Biology subjects

Jackson, A.

Publications and source records attributed to Jackson, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Restricted expression site use and extreme genome diversification drives trypanosome antigenic variation in chronic bovine infections

Trypanosoma brucei exploits an extreme form of antigenic variation to escape the mammalian immune response. This involves the progressive expression of antigenically distinct variant surface glycoproteins (VSGs) on the surface of individual parasites in the population, generating waves of parasitaemia that are successively cleared by host antibodies. Current paradigms were established using in vitro studies and acute rodent infections characterized by high parasitaemia, but natural livestock infections are characterized by low parasitaemia and chronicity. Here, we analysed the infection dynamics of isogenic parasites in mice and cattle in blood during early and chronic infections, quantitating VSG expression diversity within and between hosts, antigen type persistence in vivo and their timing of appearance. This revealed enhanced antigenic diversity in cattle but with a surprisingly reproducible temporal expression hierarchy of related VSGs between independent chronic infections. Analyses demonstrated the unexpected dominance of a single telomeric VSG expression site irrespective of host species and time of infection. Detailed prediction of mosaic VSG assembly reveals exceptional parasite genome diversification within infections involving extensive macro and micro-homology-based recombination to evolve the antigen repertoire. This diversity was restricted but not eliminated in homologous recombination mutants, which could nonetheless sustain chronic infections in mice. These data provide the first comprehensive insight into trypanosome antigenic variation in the clinically-relevant host.

microbiology↗

bioRxiv2026

Mapping immune cellular landscapes and vaccine responses across a spectrum of health and immunodeficiency.

Immune responses to infection and vaccination exhibit diversity between individuals that can be shaped by differences in their immune cell landscapes and the signalling, transcriptional, and genetic mechanisms that coordinate immune cell function. Specific antibody deficiency (SAD) and common variable immunodeficiency (CVID) are common forms of predominantly antibody deficiencies that result in poor responses to vaccination. While molecular and cellular causes of the immune dysfunction and poor vaccination responses for individuals with CVID have been reported, immune cell or molecular defects have not yet been identified in SAD. Here, we have used single-cell multi-omics to define the cellular landscapes, transcriptional states, adaptive immune repertoires and protein expression of patients with SAD and CVID before and after polysaccharide vaccination. We discovered that while SAD and CVID exhibit overlapping immune defects, including accumulation of exhausted NK memory cells and dysregulated expression of genes that mediate lipopolysaccharide sensing and clearance by monocytes, individuals with SAD have a unique expansion of cytotoxic CD4+ T cells that correlates with reduced regulatory T cells. In response to vaccination, we observed rapid changes in gene expression associated with lipopolysaccharide responses by monocytes and NF-kB pathway activation in B cells, and an apparent expansion of a CD95+ class-switched memory B cell population that does not occur in patients with lower antigen-specific responses. Together, our findings reveal cellular and molecular factors that underpin variability in vaccine responses and define SAD in a broader spectrum of immune dysfunction.

immunology↗

bioRxiv2026

Impact of Fluorophore and Epitope Position on Destabilized Reporter Performance in C. elegans

Determining spatial and temporal gene expression is crucial for understanding animal development and physiology. Promoter reporters are powerful tools used to dissect how cis-regulatory elements and trans-acting factors control gene expression. Many fluorescent proteins used in promoter reporters, however, have long half-lives (>24 hr) which limit the study of dynamic expression. Destabilizing sequences like PEST reduce the half-life of reporter proteins to provide a more representative readout of gene expression. mlt-11 is a putative protease inhibitor known to oscillate in expression at the mRNA and protein level, yet a mlt-11p::mNeonGreen::3xFLAG::PEST::tbb-2 3UTR promoter reporter did not detectably oscillate. We systematically dissected this transgene, finding that placement of a 3xFLAG tag adjacent to a PEST sequence severely hampered oscillatory expression of a mNeonGreen promoter reporter. Surprisingly, reporter designs that effectively oscillate with a GFP or mNeonGreen fluorophore fail to oscillate when mStayGold is used, with these reporters remaining detectable over 24 hours following promoter inactivation. In addition, other tested epitopes (Myc, ALFA, OLLAS) did not hamper PEST-dependent destabilization but led to varying levels of reporter expression. This study details key considerations for designing destabilized fluorescent promoter reporters.

