Biology subjects

Bakshi, S.

Publications and source records attributed to Bakshi, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Population reweighting and reporter distortion obscure ribosome regulation during translation inhibition

Bacteria dynamically adjust their ribosome allocation as growth conditions change, but how this regulation unfolds within individual cells remains poorly understood. Established relationships between ribosome allocation and growth are largely derived from population measurements under steady-state conditions, which may not capture the trajectories followed by individual cells during perturbation. Differential growth can reweight heterogeneous cellular states over time, causing population-level measurements to diverge from the regulatory trajectories followed by individual cells. Here, we use lineage-resolved single-cell microscopy to follow growth and ribosomal reporter dynamics in Escherichia coli during translation inhibition without growth-dependent reweighting of the sampled lineages. Unexpectedly, fluorescent protein fusions to the ribosomal proteins S2 and L9 decrease in fluorescence following chloramphenicol treatment, whereas transcriptional reporters driven by the ribosomal promoters initially increase. The decrease in ribosomal fusion fluorescence persists across multiple fluorescent proteins and is reproduced during tetracycline treatment, indicating that it is not specific to a particular fluorophore, ribosomal protein or translation inhibitor. Although some fusion reporters retain a relationship with cellular growth within physiological states, this relationship is itself reporter-dependent. These results reveal two independent requirements for quantitative measurements of dynamic cellular responses: longitudinal sampling is needed to distinguish within-cell regulation from growth-dependent population reweighting, and reporter behaviour must be validated when the perturbation alters the processes generating the measured signal.

systems biology↗

bioRxiv2026

Target-Specific Discovery of BMM_1567 Restores Aminoglycoside Activity Against Multidrug-Resistant Gram-Negative ESKAPE Pathogens

The global emergence of multidrug-resistant (MDR) ESKAPE pathogens has significantly reduced the effectiveness of existing antibiotics, highlighting the urgent need for new strategies to restore antimicrobial susceptibility. Here, we report the discovery and mechanism of BMM_1567, a peptide potentiator that enhances aminoglycoside efficacy against MDR pathogens. A genetically defined reporter-based screen identified BMM_1567 as a potent inhibitor of aminoglycoside resistance, potentiating spectinomycin activity against MDR Gram-negative ESKAPE isolates at low micromolar concentrations. Structural modeling and molecular dynamics simulations indicated that BMM_1567 interacts with residues lining the antibiotic-binding groove of aminoglycoside-modifying enzymes (ANT, APH, AAC), with highest affinity for ANT ({Delta}G_bind = -62.25 kcal/mol), suggesting competitive inhibition of substrate binding. Site-directed mutagenesis of key ANT residues identified critical amino acids involved in BMM_1567 binding, confirming their role in mediating spectinomycin potentiation. In murine abscess model using XDR E. coli, BMM_1567 in combination with spectinomycin significantly reduced bacterial burden and pro-inflammatory cytokine levels, comparable to colistin. Collectively, these findings establish BMM_1567 as a promising aminoglycoside potentiator that restores antibiotic activity against MDR pathogens through direct inhibition of resistance enzymes, while exhibiting in vivo efficacy and a remarkably low propensity for resistance development.

microbiology↗

bioRxiv2026

STRESS HISTORY ESTABLISHES A TRANSIENT TOLERANT STATE THAT SHAPES ANTIBIOTIC SURVIVAL UPON RESUSCITATION

Antibiotic treatment failure, often driven by non-genetic mechanisms such as tolerance and persistence, remains a major global health challenge. {beta}-lactams, the most widely prescribed antibiotic class, are particularly compromised by tolerance in dormant, non-growing cells; yet, how these drugs act on cells resuscitating from dormancy remains poorly understood. Here, we investigate the resuscitation phase at an unprecedented scale using Hi-DFA (High-throughput Dynamic Fate Analyser), a single-cell microfluidic platform integrating time-lapse imaging with machine-learning-based image analysis for dynamic cell-fate tracking. We identify a distinct survival strategy: a significant fraction of resuscitating cells transiently slow their growth, facilitating survival upon {beta}-lactam exposure. This transiently tolerant phenotype is considerably less frequent in unstressed, exponentially growing cells, indicating that prior starvation history predisposes cells to this state. Using simulated in vitro pharmacokinetic treatment profiles, we show that suboptimal dosing selectively enriches for this transient tolerance state. A population dynamics model built from this single-cell antibiotic-response data suggests that these transient-tolerant cells, not typical starvation-triggered persisters, may be the primary drivers of rapid population regrowth post-treatment under clinically relevant conditions. Together, our findings define a distinct class of antibiotic survival shaped by stress history and treatment profile, offering a quantitative framework for optimising antibiotic dosing strategy.

