Biology subjects

Natarajan, A.

Publications and source records attributed to Natarajan, A..

29 records · Page 2Linked to original sources

bioRxiv2022

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

The cell envelope of the Gram-negative Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolate B. cenocepacia K56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between {beta}-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of {beta}-lactam/{beta}-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies. Author SummaryWhile the Gram-negative cell envelope is a major barrier to antibiotic action, we have an incomplete picture of how each component contributes to antibiotic resistance. To answer this question from a genome-wide perspective in a model of antibiotic-resistant bacteria, we constructed a library of randomly-barcoded transposon mutants in Burkholderia cenocepacia and exposed it to a panel of diverse cell envelope-targeting antibiotics. We identified individual genes and whole pathways associated with antibiotic resistance, including several novel players. Focusing on clinically relevant antibiotics, our strategy dissected how the synergy of the important {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam primarily depends upon inhibition of a single {beta}-lactamase. Additionally, we are the first to characterise the molecular basis of the antibiotic mechanism of cefiderocol in Burkholderia, a new and very potent siderophore-cephalosporin conjugate drug. Encouragingly, we found that cefiderocol susceptibility was greatest at physiological iron concentrations. Overall, we highlight several avenues that can be taken to enhance or develop new therapeutic strategies against Burkholderia infection.

microbiology↗

bioRxiv2022

Heart Rate Variability During Mindful Breathing Meditation

In this article, we discuss Heart Rate Variability (HRV) measured during mindful breathing meditation. We provide a pedagogical computation of 2 commonly used HRV metrics, i.e. the root mean square of successive differences (RMSSD) and the standard deviation of RR intervals (SDRR), in terms of Fourier components. It is shown that the RMSSD preferentially weights higher frequency Fourier modes, making it unsuitable as a biosignal for mindful breathing meditation which encourages slow breathing. We propose a new metric called the autonomic balance index (ABI) which uses Respiratory Sinus Arrhythmia to quantify the fraction of HRV contributed by the parasympathetic nervous system. We apply this metric to HRV data collected during two different meditation techniques, and show that the ABI is significantly elevated during mindful breathing, making it a good signal for biofeedback during meditation sessions.

biophysics↗

bioRxiv2022

Characterization and Preclinical Treatment of Rotational Force-Induced Brain Injury

Millions of traumatic brain injuries (TBIs) occur annually. TBIs commonly result from falls, traffic accidents, and sports-related injuries, all of which involve rotational acceleration/deceleration of the brain. During these injuries, the brain endures a multitude of primary insults including compression of brain tissue, damaged vasculature, and diffuse axonal injury. All of these deleterious effects can contribute to secondary brain ischemia, cellular death, and neuroinflammation that progress for weeks to months after injury and impede neurological recovery. While the linear effects of head trauma have been extensively modeled, less is known about how rotational injuries mediate neuronal damage following injury. Here, we developed a new model of rotational head trauma in rodents and extensively characterized the pathological, behavioral, and electrophysiological effects of rotational TBI (rTBI). We identify aberrant cyclin dependent kinase 5 (Cdk5) activity as a principal mediator of rTBI and show pharmacological inhibition of Cdk5 reduces the cognitive and pathological consequences of injury. Finally, we utilize Cdk5-enriched phosphoproteomics to uncover potential downstream mediators of rTBI. These studies contribute meaningfully to our understanding of the mechanisms of rTBI and how they may be effectively treated.

neuroscience↗

bioRxiv2022

Dynamic DNA methylation contributes to carryover effects and beneficial acclimatization in geoduck clams

Given the rapidly changing global climate, there is a growing need to understand the acclimatory basis of animal response to environmental change. To investigate the role of DNA methylation in environmental acclimatization, we generated a reference genome and surveyed the physiology and DNA methylomes of juvenile geoduck clams, Panopea generosa, under differing seawater pH regimes. Juveniles were initially exposed to one of three seawater pH conditions, followed by ambient common-garden conditions, then a second reciprocal exposure to ambient pH or pH 7.4. Within 10 days of the initial low pH exposure, juvenile clams showed decreased shell size relative to ambient pH with corresponding differential DNA methylation. Following four months of ambient common-garden conditions, juveniles initially exposed to low pH compensatorily grew larger, with DNA methylation indicative of these phenotypic differences, demonstrating epigenetic carryover effects persisted months after initial exposure. Functional enrichment analysis of differentially methylated genes revealed regulation of signal transduction through widespread changes in the Wnt signaling pathways that influence cell growth, proliferation, tissue and skeletal formation, and cytoskeletal change. After 10 days of secondary exposure to pH 7.4, naive juvenile clams were more sensitive to low pH compared to those initially exposed, showing reduced growth and having nearly a 2-fold greater change in DNA methylation. Collectively, this new genomic resource and coordinated phenotypic and methylomic response support that epigenetic mechanisms underlie acclimatization to provide beneficial phenotypes.

genomics↗

bioRxiv2022

Cooperative ectodomain interaction among TCRαβ, CD3γε, and CD3δε enhances TCR mechanotransduction