molecular biology↗

bioRxiv2025

MLT-11 is necessary for C. elegans embryogenesis and conserved sequences play distinct roles in cuticle structure

Apical extracellular matrices (aECMs) are associated with many epithelia and many form a protective layer against biotic and abiotic threats in the environment. Despite their importance, we lack a deep understanding of their structure and dynamics in development and disease. C. elegans molting offers a powerful entry point to understanding developmentally programmed aECM remodeling. Here, we show that the poorly characterized putative protease inhibitor gene, mlt-11, is directly regulated by the NHR-23 transcription factor. We identify key cis-regulatory elements required for robust mlt-11 expression. An internal MLT-11::mNeonGreen translational fusion transiently localized to the aECM in the cuticle and embryo. MLT-11::mNeonGreen was also detected in lining openings to the exterior (vulva, rectum, mouth). mlt-11 is necessary to pattern all layers of the adult cuticle, and reduction of MLT-11 levels disrupted the barrier function of the cuticle. Deletion of conserved Kunitz protease inhibitor domains or intervening sequences produced a range of defects including either left or right rollers, and small separations of the cuticle along the length of the animal (microblisters). MLT-11 is processed into at least two fragments and internal and C-terminal mNeonGreen knock-ins display distinct localization patterns. Predicted mlt-11 null mutations caused fully penetrant embryonic lethality and elongation defects. Together, this work suggests that MLT-11 localizes similarly to pre-cuticle components and conserved sequences play distinct roles in promoting proper assembly of the aECM.

developmental biology↗

bioRxiv2025

TI-Toolbox: An Open-Source Software for Temporal Interference Stimulation Research

BackgroundTemporal interference stimulation is a novel non-invasive brain stimulation approach that promises selective targeting of deep brain structures while minimizing off-target cortical stimulation. Despite a growing interest in temporal interference applications, there is a need for integrated computational tools that seamlessly connect neuroimaging data preprocessing through montage optimization, field simulation, and analysis within a unified framework designed for translational and clinical research. MethodsWe developed TI-Toolbox, an open-source software platform that integrates established neuroimaging tools (dcm2niix, SimNIBS, FreeSurfer) with specialized algorithms for temporal interference research. The platform provides end-to-end workflows encompassing structural MRI preprocessing, volume conduction modeling, montage optimization, electric field simulation, and region-of-interest analysis. Both graphical user interface and command-line interface implementations ensure accessibility across user expertise levels. The platform employs containerized deployment via Docker to ensure reproducibility and cross-platform compatibility. ResultsTI-Toolbox successfully automates the complete temporal interference research pipeline, from DICOM conversion through final field analysis. The platform demonstrates robust performance across operating systems and provides standardized workflows that enhance reproducibility. Furthermore, our case studies support the validity of our HD-EEG mapping approach for montage standardization and the need for individualized modeling for exposure assessment. ConclusionsTI-Toolbox addresses critical infrastructure gaps in temporal interference research by providing researchers with a unified, validated platform that reduces technical barriers and accelerates translational research in non-invasive deep brain stimulation. HighlightsO_LIOpen-source platform unifying TI stimulation workflow end-to-end C_LIO_LIDocker deployment ensures reproducibility across operating systems C_LIO_LIHD-EEG electrode mapping preserves field characteristics C_LIO_LIStandardized model is sufficient for montage optimization C_LIO_LIAnatomical differences explain >40% of inter-individual variability C_LI

neuroscience↗

bioRxiv2025

A robust platform for recombinant production of animal venom toxin modulators of ion channels