systems biology↗

bioRxiv2026

Tmn blocks phage spread via plasmolysis and triggers synergistic defence responses

Membrane-associated phage defences remain poorly understood. Here we characterise Tmn, a YobI-family transmembrane P-loop NTPase that protects bacteria from phage infection by establishing a plasmolysis-associated antiviral state. Upon recognising phage T2 RIIB protein, Tmn enhances ATP turnover and selectively exports Mg2+, causing rapid cytoplasmic collapse that arrests phage replication without detectable membrane depolarisation or gross leakage of the cell content. Cryo-electron microscopy shows that Tmn assembles into a decameric membrane complex with extended cytosolic arms, an uncommon architecture among P-loop NTPases. The cytosolic arms, including a solenoid-like repeat domain, mediate trigger interaction and determine specificity. In addition to its primary defence function, Tmn-driven ATP collapse activates otherwise silent ATP-depletion-sensing defences, including Gabija and Septu type I, providing a mechanistic basis for synergy among defence systems and limiting secondary phage spread. These findings demonstrate that Tmn is a membrane-integrated sensor-effector that couples phage recognition to metabolic collapse and coordinated multi-layered immunity.

microbiology↗

bioRxiv2025

Hierarchical and Spatial Mapping of Whole-Brain c-Fos Activity Reveals Distinct Opioid and Withdrawal Neuronal Ensembles

How opioid exposure and withdrawal states shape brain activity at the systems and circuit level remains poorly understood. Here, we use whole-brain, cellular-resolution c-Fos mapping to define brain-wide activity patterns and neuronal ensembles associated with morphine administration and withdrawal. To account for the brains anatomically nested structure, we developed and applied a hierarchical statistical framework that detects region-specific changes in activity and outperforms conventional methods that treat brain regions as individual, unrelated units. These distributed signals formed ensembles with consistent and anatomically structured patterns of activity, both within subregions and across multiple connected brain areas. By combining TRAP2-based activity tagging with acute whole-brain c-Fos staining, we identified morphine- and withdrawal-activated ensembles and found that they are largely non-overlapping at the single-cell level, even within the same brain region. Integration with existing spatial transcriptomics datasets identified molecular markers for these state-specific ensembles in key brain areas such as the nucleus of accumbens, amygdala and ventral tegmental areas. Lastly, by integrating Allen mouse whole-brain transcriptional datasets, we identified the molecular identity of the morphine- administration and withdrawal ensembles. These findings define dissociable neuronal ensembles that encode opposing drug states and introduce a scalable framework for linking whole-brain activity to molecular and circuit-level mechanisms. HighlightsO_LIWhole-brain neural activity mapping identifies the regional and spatial difference of morphine-administration and withdrawal neuronal ensembles. C_LIO_LIBRANCH, a hierarchical statistical testing framework, offers increased sensitivity and anatomical interpretability for whole-brain datasets. C_LIO_LITRAP2-based activity tagging reveals brain-wide separation of acute morphine and withdrawal ensembles at the cellular level. C_LIO_LIA systematic analysis of the whole brain neural activity data combined with spatial transcriptomic data revealed molecular features of opioid-related neural ensembles. C_LI

neuroscience↗

bioRxiv2025

Quantitative Single-Cell Imaging of Efflux Pump Heterogeneity and Antibiotic Response Dynamics

