Biology subjects

Marshall, M.

Publications and source records attributed to Marshall, M..

8 recordsLinked to original sources

bioRxiv2024

Spatial transcriptomics identifies novel P. aeruginosa virulence factors

To holistically unravel the complexity of pathogen-host interactions within infected tissues we leverage a dual spatial transcriptomic approach that, for the first time, simultaneously captures the expression of Pseudomonas aeruginosa genes alongside the entire host transcriptome in a model of ocular infection. This innovative method reveals differential pathogen and host-specific gene expression patterns across specific anatomical regions generating a unified transcriptional map of infection. By integrating these data, we developed a predictive ridge regression model trained on images from infected tissues. The model achieved an R{superscript 2} score of 0.923 in predicting bacterial burden distributions by using host features thereby predicting novel biomarkers associated with disease severity. Our analysis revealed a complex interplay between P. aeruginosa nutritional requirements and protective host responses and identified novel interactions between bacterial metabolite transport proteins and host autophagy. Among an array of iron acquisition gene transcripts that showed significant enrichment at the host-pathogen interface, we discovered a novel virulence mediator PA2590. This study highlights the power of spatial transcriptomics, particularly in combining bacterial and host transcriptomes, to uncover novel host-pathogen interactions, advance our understanding of bacterial virulence mechanisms, and point to druggable molecules.

microbiology↗

bioRxiv2024

Two-hit mouse model of heart failure with preserved ejection fraction combining diet-induced obesity and renin-mediated hypertension

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We have developed a novel "2-hit" model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57BL6/NJ mice fed a high fat diet for >10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d. Control mice, HFD only, Renin only and HFD-Renin (aka "HFpEF") littermates underwent a battery of cardiac and extracardiac phenotyping. HFD-Renin mice demonstrated obesity and insulin resistance, a 2-3-fold increase in circulating renin levels that resulted in 30-40% increase in left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by altered E/e, IVRT, and strain measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to findings in human HFpEF. Treatment of these mice with the sodium-glucose cotransporter 2 inhibitor empagliflozin, an effective but incompletely understood HFpEF therapy, improved exercise tolerance, left heart enlargement, and insulin homeostasis. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Addition of HFD-Renin mice to the preclinical HFpEF model platform allows for orthogonal studies to increase validity in assessment of interventions. NEW & NOTEWORTHYHeart failure with preserved ejection fraction (HFpEF) is a complex disease to study due to limited preclinical models. We rigorously characterize a new two-hit HFpEF mouse model, which allows for dissecting individual contributions and synergy of major pathogenic drivers, hypertension and diet-induced obesity. The results are consistent and reproducible in two independent laboratories. This high-fidelity pre-clinical model increases the available, orthogonal models needed to improve our understanding of the causes and assessment treatments for HFpEF.

physiology↗

bioRxiv2022

mRNA-mediated induced regeneration of the corneal endothelium

Loss of vision due to corneal endothelial dysfunction affects millions worldwide. The development of new treatments is hampered by the incomplete knowledge of the regenerative capacity of corneal endothelial cells in vivo. Herein, we developed a mouse model to directly monitor corneal endothelial regeneration in real time, and at the single cell level, by two-photon microscopy. We show that the mouse corneal endothelium recapitulates the main features of human endothelial physiology, including complete cellular quiescence and a decline in cell density with aging. Critically, we demonstrate the endogenous regenerative potential of the tissue by capturing the proliferation of corneal endothelial cells during repair of large injuries. By single cell lineage tracing analysis, we provide evidence that corneal endothelial cells are equipotent in their ability to activate the cell cycle and contribute to tissue regeneration. Based on these findings we developed a feasible therapeutic approach to stimulate the regeneration of the corneal endothelium, using modified mRNA technology. To reprogram corneal endothelial cells in vivo and unlock their ability to escape quiescence, we combined five modified mRNAs encoding for proteins involved in cell cycle activation. Injection of the encapsulated mRNAs directly into the eye of older mice induced transient proliferation of corneal endothelial cells that led to an increase in endothelial cell density, effectively reversing the effect of aging. This therapeutic strategy offers a compelling paradigm for treating ocular disease and modulating tissue regeneration in organs with limited endogenous ability.

cell biology↗

bioRxiv2022

Stress vesicles are induced by acute mechanical force and precede the commitment of epidermal stem cells to terminal differentiation

The skin has a pronounced ability to adapt to physical changes in the environment by exhibiting plasticity at the cellular level. Transient mechanical deformations applied to the skin are accommodated without permanent changes to tissue structure. However, sustained physical stress induces long-lasting alterations in the skin, which are mediated by shifts in the fates of epidermal stem cells. To investigate this phenomenon, we implemented two-photon intravital imaging to capture the responses of epidermal cells when an acute mechanical force is applied to the live skin. We show that mechanical stress induces the formation of intracellular vesicles in epidermal stem cells, which are filled with extracellular fluid and gradually enlarge, causing the deformation of the cell nucleus. By lineage tracing analysis we demonstrate that the degree of nuclear deformation is linked to cell fate. Utilizing a fluorescent in vivo reporter, to capture intracellular calcium dynamics, we show that mechanical force induces a sustained increase in intracellular calcium within basal epidermal stem cells. Conditional deletion of Piezo1, a mechanosensitive ion channel, alters intracellular calcium dynamics and increases the number of stress vesicles in epidermal stem cells. Using a human skin xenograft model, we show that stress vesicles are a conserved phenomenon in mammalian skin. This study uncovers stress vesicles as key manifestations of the mechanism that regulates the fate of epidermal stem cells under conditions of mechanical stress, in which Piezo1 and calcium dynamics are also involved.