Background and PurposePeptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions. Experimental Approach13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca2+ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding. Key ResultsMonovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK) toxins from spiders, and -toxins from scorpions, substantially surpassing the performance of unfused toxins or bivalent Fc-toxin fusions which gave low or no yield. Snake toxins targeting nicotinic acetylcholine receptors retained high single digit nanomolar inhibitory potency. Spider and scorpion toxins targeting the voltage-gated Na+ channel Nav1.7 retained pharmacological function and selectivity across a panel of five Nav subtypes, albeit with reduced potencies that did not exceed [~]70 nM. Conclusions and ImplicationsWe present a strategy for straightforward robust production of pure, monodisperse, and functional animal venom-derived toxins. This lowers the barrier to toxin production in a standard laboratory setting for follow-on engineering purposes.

pharmacology and toxicology↗

bioRxiv2025

Generation of a Synthetic Single Domain Antibody Library for Radiopharmaceutical Ligand Discovery

Single domain antibodies, often known as nanobodies, are versatile molecules with therapeutic and diagnostic applications, but they are primarily developed through immunization of camelids. This approach is not scalable by automation, not effective for non-immunogenic or toxic antigens, and prevents the use of modified scaffolds for altered pharmacokinetic properties. Synthetic libraries allow for pre-selection of a single domain framework tailored to its intended downstream use. One area of interest for these biologic vectors is radiopharmaceuticals. Ideal radiopharmaceutical pharmacokinetic properties differ from most traditional therapeutics, as short plasma circulation and rapid kidney clearance are necessary to avoid dose-limiting organ radiation. Although there are a growing number of nanobody radiopharmaceuticals in clinical trials, their frameworks and corresponding pharmacokinetic properties vary. One potential method for improving the development of novel single domain antibody radiopharmaceuticals is through synthetic libraries based on nanobodies with proven clinically acceptable pharmacokinetics. We developed a modular synthetic nanobody phage display vector based on the scaffold of the 2Rs15d nanobody that allows for manipulation of the binding and framework regions. Using this vector, we created a library of nanobodies with a randomized CDR2 containing over 1.7x106 unique sequences/{micro}L. As a proof-of-concept, we panned the library for nanobodies binding calreticulin (CALR), a protein critical in immunogenic cell death. One isolated clone, Cal3, has a measured affinity of 140 nM for CALR and is cross-reactive with mouse and human CALR. Using positron emission tomography (PET) imaging, the radiolabeled 64Cu-NOTA-Cal3 demonstrated CALR binding in vivo, representing the first reported synthetic nanobody characterized by PET imaging. This study demonstrates the feasibility of building and panning synthetic libraries for high-affinity radiopharmaceutical nanobodies as an alternative to immunized camelid libraries.

bioengineering↗

bioRxiv2025

Epigenome and transcriptome changes in KMT2D-related Kabuki syndrome Type 1 iPSCs, neuronal progenitors and cortical neurons

Kabuki syndrome type 1 (KS1) is a neurodevelopmental disorder caused by loss-of-function variants in KMT2D which encodes a H3K4 methyltransferase. The mechanisms underlying neurodevelopmental problems in KS1 are still largely unknown. Here, we track the epigenome and transcriptome across three stages of neuronal differentiation using patient-derived induced pluripotent stem cells (iPSCs) to gain insights into the disease mechanism of KS1. In KS1 iPSCs we detected significantly lower levels of functional KMT2D transcript and KMT2D protein, and lower global H3K4me1 and H3K4me2 levels. We identify loss of thousands of H3K4me1 peaks in iPSCs, neuronal progenitors (NPs) and early cortical neurons (CNs) in KS1. We show that the number of lost peaks increase as differentiation progresses. We also identify hundreds of differentially expressed genes (DEGs) in iPSCs, NPs and CNs in KS1. In contrast with the epigenomic changes, the number of DEGs decrease as differentiation progresses. Our analysis reveals significant enrichment of differentially downregulated genes in areas containing putative enhancer regions with H3K4me1 loss. We also identify a set of distinct transcription factor binding sites in differentially methylated regions and a set of DEGs related to KS1 phenotypes. We find that genes regulated by SUZ12, a subunit of Polycomb Repressive complex 2, are over-represented in KS1 DEGs at early stages of differentiation. In conclusion, we present a disease-relevant human cellular model for KS1 that provides mechanistic insights for the disorder and could be used for high throughput drug screening for KS1.