TCR signaling poses a mechanical problem: pMHC binding occurs at the TCR{beta} ectodomain (ECD) head, whereas ITAM phosphorylation occurs on CD3 cytoplasmic tails. Chemistry cannot bridge this >10 nm gap, requiring the two events to be coupled through the TCR-CD3 interface, thus involving conformational allostery and being regulatable by force. Although weak ECD cis-interactions between TCR{beta} and CD3 have been proposed to contribute to this coupling, their kinetics and mechanical competence remain elusive. Here, we quantify TCR{beta}-CD3 ECD cis-interactions in a pseudo-cis configuration using two-dimensional binding and single-bond force spectroscopy, finding that TCR{beta}-CD3{gamma}{varepsilon} and TCR{beta}-CD3{delta}{varepsilon} interactions have low affinity and rapid kinetics, yet form catch bonds. Critically, concurrent engagement of CD3{gamma}{varepsilon} and CD3{delta}{varepsilon} produces high cis-cooperativity, yielding a stronger and longer-lasting CD3{gamma}{varepsilon}-TCR{beta}-CD3{delta}{varepsilon} trimolecular catch bond than the sum of the two dimeric bonds, with force-stabilized lifetimes matching those of agonist TCR-pMHC trans-interaction. Molecular dynamics simulations reveal an expanded, cooperative, and asymmetric contact network, making CD3{delta}{varepsilon} more force-responsive and susceptible to conformational change than CD3{gamma}{varepsilon}. Interface mutations do not alter force-free affinities but remodel cooperative cis-bond profiles, leading to an inverse correlation with trans-bond profiles and T cell signaling. These results identify cooperative ECD cis-interaction as a mechanically regulatable allosteric coupling element at the TCR-CD3 junction important to antigen recognition and signal initiation.

immunology↗

bioRxiv2022

In situ cell-surface conformation of the TCR-CD3 signaling complex

T cells play a vital role in adaptive immune responses to infections, inflammation and cancer and are dysregulated in autoimmunity. Antigen recognition by T cells - a key step in adaptive immune responses - is performed by the T cell receptor (TCR)-CD3 complex. The extracellular molecular organization of the individual CD3 subunits (CD3{delta}{varepsilon} and CD3 {gamma}{varepsilon}) around the {beta}TCR is critical for T cell signaling. Here, we incorporated unnatural amino acid (UAA) photo-crosslinkers at specific mouse TCR, TCR{beta}, CD3{delta} and CD3{gamma} sites, based on previous mutagenesis, NMR spectroscopy and cryo-EM evidence, and crosslinking allowing us to identify nearby interacting CD3 or TCR subunits on the mammalian cell surface. Using this approach, we show that CD3{gamma} and CD3{varepsilon}, belonging to CD3{gamma}{varepsilon} heterodimer crosslinks to C{beta} FG loop and C{beta} G strand, respectively and CD3{delta} crosslinks to C{beta} CC loop and C DE loop. Together with computational docking, we identify that in in situ cell-surface conformation, the CD3 subunits exists in CD3{varepsilon}-CD3{gamma}-CD3{varepsilon}-CD3{delta} arrangement around the {beta} TCR. This unconventional technique, which uses the native mammalian cell surface microenvironment, includes the plasma membrane and excludes random, artificial crosslinks, captures a dynamic, biologically relevant, cell-surface conformation of the TCR-CD3 complex, which is compatible with the reported static cryo-EM structures overall CD3 subunits arrangement, but with key differences at the TCR-CD3 interface, which may be critical for experiments in T cell model systems.

immunology↗

bioRxiv2022

Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Tumor

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers. HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5. C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade. C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma. C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinsons disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. Compared to WT cells, CD4+ T cells with reduced Fbxo7 expression show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

cell biology↗

bioRxiv2019

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer protein glycosylation remains a bottleneck. To address this limitation, we describe a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude cell lysates are enriched with glycosyltransferases by cell-free protein synthesis and then glycosylation pathways are assembled in a mix-and-match fashion to elaborate a single glucose priming handle installed by an N-linked glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs. We then use selected pathways to design a one-pot cell-free system to synthesize a vaccine protein with an -galactose motif and engineered Escherichia coli strains to produce human antibody constant regions with minimal sialic acid motifs. We anticipate that our work will facilitate glycoscience and make possible new glycoengineering applications.

synthetic biology↗

bioRxiv2019

Heart Rate Variability with Photoplethysmography in 8 Million Individuals: Results and Scaling Relations with Age, Gender, and Time of Day

Heart rate variability, or the variation in the time interval between consecutive beats, is a non-invasive dynamic metric of the autonomic nervous system and an independent risk factor for cardiovascular death. Prior limitations of use include requirements for continuous electrocardiography and lack of reference standards. Consumer wrist-worn tracking devices using photoplethysmography now provide the unique potential of continuously measuring surrogates of sympathetic and parasympathetic activity through the analysis of interbeat intervals. Here we leverage wrist-worn trackers to present the largest, to our knowledge, analysis of heart rate variability in humans across the time, frequency, and graphical domains. We derive diurnal parasympathetic and sympathetic measures and provide scaling parameters by age, sex, and time of day. Poincare plots graphically summarize heart rate variability metrics and may detect common arrhythmias. Lastly, we observe a strong dose-dependent correlation between daily steps and optimal heart rate variability metrics. Our results provide the ability to interpret continuous heart rate variability for tens of millions of wrist-worn trackers already in use.

physiology↗

bioRxiv2017

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 {micro}g/mL for the single-subunit OST enzyme, Protein glycosylation B (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

synthetic biology↗