Biology subjects

Holland, S.

Publications and source records attributed to Holland, S..

16 recordsLinked to original sources

bioRxiv2026

From Magnetosomes to Vesicles: LemA Proteins as Drivers of Bacterial Membrane Reorganization

The cellular machineries that remodel bacterial membranes during organelle and vesicle formation remain poorly defined. Herein, we report MamQ as a key determinant of the lipid invaginations that enclose the magnetosome. MamQ belongs to the broadly conserved LemA protein family, whose members localize to either the inner or outer membrane. Heterologous overproduction of 13 different LemA proteins in Escherichia coli induced extensive membrane restructuring and vesiculation, indicating a conserved membrane-remodelling activity. In Pseudomonas aeruginosa, overproduction of the inner membrane protein LemA1 (PaLemA1) promoted cytoplasmic membrane vesicle (CMV) formation, whereas the periplasmic-located outer membrane-anchored LemA2 (PaLemA2) drove the formation of bacterial outer-membrane vesicles (OMVs). Conversely, deletion of the corresponding genes reduced extracellular vesicle production. Interactome analyses identified 256 protein partners of PaLemA1 and PaLemA2, including multiple components of the cellular divisome, such as CpoB, FtsE, FtsI, FtsK, FtsN, FtsW, MinE, ParB, ZapG, ZipA. Bacterial two-hybrid assays confirmed interactions between both LemA proteins and the cell division regulator CpoB, whose perturbation markedly altered vesicle formation. Additional interaction partners included PspA, an ESCRT-III-like protein implicated in membrane remodelling. Proteins involved in cell wall metabolism, membrane turnover, lipopolysaccharide (LPS) biogenesis, protein export and maturation, energy metabolism, and stress-responsive gene regulation were also enriched, suggesting a supporting role for PaLemA proteins in coordinating membrane-associated processes. Consistent with this, LemA deletion mutants displayed altered vesicle composition and global changes in gene expression. Furthermore, PalemA2 expression was induced in stationary phase and under envelope stress via the extracytoplasmic sigma factor AlgU, itself an interaction partner of the LemA proteins. Together, our findings indicate the LemA family act as a conserved membrane-remodelling system that, in conjunction with PspA, CpoB and divisome components, organises membrane-localized protein complexes required for bacterial organelle biogenesis and vesicle formation while executing a controlled, protein-machinery-driven mechanism that operates independently of passive chemical blebbing (such as hydrophobic PQS intercalation) or lytic membrane fragmentation.

microbiology↗

bioRxiv2025

State-dependent release of extracellular particles with distinct α2,6-sialylation patterns and small RNA cargo related to neuroinflammation

Neuroinflammation is a significant contributor to neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, and related dementias; yet peripheral biomarkers for neuroinflammation remain an unmet medical need. Microglia, the resident immune cells of the central nervous system, play a dual role in maintaining homeostasis under physiological conditions and driving neuronal damage when chronically dysregulated. One mechanism by which microglia influence their environment is through the release of extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs), which can serve as biomarkers the reflect cellular states. Here, we systematically isolated and characterized microglia-derived EVs and NVEPs under pro- and anti-inflammatory conditions and profiled their small RNA cargo by small RNA sequencing. We validated these findings in human iPSC-derived microglia and further recapitulated them in EVs and NVEPs from mouse brain and plasma. Using an engineered mouse model, we were able to isolate plasma microglia-specific EVs in vivo and demonstrated that their RNA cargo reflects their inflammatory state. Importantly, microglial EVs and NVEPs display distinct 2,6-sialylation patterns and small RNA signatures implicated in neurological diseases. These findings demonstrate that microglia-derived EVs and NVEPs cargo reflect microglial cellular state and establish them as putative minimally non-invasive biomarkers of early-stage neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Co-circulation of distinct high pathogenicity avian influenza virus (HPAIV) subtypes in a mass mortality event in wild seabirds and co-location with dead seals

H5Nx clade 2.3.4.4b high pathogenicity avian influenza viruses (HPAIV) have been detected repeatedly in Great Britain (GB) since autumn 2020, with H5N1 dominating detections but with low level detection of H5N5 during 2025. Globally, these viruses have caused mass mortalities in captive and wild avian and mammalian populations, including terrestrial and marine mammals. H5N1 has been the dominant subtype, and whilst incursions have overlapped temporally, occurrences have often been spatially distinct. Here, we report the detection of a mortality event in wild birds on the Norfolk coastline in the east of England, where H5N1 HPAIV was detected in five Great Black-backed Gulls (Larus marinus) and a Northern Fulmar (Fulmarus glacialis). Interestingly, at the same site, and as part of the same mortality event, a total of 17 Great Black-backed Gulls, one Herring Gull (Larus argentatus), one Atlantic Puffin (Fratercula arctica) and one Northern Fulmar tested positive for H5N5 HPAIV. Additionally, H5N5 was also detected in 17 co-located Grey Seal carcases (Halichoerus grypus). The H5N1 HPAIV from an infected bird belonged to genotype DI.2, closely related to contemporaneous detections in GB wild birds and poultry. In contrast, all H5N5 HPAIVs from birds and seals were genotype I with a 22-amino acid stalk deletion in NA and the 627K polymorphism in PB2. This represents the first recorded instance in GB of two subtypes being detected within the same avian population at the same location. It is also the first mass detection of HPAIV H5N5 in mammals within GB. Potential infection mechanisms are discussed.