genetics↗

bioRxiv2025

H-NS is a conserved repressor of the type VI secretion system in Vibrio fischeri

The type VI secretion system (T6SS) is a broadly distributed interbacterial weapon found in both beneficial and pathogenic bacteria and can enhance a microbes ability to colonize a host. Vibrio fischeri is a beneficial symbiont of fish and squid and a model organism for T6SS function, which is activated in high-viscosity conditions. Previously, we isolated an hns mutant in a transposon screen to identify regulators of the T6SS in the fish symbiont V. fischeri MJ11. The hns gene encodes the DNA-binding protein, H-NS, a conserved global regulator of gene expression that aids in adaptation to changing environments. Quantitative transcriptomes of the hns mutant and parent strains grown in liquid or hydrogel media revealed hns is required for the global transcriptional changes that occur during transition from lower to higher viscosity conditions. Furthermore, T6SS gene transcripts are more abundant in the hns mutant in both conditions, suggesting H-NS represses T6SS in the parent. Single-cell fluorescence microscopy confirmed hns mutant cells make more T6SS weapons in both liquid and hydrogel medium, where the hns mutant is more proficient at killing a competitor strain, compared to the wild-type parent. Finally, disrupting the hns gene in additional light organ isolates resulted in a similar derepression of T6SS, indicating H-NS is a conserved repressor of this interbacterial weapon. This work furthers our understanding of the role of H-NS as a global regulator during environmental shifts in a host-associated bacterial symbiont and expands the list of species where H-NS represses T6SS to include V. fischeri.

microbiology↗

bioRxiv2025

DMEM and EMEM are suitable surrogate media to mimic host environment and expand leptospiral pathogenesis studies using in vitro tools

Pathogenic Leptospira species can survive and thrive in a wide range of environments. Distinct environments expose the bacteria to different temperatures, osmolarities, and amounts and sources of nutrition. However, leptospires are mostly cultured, in a laboratory setting under in vitro conditions that do not reflect natural environments. This constraint on laboratory cultures limits the applicability of in vitro studies to the understanding of even simple pathogenic processes. Here we report, investigate, and identify a medium and conditions that mimic the host environment during leptospirosis infection, expanding the available in vitro tools to evaluate leptospiral pathogenesis. We quantified genome-wide gene expression of pathogenic Leptospira interrogans cultured in different in vitro media compositions (EMJH, DMEM, EMEM, and HAN). Using EMJH as standard, we compared gene expression in these compositions to genome-wide gene expression gathered in a host environment: whole blood (WB) of hamsters after infection with pathogenic leptospires. Leptospires cultured in DMEM and EMEM media shared 40% and 47% of all differentially expressed genes (DEGs) of leptospires present within WB (FDR<0.01), while leptospires cultured in HAN media only shared 20% of DEGs with those from WB. Furthermore, gene and pathway expression of leptospires cultured on DMEM and EMEM media exhibited a better correlation with leptospires grown in WB, including promoting expression of a similar leptospiral lipid A profile to the one identified directly in host tissues. Taken together, these results indicate that commercial cell-culture media EMEM or DMEM are better surrogates for in vivo pathogenic studies than EMJH or HAN media in Leptospira. These alternative culture conditions, using media that are a standard supply worldwide, provide a reproducible and cost-effective approach that can accelerate research investigation and reduce the number of animal infections necessary for basic research of leptospirosis.

microbiology↗

bioRxiv2025

The fractionation dependence of tumor control in proton therapy for early-stage non-small cell lung cancer