Understanding how bacterial cells survive antibiotic treatment requires tools capable of resolving dynamic physiological heterogeneity at single-cell resolution. Here, we present a high-throughput, lineage-resolved microfluidic imaging platform for quantifying the expression and spatial distribution of the AcrAB-TolC efflux pump in Escherichia coli during antibiotic exposure. The system is built around a custom-designed multilayer microfluidic device, fabricated using a direct-write photolithography protocol, which ensures uniform delivery of nutrients and antibiotics while confining thousands of individual cells in dead-end trenches. Brightfield imaging provides a label-free, high-temporal-resolution method for tracking cell growth and division, enabling accurate reconstruction of lineages over time. Efflux pump abundance is quantified using GFP-tagged AcrB, while a red fluorescent reporter driven by a constitutive promoter serves as a control to account for growth-dependent fluorescence artifacts. To analyse the sources of heterogeneity in efflux abundance and its consequences for antibiotic response dynamics, we developed a lineage-resolved analytical framework that compares cells of similar replicative age but differing spatial positions, and vice versa. This analysis was made possible by a novel machine-learning-based image processing pipeline, which enables robust cell segmentation, tracking, and lineage reconstruction across multiple fields of view. By extracting time-resolved single-cell data, this platform allows precise dissection of non-genetic variability in efflux activity and its role in determining survival outcomes, offering a powerful foundation for future quantitative studies of bacterial physiology under antibiotic stress.

systems biology↗

bioRxiv2025

Live and let die: lysis time variability and resource limitation shape lytic bacteriophage fitness

Bacteriophages (phages) play a critical role in controlling bacterial populations, both in nature and as potential therapeutic agents. Their ability to replicate, compete against each other, and eradicate target cell populations is usually understood through a number of life history parameters, traditionally measured by population-level assays, which implicitly average the parameters value across a large number of infection events. Recent experiments suggest that bacteriophage life history parameters are subject to considerable heterogeneity, raising the question of whether experimental and modelling efforts that do not account for this variability may overlook important factors in phages behaviour, competitive fitness or therapeutic viability. Here, using agent-based simulations, we investigate the importance of stochasticity in lysis time and burst size of lytic bacteriophages in two common laboratory competition experiments: serial passage of well-mixed populations and plaque expansion across a bacterial lawn. We find that a phages analytic growth rate in isolation can be a poor predictor of its fitness advantage in simulated competition experiments. Specifically, when lysis times are tightly distributed, we identify a novel effect we name "population resonance", through which a bacteriophage can display a significant fitness advantage over a competitor with a much greater growth rate in isolation. Our simulations also show that both serial passage and plaque expansion reward variability in lysis time more than expected, by increasing the phage resilience when resources are scarce. Author SummaryBacteriophages (viruses that infect bacteria) can be described by a set of attributes called life history parameters. Historically, these parameters could only be measured on-average over large populations. Recent experimental advancements, however, have enabled their quantification at the single-cell-single-virus level, and have revealed that these traits are subject to inherent variability, even for genetically identical viruses infecting genetically identical cells. Here, we explore whether the degree of variability in one particular life history parameter, the lysis time (the time taken for a bacteriophage to infect and kill its host bacteria) might be subject to natural selection, by quantifying its effects on phages fitness in two simulated competition experiments: serial passage and plaque expansion. Surprisingly, we find that lysis time variability is advantageous in competition even when it reduces the phages growth rate in isolation. This finding opens the door to future computational and experimental work, as it demonstrates that mean values alone are not sufficient to describe or predict a bacteriophage efficacy, and that models that ignore this variability can overlook viable evolutionary strategies.

evolutionary biology↗

bioRxiv2025

A Transient Increase in Cardiomyocyte Protein O-GlcNAcylation Enhances Susceptibility to Pressure Overload-Induced Cardiac Remodeling