genetics↗

bioRxiv2025

Gut microbiota phospholipids regulate intestinal gene expression and can counteract the effects of antibiotic treatment

The gut microbiome influences immune and metabolic homeostasis. Our research using non-obese diabetic (NOD) mice revealed that early-life antibiotic exposure remodels the gut microbiome affecting metabolism and accelerating type 1 diabetes (T1D) incidence, with cecal material transplant (CMT) mitigating the damage. Now examining murine intestinal lipidomic profiles, we identified 747 compounds. Comparing the lipidomic profiles of cecal contents of conventional and germ-free mice and their diets, we identified 87 microbially-produced lipids reduced by antibiotic exposure but CMT-restored. Parallel analysis of human fecal lipid profiles after azithromycin-exposure showed significant alterations with substantial overlap with mice. In vitro co-culture with mouse macrophages or small intestinal epithelial cells and human colonic epithelial cells identified phospholipids that repress inflammation through the NF{kappa}B pathway. Oral administration of these phospholipids to antibiotic-treated NOD mice reduced expression of ileal genes involved in early stages of T1D pathogenesis. These findings indicate potential therapeutic anti-inflammatory roles of microbially-produced lipids.

microbiology↗

bioRxiv2024

The LYCOPENE EPSILON CYCLASE untranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

Metabolic feedback is proposed to modulate nuclear gene expression and carotenoid biosynthesis in plastids, however few mechanisms have been identified so far in plants. Utilising mutants, overexpression lines, and chemical inhibitors, we demonstrate that Arabidopsis LYCOPENE EPSILON CYCLASE ({varepsilon}LCY) mRNA levels correlate with changes in {beta}-carotenoid accumulation. Transgenic seedlings harbouring the{varepsilon} LCY 5 leader sequence fused to FIREFLY LUCIFERASE (FiLUC) showed reporter responsiveness to metabolic feedback triggered by norflurazon or loss-of-function in the CAROTENOID ISOMERASE (CRTISO). The{varepsilon} LCY 5UTR harboured three alternative transcription start sites (TSS). The most abundant -133bp sequence generated in dark and light grown seedlings harboured a 5 conserved domain (CD) with other Brassicaceae species and a viral internal ribosome entry site (IRES) proximal to the start codon. In silico modelling predicted the 5UTR formed two energetically separated RNA structural probabilities having a minimal free energy consistent with metabolite-binding RNA riboswitches that was distinguished by hairpin structures within the CD. Site-specific mutations were used to stabilize the 5UTR into a single RNA shape definition having negligible separation between the mountain plot structure prediction curves and a distal terminator-like hairpin structure. Stabilizing the 5UTR shape triggered the posttranscriptional repression of FiLUC activity enabled by the CaMV35S promoter in tobacco transient assays and stable transgenic Arabidopsis lines. The stabilised shape fragment became responsive to metabolic feedback induced by norflurazon and in crtiso mutant etiolated and de-etiolated seedlings. The{varepsilon} LCY 5UTR resembles a conformational RNA regulatory switch harbouring a posttranscriptional expression platform and aptamer domain responsive to carotenoid-mediated feedback signalling.

plant biology↗

bioRxiv2024

Bacterial membrane vesicles of Pseudomonas aeruginosa activate AMPK signaling through inhibition of mitochondrial complex III

Bacterial membrane vesicles (BMVs) are secreted by many pathogenic bacteria and known to stimulate various host responses upon infection, thereby contributing to the pathogenicity of bacterial pathogens like Pseudomonas aeruginosa. While the effects of BMVs on host immune responses are well studied, little is known about their impact on cell metabolism and mitochondrial respiration. Here, we show that P. aeruginosa BMVs (1) reprogram cell metabolism of human lung cells, (2) negatively affect mitochondrial respiration by (3) specifically inhibiting complex III of the electron transport chain leading to (4) the activation of AMP-activated protein kinase (AMPK) signaling which in turn results in (5) AMPK-dependent inhibition of global protein synthesis.

cell biology↗

bioRxiv2024

In situ, High-Resolution Quantification of CO2 Uptake Rates via Automated Off-Gas Analysis Illuminates Carbon Uptake Dynamics in Cyanobacterial Cultures