PurposeThe relative biological effectiveness (RBE) of tumor control for proton beam therapy (PBT) compared to photon radiotherapy (RT) is typically assumed to be independent of fractionation. To test this, we modeled published PBT outcome results for early-stage non-small cell lung cancer (NSCLC) treatments across a range of fractionation schedules. Materials and MethodsAll published and analyzable cohorts were included (399 patients, 413 treated lesions). Two models were used to fit the data: a previously published tumor simulation model that fits photon RT results of NSCLC across all fractionation regimes and the Fowler LQ model with a kick-off time term. The treatment effect of each cohort was referenced to the photon equivalent dose through mechanistic model simulations in a 2 Gy/weekday scenario, with radiobiological parameters determined to simultaneously best-fit all fractionation results. The tumor control RBE of each published treatment schedule, compared to the modeled photon RT effect of the same schedule, was then estimated. ResultsFor cohorts whose treatments lasted less than three weeks (i.e., 12 fractions or less), the RBE of PBT was in the range of 1.08 to 1.11. However, for fractionated treatments stretching over four weeks or more (20-25 fractions), the relative effectiveness was much lower, with RBEs in the range of 0.82-0.89. This conclusion was unchanged using the simpler Fowler LQ + time model. ConclusionsThe proton RBE for hypo-fractionated schedules was 20-30% higher than for conventional schedules. The derived radiobiological parameters of PBT differ significantly from those of photon RT, indicating that PBT is influenced differentially by radiobiological mechanisms which require further investigation.

biophysics↗

bioRxiv2025

The SinR*SlrR heteromer attenuates transcription of a long operon of flagellar genes in Bacillus subtilis

During growth, Bacillus subtilis differentiates into subpopulations of motile individuals and non-motile chains, associated with dispersal and biofilm formation respectively. The two cell types are dictated by the activity of the alternative sigma factor SigD encoded as the penultimate gene of the 27 kb long fla/che flagellar operon. The frequency of SigD-ON motile cells is increased by the heteromeric transcription factor SwrA*DegU that activates the fla/che promoter. Conversely, the frequency of motile cells is decreased by the heteromeric transcription factor SinR*SlrR, but the mechanism and location of inhibition is poorly understood. Here, using ChIP-Seq analysis, we determine the binding sites of the SinR*SlrR heteromer on the genome. We identified two sites within the fla/che operon that were both necessary and sufficient to attenuate transcript abundance by causing premature termination upstream of the gene that encodes SigD. Thus, cell motility and the transition to biofilm formation depend on the expression of a long operon governed by two opposing heteromeric transcription factors that operate at two different stages of the transcription cycle. More broadly, our study serves as a model for transcription factors that control transcriptional elongation and the regulation of long operons in bacteria.

microbiology↗

bioRxiv2024

The conserved global regulator H-NS has a strain-specific impact on biofilm formation in Vibrio fischeri symbionts

Strain-level variation among host-associated bacteria often determines host range and the extent to which colonization is beneficial, benign, or pathogenic. Vibrio fischeri is a beneficial symbiont of the light organs of fish and squid with known strain-specific differences that impact host specificity, colonization efficiency, and interbacterial competition. Here, we describe how the conserved global regulator, H-NS, has a strain-specific impact on a critical colonization behavior: biofilm formation. We isolated a mutant of the fish symbiont V. fischeri MJ11 with a transposon insertion in the hns gene. This mutant formed sticky, moderately wrinkled colonies on LBS plates, a condition not known to induce biofilm in this species. A reconstructed hns mutant displayed the same wrinkled colony, which became smooth when hns was complemented in trans, indicating the hns disruption is causal for biofilm formation in MJ11. Transcriptomes revealed differential expression for the syp biofilm locus in the hns mutant, relative to the parent, suggesting biofilm may in part involve SYP polysaccharide. However, enhanced biofilm in the MJ11 hns mutant was not sufficient to allow colonization of a non-native squid host. Finally, moving the hns mutation into other V. fischeri strains, including the squid symbionts ES114 and ES401, and seawater isolate PP3, revealed strain-specific biofilm phenotypes: ES114 and ES401 hns mutants displayed minimal biofilm phenotypes while PP3 hns mutant colonies were more wrinkled than the MJ11 hns mutant. These findings together define H-NS as a novel regulator of V. fischeri symbiotic biofilm and demonstrate key strain specificity in that role. ImportanceThis work, which shows how H-NS has strain-specific impacts on biofilm in Vibrio fischeri, underscores the importance of studying multiple strains, even when examining highly conserved genes and functions. Our observation that knocking out a conserved regulator can result in a wide range of biofilm phenotypes, depending on the isolate, serves as a powerful reminder that strain-level variation is common and worthy of exploration. Indeed, uncovering the mechanisms of strain-specific phenotypic differences is essential to understand drivers of niche differentiation and bacterial evolution. Thus, it is important to carefully match the number and type of strains used in a study with the research question to accurately interpret and extrapolate the results beyond a single genotype. The additional work required for multi-strain studies is often worth the investment of time and resources, as it provides a broader view of the complexity of within-species diversity in microbial systems.