BACKGROUNDThe observation that diabetic patients always under tight-glycemic control consistently show better cardiovascular disease outcomes compared to patients who transition to tight-glycemic control after prior conventional glycemic control lead to the concept of metabolic memory. Mechanisms such as epigenetics possibly mediate the lasting metabolic memory effects, our understanding of the underlying mechanisms remains limited. Increased cardiac protein posttranslational O-linked {beta}-N-acetylglucosamine (O-GlcNAc) modification is implicated in cardiac remodeling observed in diabetes, and our previous work shows chronically elevated cardiomyocyte O-GlcNAc causes adverse cardiac changes. Therefore, the current study hypothesized that transiently increased cardiomyocyte O-GlcNAcylation leads to exacerbated adverse cardiac remodeling after subsequent pressure-overload. METHODS AND RESULTSUsing our previously described inducible cardiomyocyte specific, dominant-negative O-GlcNAcase (dnOGAh) mouse and single transgenic littermate controls (Con), we induced O-GlcNAc levels for 2wk (ON), followed by a 2wk washout (OFF); mice then underwent transverse-aortic constriction (TAC) or Sham surgery. We observed the expected cardiac remodeling in TAC groups, including decreased cardiac function, and increased hypertrophy and fibrosis. Moreover, these pathologic measures were exacerbated in the ON/OFF-TAC vs. Con-TAC mice; additionally, transcriptomic analysis of LV-tissue from each experimental group showed pathways which not only supported our fibrosis, hypertrophy and functional results of exacerbated cardiac remodeling, but also, revealed potential novel molecular pathways underlying this pathologic remodeling. CONCLUSIONSWe observed exacerbated cardiac pathology between ON/OFF-TAC vs. Con-TAC groups supporting the concept of "O-GlcNAc memory" as a component of metabolic memory. Moreover, transcriptomic analysis provides insight into potential molecular pathways underpinning this metabolic/O-GlcNAc memory such as Ccn2/CTGF-driven fibrosis, and/or Nox4-driven oxidative stress. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/657956v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@717ea9org.highwire.dtl.DTLVardef@132bdcaorg.highwire.dtl.DTLVardef@4a2bdaorg.highwire.dtl.DTLVardef@18508d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIWe provide a novel paradigm to study phenotypic and molecular effects of specific, transiently increased cardiomyocyte O-GlcNAcylation on the heart. C_LIO_LIOur results show exacerbated adverse cardiac remodeling due to transiently increased cardiomyocyte O-GlcNAc with pressure-overload, supporting the concept of "O-GlcNAc memory" as a component of metabolic memory. C_LIO_LITranscriptomic insights show gene expression basis for not only observed exacerbated adverse cardiac remodeling (e.g., hypertrophy, fibrosis, cardiac dysfunction), but also potential molecular pathways that could drive cardiac pathology exacerbation of O-GlcNAc memory. C_LI What are the clinical implications?O_LIThis study supports a concept of "O-GlcNAc memory", where previously increased cardiomyocyte protein O-GlcNAcylation can impact the later development of differential cardiac pathology--like the pathology seen in metabolic memory research. C_LIO_LIThe potential role of O-GlcNAc in mediating metabolic memory will help focus future translational research on this modification and downstream cardiac effects in diabetes. C_LIO_LITranscriptomic profiling of cardiac remodeling in this model provides an investigational roadmap for future molecular and functional studies to identify novel therapeutics that ameliorate heart disease induced by differential metabolic memory. C_LI

molecular biology↗

bioRxiv2024

POLYGENIC DETERMINANTS OF H5N1 ADAPTATION TO BOVINE CELLS

Avian influenza H5N1 clade 2.3.4.4b viruses caused a global panzootic and, unexpectedly, widespread outbreaks in dairy cattle, therefore representing a pandemic threat. To inform effective control strategies, it is critical to determine whether the potential to adapt to bovine cells is a generalised feature of H5N1 viruses, or is specific to clade 2.3.4.4b, or even more restricted to specific genotypes within this clade (e.g., B3.13 and D1.1). Using a large panel of H5N1 viruses representing >60 years of their natural history and other IAV for comparative purposes, we demonstrate that virus adaptation to bovine cells is: (i) highly variable across 2.3.4.4b genotypes, (ii) limited in viruses predating the global expansion of this clade, (iii) determined by the viral internal gene cassette, and (iv) not restricted to udder epithelial cells. Mutations in the PB2 polymerase subunit, particularly M631L, emerge as key determinants of adaptation, although their phenotypic effects are context dependent and have limited enhanced viral polymerase activity in human cells. Bovine B3.13 and some avian genotypes also exhibit enhanced modulation of bovine interferon-induced antiviral responses, determined by at least the viral PB2, nucleoprotein, and the non-structural protein NS1. Our results highlight the polygenic nature of IAV host range and reveal that the potential to cross the species barrier varies during the evolutionary trajectory of H5N1, with some avian viruses more predisposed to spillover than others.

microbiology↗

bioRxiv2024

Pulmonary Fibrosis Ferret Model Demonstrates Sustained Fibrosis, Restrictive Physiology, and Aberrant Repair

RationaleThe role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. MethodsBleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with {micro}CT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. ResultsBleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric {micro}CT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant proximalization repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. ConclusionsBleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

physiology↗

bioRxiv2024

Single-cell imaging of the lytic phage life cycle dissects the timing and noise in individual infection steps