Quantification of CO2 fixation rates by cyanobacteria is vital to determining their potential as industrial strains in a circular bioeconomy. Currently, however, CO2 fixation rates are most often determined through indirect and/or low-resolution methods, resulting in an incomplete picture of both dynamic behaviors and total carbon fixing potential. To address this, we developed a novel, low-cost system for in situ off-gas analysis which supports the automated acquisition of high-resolution and CO2 uptake rates from cyanobacterial cultures. Carbon fixation data obtained via this system was independently verified by elemental analysis of cultivated biomass. Using Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, we demonstrate that phototrophic CO2 uptake rates accelerate linearly to a maximum before then decaying monotonically to cessation by stationary phase. Furthermore, consistent with the expected stoichiometry, we found that strong correlations exist between both the rates and total levels of cell growth and carbon fixation. This system simultaneously provides a high-resolution growth curve, accurate carbon fixation rates, as well as the total amount of carbon fixed in a cyanobacterial batch culture, thus illuminating the parameters with which cyanobacterial researchers can exploit use to realize their full potential in industrial applications.

microbiology↗

bioRxiv2024

Microbial aerotrophy enables continuous primary production in diverse cave ecosystems

Most aerated cave ecosystems are assumed to be oligotrophic given they receive minimal inputs of light energy. Diverse microorganisms have nevertheless been detected within caves, though it remains unclear what strategies enable them to meet their energy and carbon needs. Here we determined the processes and mediators of primary production in aerated limestone and basalt caves through paired metagenomic and biogeochemical profiling. Based on 1458 metagenome-assembled genomes, over half of microbial cells in caves encode enzymes to use atmospheric trace gases as energy and carbon sources. The most abundant microbes in these systems are chemosynthetic primary producers, notably the novel gammaproteobacterial methanotrophic order Ca. Methylocavales and two uncultivated actinobacterial genera predicted to grow on atmospheric hydrogen, carbon dioxide, and carbon monoxide. In situ and ex situ biogeochemical and isotopic measurements consistently confirmed that these gases are rapidly consumed at rates sufficient to meet community-wide energy needs and drive continual primary production. Conventional chemolithoautotrophs, which use trace lithic compounds such as ammonium and sulfide, are also enriched and active alongside these trace gas scavengers. These results indicate that caves are unique in both their microbial composition and the biogeochemical processes that sustain them. Based on these findings, we propose caves are the first known ecosystems where atmospheric trace gases primarily sustain growth rather than survival and define this process as aerotrophy. Cave aerotrophy may be a hidden process supporting global biogeochemistry.

microbiology↗

bioRxiv2024

Evolution towards extremely high β-lactam resistance in Mycobacterium abscessus outbreak strains

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous {beta}-lactams (e.g., imipenem, meropenem). M. abscessus produces a {beta}-lactamase (BlaMab) that inactivates {beta}-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 {micro}g/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the {beta}-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in {beta}-lactamase activity and increased blaMab mRNA level. Concurrent increase in transcription of preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 {micro}g/ml). A frameshift mutation in msp2 responsible for the rough colony morphology, and a SNP in ATP-dependent helicase hrpA co-occurred with the second increase in MIC (from 32 to 256 {micro}g/ml). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-blaMab operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/{beta}-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem resistant strains.

microbiology↗

bioRxiv2024

Peptide Amphiphiles Hitchhike on Endogenous Biomolecules for Enhanced Cancer Imaging and Therapy

The interactions of nanomaterials with biomolecules in vivo determine their biological fate. Here, we show that self-assembled peptide amphiphile (PA) nanostructures can dynamically interact with endogenous biomolecules and take advantage of naturally occurring processes to target a broad range of solid tumors. In circulation, self-assembled PA nanostructures disassemble and reassemble mainly with lipoproteins, which prolongs blood circulation and dramatically improves tumor accumulation and retention. Mechanistic studies suggested that PAs internalize into cancer cells by assembling with their cell membranes and independently of specific receptors. By exploiting these interactions, a PA developed in this study (namely SA-E) demonstrated specific accumulation in various xenograft, syngeneic, patient-derived xenograft, or transgenic rodent models. In addition, SA-E enabled the effective delivery of highly potent chemotherapy to different syngeneic and xenografted tumors with reduced side effects. With its simple and modular design and universal tumor accumulation mechanism, SA-E represents a promising platform for broad applications in cancer imaging and therapy.

bioengineering↗

bioRxiv2023

Synergistic Effects of Sulopenem in Combination with Cefuroxime or Durlobactam against Mycobacterium abscessus