microbiology↗

bioRxiv2024

Brain-responsive music enables non-invasive, targeted and unobtrusive neurostimulation

ObjectiveWe are developing a new closed-loop brain stimulation method by embedding, within music, auditory elements that respond to the listeners brain activity. Here we show that this brain-responsive music has systematic and targeted effects on neural oscillations implicated in a variety of neurological and mental health disorders. ApproachWe recorded magnetoencephalogram (MEG) or electroencephalogram (EEG) signals from participants as they listened to music synthesized by commercial audio software. Brain signals were bandpass filtered, phase-shifted and used to control the timbre and/or timing of notes within the music. Main resultsListening to brain-responsive music induced peaks and troughs in spectral power at frequencies that depended systematically on the phase-shift applied to the brain signal. Phase-dependent modulation was greatest at the centre frequency of the filter. As a result, by calibrating these parameters we could achieve selective enhancement or suppression of either theta (5 Hz) or alpha (10 Hz) oscillations. Moreover, by chosing different sensor locations we could target power modulation to either frontal or temporal cortex. The phase-dependent power modulation observed with brain-responsive music was significantly attenuated when participants listened to identical music as a conventional, open-loop stimulus. Finally, we demonstrate that brain activity could be modulated by more complex compositions combining a variety of brain-responsive musical elements controlled by a wireless, wearable EEG headband suitable for home use. SignificanceBrain-responsive music provides an unobtrusive and targeted method of modulating neural oscillations in the listeners brain, and may enable both creative and therapeutic applications of Brain Computer Interface technologies.

neuroscience↗

bioRxiv2024

Development of antisense tools to study Bodo saltans and its intracellular symbiont

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non-model species with poor tools for genetic manipulation and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here we investigated the potential for using antisense inhibition to analyse host and symbiont gene function within an obligate aquatic symbiosis. We focused on the kinetoplastid host Bodo saltans and its bacterial symbiont, Candidatus Bodocaedibacter vickermanii, a member of Rickettsiales. We conclude that antisense inhibition is not feasible in the B. saltans and its symbiont, as the holobiont feeds on the antisense molecules - and increases in numbers - upon treatment with the antisense construct. Although our approach has proven unsuccessful, we have developed an array of protocols which can be used to study the biology of this microeukaryote and its microbial associates.

molecular biology↗

bioRxiv2024

MLT-11 is a transient apical extracellular matrix component required for cuticle patterning and function