When a lytic bacteriophage infects a bacterial cell, it commandeers the cells resources to replicate, ultimately causing cell lysis and the release of new virions. As phages function as obligate parasites, each stage of the infection process depends on the physiological parameters of the host cell. Given the inherent physiological variability within a population of genetically identical bacterial cells, we ask how the phage infection dynamic reflects such heterogeneity. Here, we introduce a timelapse imaging assay for investigating the dynamics of individual infection steps by a single T7 phage on a single bacterium. This high-throughput, time-resolved assay enables us to monitor the infection progression simultaneously in multiple cells, uncovering substantial heterogeneity at each step and revealing correlations between infection dynamics and the physiological state of the infected cell. Simulations of competing phage populations with different lysis time distributions reveal that heterogeneity in infection dynamics can significantly impact phage fitness, highlighting it as a potential evolutionary driver of phage-bacteria interactions.

microbiology↗

bioRxiv2024

Protein Translation Can Fluidize Bacterial Cytoplasm

The cytoplasm of bacterial cells is densely packed with highly polydisperse macromolecules that exhibit glassy dynamics. Research has revealed that metabolic activities in living cells can counteract the glassy nature of these macromolecules, allowing the cell to maintain critical fluidity for its growth and function. While it has been proposed that the crowded cytoplasm is responsible for this glassy behavior, a detailed explanation for how cellular activity induces fluidization remains elusive. In this study, we introduce and validate a novel hypothesis through computer simulations: protein synthesis in living cells contributes to the metabolism-dependent fluidization of the cytoplasm. The main protein synthesis machinery, ribosomes, frequently shift between fast and slow diffusive states. These states correspond to the independent movement of ribosomal subunits and the actively translating ribosome chains called polysomes, respectively. Our simulations demonstrate that the frequent transitions of the numerous ribosomes, which constitute a significant portion of the cell proteome, greatly enhance the mobility of other macromolecules within the bacterial cytoplasm. Considering that ribosomal protein synthesis is the largest consumer of ATP in growing bacterial cells, the translation process likely serves as the primary mechanism for fluidizing the cytoplasm in metabolically active cells.

biophysics↗

bioRxiv2023

Enabling AI in Synthetic Biology through Construction File Specification

The Construction File (CF) specification establishes a standardized interface for molecular biology operations, laying a foundation for automation and enhanced efficiency in experiment design. It is implemented across three distinct software projects: PyDNA_CF_Simulator, a Python project featuring a ChatGPT plugin for interactive parsing and simulating experiments; ConstructionFileSimulator, a field-tested Java project that showcases Experiment objects expressed as flat files; and C6-Tools, a JavaScript project integrated with Google Sheets via Apps Script, providing a user-friendly interface for authoring and simulation of CF. The CF specification not only standardizes and modularizes molecular biology operations but also promotes collaboration, automation, and reuse, significantly reducing potential errors. The potential integration of CF with artificial intelligence, particularly GPT-4, suggests innovative automation strategies for synthetic biology. While challenges such as token limits, data storage, and biosecurity remain, proposed solutions promise a way forward in harnessing AI for experiment design. This shift from human-driven design to AI-assisted workflows, steered by high-level objectives, charts a potential future path in synthetic biology, envisioning an environment where complexities are managed more effectively.

synthetic biology↗

bioRxiv2023

Quantitative Microbiology with Microscopy: Effects of Projection and Diffraction

Time-resolved live-cell imaging using widefield microscopy is instrumental in quantitative microbiology research. It allows us to track and measure the size, shape, and content of individual microbial cells over time. However, the small size of microbial cells poses a significant challenge in interpreting image data, as it approaches the dimensions of the microscopes depth of field and experiences diffraction effects. As a result, 2D widefield images of microbial cells contain projected 3D information, blurred by the 3D point spread function. In this study, we employ computer simulations and targeted experiments to investigate the impact of diffraction and projection on our ability to quantify the size and content of microbial cells from 2D microscopic images. This study points to some new and often unconsidered artefacts resulting from the interplay of projection and diffraction effects, within the context of quantitative microbiology. These artefacts introduce substantial errors and biases in size and fluorescence quantification, and even single-molecule counting, making the elimination of these errors a complex task. Awareness of these artefacts is crucial for designing strategies to accurately interpret micrographs of microbes. To address this, we present new experimental designs and machine learning based analysis methods that account for these effects, resulting in accurate quantification of microbiological processes.