Mycobacterium abscessus (Mab) affects patients with immunosuppression, Cystic Fibrosis (CF), or underlying structural lung diseases. Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual {beta}-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with a {beta}-lactamase inhibitors [BLI; avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration MIC values for 54 clinical Mab isolates and ATCC19977 (MIC50 and MIC90 [≤] 0.25 g/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding [~]1.5 Log10 CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in additional reduction of CFUs ([~]3 Log10 for sulopenem-CXM and AVI and [~]4 Log10 for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (LDTs; LDTMab1-LDTMab4), Penicillin-Binding-Protein B (PBP-B), and D,D-Carboxypeptidase (DDC). Acyl complexes identified via mass spectrometry analysis, demonstrated the binding of sulopenem with LdtMab2-LdtMab4, DDC, and PBP B, and CXM with LdtMab2 and PBP-B. Molecular docking suggested formation of a covalent adduct between sulopenem and LdtMab2 after the nucleophilic attack of the cysteine residue at the {beta}-lactam carbonyl carbon, leading to the cleavage of the {beta}-lactam ring, and the establishment of a thioester bond linking the LdtMab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLI. These findings potentially broaden selection of oral therapeutic agents to combat Mab.

microbiology↗

bioRxiv2023

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

microbiology↗

bioRxiv2022

ImmunoTyper-SR: A Novel Computational Approach for Genotyping Immunoglobulin Heavy Chain Variable Genes using Short Read Data

Human immunoglobulin heavy chain (IGH) locus on chromosome 14 includes more than 40 functional copies of the variable gene (IGHV), which, together with the joining genes (IGHJ), diversity genes (IGHD), constant genes (IGHC) and immunoglobulin light chains, code for antibodies that identify and neutralize pathogenic invaders as a part of the adaptive immune system. Because of its highly repetitive sequence composition, the IGH locus has been particularly difficult to assemble or genotype through the use of standard short read sequencing technologies. Here we introduce ImmunoTyper-SR, an algorithmic method for genotype and CNV analysis of the germline IGHV genes using Illumina whole genome sequencing (WGS) data. ImmunoTyper-SR is based on a novel combinatorial optimization formulation that aims to minimize the total edit distance between reads and their assigned IGHV alleles from a given database, with constraints on the number and distribution of reads across each called allele. We have validated ImmunoTyper-SR on 12 individuals with Illumina WGS data from the 1000 Genomes Project, whose IGHV allele composition have been studied extensively through the use of long read and targeted sequencing platforms, as well as nine individuals from the NIAID COVID Consortium who have been subjected to WGS twice. We have then applied ImmunoTyper-SR on 585 samples from the NIAID COVID Consortium to investigate associations between distinct IGHV alleles and anti-type I IFN autoantibodies which have been linked to COVID-19 severity.

bioinformatics↗

bioRxiv2022

ARZIMM: A Novel Analytic Platform for the Inference of Microbial Interactions and Community Stability from Longitudinal Microbiome Study

Dynamic changes of microbiome communities may play important roles in human health and diseases. The recent rise in longitudinal microbiome studies calls for statistical methods that can model the temporal dynamic patterns and simultaneously quantify the microbial interactions and community stability. Here, we propose a novel autoregressive zero-inflated mixed-effects model (ARZIMM) to capture the sparse microbial interactions and estimate the community stability. ARZIMM employs a zero-inflated Poisson autoregressive model to model the excessive zero abundances and the non-zero abundances separately, a random effect to investigate the underlining dynamic pattern shared within the group, and a Lasso-type penalty to capture and estimate the sparse microbial interactions. Based on the estimated microbial interaction matrix, we further derive the estimate of community stability, and identify the core dynamic patterns through network inference. Through extensive simulation studies and real data analyses we evaluated ARZIMM in comparison with the other methods.

bioinformatics↗

bioRxiv2021

Genetically diverse mouse models of SARS-CoV-2 infection model clinical variation and cytokine responses in COVID-19

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity. One Sentence SummaryGenetically diverse mice model a spectrum of clinically relevant innate immune responses to SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17

After optic nerve crush (ONC), the cell bodies and distal axons of most retinal ganglion cells (RGCs) degenerate. RGC somal and distal axon degeneration were previously thought to be controlled by two distinct pathways, involving activation of the kinase DLK and loss of the axon survival factor NMNAT2, respectively. However, we found that mutual palmitoylation by the palmitoyl acyltransferase ZDHHC17 couples the DLK and NMNAT2 signals, which together form a "trust, but verify system". In healthy optic nerves, ZDHHC17-dependent palmitoylation ensures NMNAT-dependent distal axon integrity, while following ONC, ZDHHC17-dependent palmitoylation is critical for DLK-dependent somal degeneration. We found that ZDHHC17 also controls survival-versus-degeneration decisions in sensory neurons and identified motifs in NMNAT2 and DLK that govern their ZDHHC17-dependent regulation. These findings suggest that the control of somal and distal axon integrity should be considered as a single, holistic process, involving two palmitoylation-dependent pathways acting in concert.

neuroscience↗