C. elegans molting offers a powerful entry point to understanding developmentally programmed apical extracellular matrix remodeling. However, the gene regulatory network controlling this process remains poorly understood. Focusing on targets of NHR-23, a key transcription factor that drives molting, we confirmed the Kunitz family protease inhibitor gene mlt-11 as an NHR-23 target. Through reporter assays, we identified NHR-23-binding sites that are necessary and sufficient for epithelial expression. We generated a translational fusion and demonstrated that MLT-11 is localized to the cuticle and lined openings to the exterior (vulva, rectum, mouth). We created a set of strains expressing varied levels of MLT-11 by deleting endogenous cis-regulatory element sequences. Combined deletion of two cis-regulatory elements caused developmental delay, motility defects, and failure of the cuticle barrier. Inactivation of mlt-11 by RNAi produced even more pronounced defects. mlt-11 is necessary to pattern every layer of the adult cuticle, suggesting a broad patterning role prior to the formation of the mature cuticle. Together these studies provide an entry point into understanding how individual cis-regulatory elements function to coordinate expression of oscillating genes involved in molting and how MLT-11 ensures proper cuticle assembly.

developmental biology↗

bioRxiv2024

Flagella are required to coordinately activate competition and host colonization factors in response to a mechanical signal

Bacteria employ antagonistic strategies to eliminate competitors of an ecological niche. Contact-dependent mechanisms, such as the type VI secretion system (T6SS), are prevalent in host-associated bacteria, yet we know relatively little about how T6SS+ strains make contact with competitors in highly viscous environments, such as host mucus. To better understand how cells respond to and contact one another in such environments, we performed a genome-wide transposon mutant screen of the T6SS-wielding beneficial bacterial symbiont, Vibrio fischeri, and identified two sets of genes that are conditionally required for killing. LPS/capsule and flagellar-associated genes do not affect T6SS directly and are therefore not required for interbacterial killing when cell contact is forced yet are necessary for killing in high-viscosity liquid (hydrogel) where cell-cell contact must be biologically mediated. Quantitative transcriptomics revealed that V. fischeri significantly increases expression of both T6SS genes and cell surface modification factors upon transition from low-to high-viscosity media. Consistent with coincubation and fluorescence microscopy data, flagella are not required for T6SS expression in hydrogel. However, flagella play a key role in responding to the physical environment by promoting expression of the surface modification genes identified in our screen, as well as additional functional pathways important for host colonization including uptake of host-relevant iron and carbon sources, and nitric oxide detoxification enzymes. Our findings suggest that flagella may act as a mechanosensor for V. fischeri to coordinately activate competitive strategies and host colonization factors, underscoring the significance of the physical environment in directing complex bacterial behaviors. SignificanceThe physical environment can have dramatic effects on bacterial behavior, but little is known about how mechanical signals impact antagonistic interactions. Symbiotic bacteria use molecular weapons to eliminate competitors for limited space within highly viscous host tissue and mucus. To better understand how the physical environment affects competition and adhesion within eukaryotic hosts, we used quantitative transcriptomics to reveal the flagella-dependent transcriptional response to bacterial transition from lower to a higher viscosity environment. This work revealed the T6SS interbacterial weapon is coordinately activated with host colonization factors, emphasizing the importance of integrating activation of interbacterial weapons into host colonization pathways to enhance a symbionts ability to successfully colonize the host while efficiently eliminating potential competitors from the host niche.

microbiology↗

bioRxiv2023

Real time quantification of apical polarity protein reveals novel dynamic processes in luminal network establishment and remodeling in the pancreas

During embryogenesis dynamic changes in tissue architecture transform primitive anlages to functional organs. Here we document in real time how pancreatic lumens are derived and transformed using a new apical-polarity mouse reporter. Our 4D imaging data reveals dynamic remodeling of apical proteins and lumens to primarily drive each stage of pancreatic duct development. Furthermore, we pinpoint two unique transitions during lumenogenesis. Contrary to current "de novo" models of polarity acquisition, we show that expansion and rearrangement of the pre-existing central primary lumen drives early network growth. We also document how the endocrine promoting niche - a "plexus" of interconnected ducts - is resolved. We show that an arborized network forms by gradual closing of ductal loops, rather than via pruning. These novel tissue dynamics provide a new framework within which cell and molecular signaling can be investigated to better understand the interplay between organ architecture and cell fate.

developmental biology↗