microbiology↗

bioRxiv2022

Timing and magnitude of the type-I interferon response are determinants of disease tolerance in arbovirus infection

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: (i) "resistance", directed to abrogate virus replication, or (ii) "disease tolerance", aimed to avoid organ and tissue damage without overly controlling viral burden. The overall principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of a major disease of ruminants, bluetongue, as a model system to investigate the mechanisms of disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Here, we show that BTV consistently reaches higher titres in ovine primary cells, compared to cells derived from cattle. The variable replication kinetics of BTV in sheep and cattle cells were mostly abolished by abrogating the cell type-I interferon (IFN) response. By screening a library of bovine interferon stimulated genes (ISGs), we identified restriction factors blocking BTV replication, however both sheep and cattle orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells, compared to cattle cells, which results in an earlier downregulation of antiviral proteins. Moreover, by RNAseq we also show a more pronounced expression of ISGs in BTV infected cattle cells compared to sheep cells. Our data provide a new perspective on how the type-I IFN response in reservoir species can have overall positive effects on both virus and host evolution.

microbiology↗

bioRxiv2022

Zoonotic avian influenza viruses evade human BTN3A3 restriction

Cross-species transmission of avian influenza A viruses (IAVs) into humans could represent the first step of a future pandemic1. Multiple factors limiting the spillover and adaptation of avian IAVs in humans have been identified, but they are not sufficient to explain which virus lineages are more likely to cross the species barrier1,2. Here, we identified human BTN3A33 (butyrophilin subfamily 3 member A3) as a potent inhibitor of avian but not human IAVs. We determined that BTN3A3 is constitutively expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts at the early stages of virus replication by inhibiting avian IAV vRNA transcription. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F, or rarely 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, several serotypes of avian IAVs that spilled over into humans in recent decades evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure4. Importantly, we identified more than 150 avian IAV lineages with a BTN3A3-resistant genotype. In conclusion, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

microbiology↗

bioRxiv2021

Accurate Segmentation of Bacterial Cells using Synthetic Training Data

We present a novel method of bacterial image segmentation using machine learning models trained with Synthetic Micrographs of Bacteria (SyMBac). SyMBac is a tool that allows for rapid, automatic creation of arbitrary amounts of training data, combining detailed models of cell growth, physical interactions, and microscope optics to create synthetic images which closely resemble real micrographs. The major advantages of our approach are: 1) synthetic training data can be generated virtually instantly, and on demand; 2) these synthetic images are accompanied by perfect ground truth positions of cells, meaning no data curation is required; 3) different biological conditions, imaging platforms, and imaging modalities can be rapidly simulated, meaning any change in ones experimental setup no longer requires the laborious process of manually generating new training data for each change. Our benchmarking results demonstrate that models trained on SyMBac data generate more accurate and precise cell masks than those trained on human annotated data, because the model learns the true position of the cell irrespective of imaging artefacts. Machine-learning models trained with SyMBac data are capable of analysing data from various imaging platforms and are robust to drastic changes in cell size and morphology.

microbiology↗

bioRxiv2021

Capsule protects against intracellular killing and enables vascular endothelial cell translocation during invasive pneumococcal disease

Streptococcus pneumoniae (Spn) is a leading cause of invasive disease. Chief among its virulence determinants is capsular polysaccharide which protects the bacterium from phagocytosis. While 100 antigenically distinct capsule types are produced by Spn, i.e. serotypes, only 20-30 are commonly associated with invasive disease. A frequency that suggests serotypespecific properties of the capsule influence virulence. Herein, we show capsule has strong antioxidant properties. Moreover, that this property promotes invasive disease by protecting Spn taken up by vascular endothelial cells during bacteremia from endosome-killing and enhancing the translocation rate into organs. Crucially, isogenic capsule-switch mutants of Spn varied considerably in their resistance to H2O2-killing in culture and measured levels correlated positively with intracellular survival rates in vitro, organ invasion rates in vivo, and epidemiologically-established human attack rates for the corresponding serotype. The amount of capsule produced and specific biochemical features of a serotype, such as acetylation, also influenced Spn resistance to oxidative stress. Autolysin-mediated shedding was also found to be necessary, indicating that capsule worked as a distal sink for reactive oxygen species. Our results outline a new role for capsular polysaccharide, as an intracellular antioxidant. They help to explain why certain serotypes of Spn have greater propensity for human disease.

microbiology↗