

Measuring RBC deformability and its heterogeneity using a fast microfluidic device

We report a high-throughput microfluidic device to determine the Youngs modulus of single red blood cells (RBCs). Our device consists of a single channel opening into a funnel, with a semi-circular obstacle placed at the mouth of the funnel. As a RBC passes the obstacle, it deflects from its original path. Using populations of artificially-stiffened RBCs, we show that the stiffer RBCs deflect more compared to the healthy RBCs. We then generate a calibration curve that maps each RBC trajectory to its Youngs modulus obtained using an atomic force microscope. Finally, we sort a mixed population of RBCs based on their deformability alone. Our device could potentially be further miniaturized to sort and obtain the elastic constants of nanoscale objects, such exosomes, whose shape change is difficult to monitor by optical microscopy.

bioengineering↗

bioRxiv2023

Chemical inhibition of histidine biosynthesis curtails M. tuberculosis infection

To overcome the drug resistance crisis and shorten the current duration of human tuberculosis (TB) therapy, new anti-TB molecules is required. In an earlier study, we have shown that Mycobacterium tuberculosis (Mtb), the causative agent of TB, with a fractured de novo histidine biosynthesis fails to mount TB infection in mouse model, emboldening that disrupting the function of this pathway may constitute a novel strategy to curtailing TB infection. In this study, through a target based approach we have designed a number of triazole scaffold molecules specific to imidazole glycerol phosphate dehydratase (IGPD; HisB) of this pathway and have delineated atomic level interactions between the enzyme and inhibitors which pinpointed the specificity and the inhibitory mechanism. Importantly, these molecules exhibited significant potency against free as well as macrophage-internalized wild-type and drug-resistant clinical isolates in culture medium. Notably, a couple of these compounds showed efficacy in reducing the bacterial burden in Mtb-infected mouse model. The chemical inhibition of IGPD induces histidine auxotrophy in Mtb and brings in new prospects to the area of anti-TB drug discovery.

biochemistry↗

bioRxiv2023

Iron chelation by deferoxamine as useful adjunct therapeutics in murine tuberculosis.

Dysregulated iron metabolism is reported in tuberculosis patients; therefore, it represents an opportunity for developing host-directed therapeutics. This study monitored the antimycobacterial properties of an iron chelator, i.e., Deferoxamine (DFO/D), and its impact on the transcript and metabolite levels of Mycobacterium tuberculosis (Mtb) in vitro. For in vivo validation, a group of mice received ferric carboxymaltose to create an iron overload condition, and controls were aerosol-infected with Mtb H37Rv. Mtb-infected mice received isoniazid (INH/H) and rifampicin (RIF/R) in combination with or without DFO before tissue-specific CFU assay, liver metabolite screening and iron quantification using mass spectrometry. DFO showed antimycobacterial properties comparable to INH in vitro. DFO treatment deregulated (log2DFO/control>{+/-}1.0) Mtb transcript (n=137) levels, the majority of which encode for iron-containing proteins and proteins involved in stress response. DFO treatment up-regulated Rv3622c (PE32), Rv2353c (PPE39) and Rv3022A (PE29) genes and conditional knocking down of ABC transporter like irtA by anhydrotetracycline (Atc) inducible CRISPR interference (CRISPRi) approach compromised Mtb growth showing their potential involvement in iron metabolism. Global Mtb metabolite analysis using GC-MS identified a set of 5 deregulated metabolites indicating a perturbed pentose phosphate pathway and inositol phosphate metabolism in the host upon DFO treatment. Iron-overloaded mice exhibited significantly higher tissue mycobacterial burden at two weeks post-infection, and the efficacy of INH and RIF were compromised, corroborating with previous reports. Iron chelation by DFO or combined with/adjunct to RIF and INH significantly reduced the lung/tissue mycobacterial burden at four weeks post-treatment, specifically in the first ([~]0.5 log) and second weeks ([~]0.5 log) of treatment. The intracellular pro-inflammatory cytokine levels in the lung CD4+ T cells of INH and RIF-treated groups with or without DFO were similar, suggesting DFO has a direct role in Mtb survival and metabolism rather than improved infection, and the efficacy of INH and RIF were compromised, corroborating with previous reports. DFO adjunct to RIF and INH treatment significantly altered liver arginine biosynthesis, which directly neutralizes ammonia and is immune-supportive. Conventionally, DFO is used for treating acute iron toxicity that is common in thalassemic patients, and this study demonstrates DFO has potential as adjunct therapeutics for tuberculosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/543389v3_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@5066fdorg.highwire.dtl.DTLVardef@99a292org.highwire.dtl.DTLVardef@80426dorg.highwire.dtl.DTLVardef@892436_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

The deamidases secreted by Burkholderia pseudomallei and Enteropathogenic Ecoli modify the residue 40 in ubiquitin from a Glutamine (Q) to Glutamate (E), triggering several downstream processes to cause cell cycle arrest and activate immune responses. Deamidation hampers the activity of ubiquitin and its interaction with ubiquitin chain receptors by an unknown mechanism. Here, we study the effect of deamidation on ubiquitin structure and dynamics. We report the crystal structure of the deamidated ubiquitin, supported by NMR and molecular dynamics simulations. The structure reveals a new intra-molecular salt bridge between the deamidated region and the C-terminal tail of ubiquitin. The salt bridge perturbs the dynamics of the ubiquitin tail to reduce affinity for ubiquitin receptors like the p62 ubiquitin-associated domain. The salt bridge disrupts the transition to catalytically active E2~Ub closed conformation. Consequently, RING E3s fail to interact with E2~Ub, reducing ubiquitination activity. Our findings reveal that deamidation-induced intramolecular salt bridges in ubiquitin modulate conformational ensembles to inactivate ubiquitination.

biochemistry↗