cell biology↗

bioRxiv2022

Neutrophil IL-1β secretion induced by ExoS expressing Pseudomonas aeruginosa is dependent on NLRP3 and Gasdermin D

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1{beta} secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1{beta} secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ({Delta}exoST) stimulated NLRC4 for IL-1{beta} secretion. While IL-1{beta} release from{Delta} exoST infected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model of P. aeruginosa corneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

microbiology↗

bioRxiv2020

Control of ss-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence

Candida albicans is a major cause of invasive candidiasis, which has a high mortality rate. The hyphal form of C. albicans is virulent and activates the host innate immune response, while the yeast form is hypovirulent and less immunogenic. The innate immune response is critical for host defense, but overactivation can cause tissue damage and sepsis. The innate immune response can be triggered when the C-type lectin receptor Dectin-1 recognizes {beta}-glucans, which is protected by the outer mannan layer of the cell wall on C. albicans. Here, we demonstrate that there is low level of Dectin-1 binding at the septum of yeast cells, but high level of Dectin-1 binding over the entire surface of hyphae. We find that {beta}-glucan masking in yeast is controlled by two highly expressed yeast proteins, the endo-1,3-{beta}-glucanase Eng1 and the Yeast Wall Protein Ywp1. An eng1 deletion mutant shows enhanced Dectin-1 binding at the septa, while an eng1 ywp1 double mutant, but not an ywp1 single mutant, shows strong overall Dectin-1 binding. Thus, Eng1-mediated {beta}-glucan trimming and Ywp1-mediated {beta}-glucan masking are two parallel mechanisms utilized by C. albicans yeast to minimize recognition by Dectin-1. In the model of disseminated candidiasis, mice infected with the eng1 deletion mutant showed delayed mortality with an increased renal immune response in males compared to mice infected with the wild-type strain, but earlier mortality with a higher renal immune response in females. Using the eng1 mutant that is specifically defective in {beta}-glucan masking in yeast, this study demonstrates that the level of {beta}-glucan exposure is important for modulating the balance between immune protection and immunopathogenesis. Abstract ImportanceCandida albicans is a major opportunistic fungal pathogen of humans. Systemic Candidiasis has high mortality rates. C. albicans is also a constituent of the human microbiome and found in gastrointestinal and genitourinary tracts of most healthy individuals. C. albicans is able to switch reversibly between yeast and hyphae in response to environmental cues. The hyphal form is virulent, while the yeast form is hypovirulent and less immunogenic. This study demonstrates that {beta}-glucan exposure in yeast is protected by two highly expressed yeast proteins, the endo-1,3-{beta}-glucanase Eng1 and the Yeast Wall Protein Ywp1. Eng1-mediated {beta}-glucan trimming and Ywp1-mediated {beta}-glucan masking are two parallel mechanisms utilized by C. albicans yeast to minimize recognition by the host C-type lectin receptor Dectin-1. The eng1 mutant triggers a higher immune response and leads to earlier mortality compared to the wild-type strain. Thus, {beta}-glucan masking in yeast keeps yeast cells less immunogenic and hypovirulent.

microbiology↗

bioRxiv2020

A novel mutation in KCNK16 causing a gain-of-function in the TALK-1 potassium channel: a new cause of maturity onset diabetes of the young.

BackgroundMaturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired glucose-stimulated insulin secretion (GSIS). Mechanisms include {beta}-cell KATP channel dysfunction (e.g., KCNJ11 (MODY13) or ABCC8 (MODY12) mutations); however, no other {beta}-cell channelopathies have been identified in MODY. MethodsA four-generation family with autosomal dominant non-obese, non-ketotic antibody-negative diabetes, without mutations in known MODY genes, underwent exome sequencing. Whole-cell and single-channel K+ currents, Ca2+ handling, and GSIS were determined in cells expressing either mutated or wild-type (WT) protein. ResultsWe identified a novel non-synonymous genetic mutation in KCNK16 (NM_001135105: c.341T>C, p.Leu114Pro) segregating with MODY. KCNK16 is the most abundant and {beta}-cell-restricted K+ channel transcript and encodes the two-pore-domain K+ channel TALK-1. Whole-cell K+ currents in transfected HEK293 cells demonstrated drastic (312-fold increase) gain-of-function with TALK-1 Leu144Pro vs. WT, due to greater single channel activity. Glucose-stimulated cytosolic Ca2+ influx was inhibited in mouse islets expressing TALK-1 Leu114Pro (area under the curve [AUC] at 20mM glucose: Leu114Pro 60.1 vs. WT 89.1; P=0.030) and less endoplasmic reticulum calcium storage (cyclopiazonic acid-induced release AUC: Leu114Pro 17.5 vs. WT 46.8; P=0.008). TALK-1 Leu114Pro significantly blunted GSIS compared to TALK-1 WT in both mouse (52% decrease, P=0.039) and human (38% decrease, P=0.019) islets. ConclusionsOur data identify a novel MODY-associated gene, KCNK16; with a gain-of-function mutation limiting Ca2+ influx and GSIS. A gain-of-function common polymorphism in KCNK16 is associated with type 2 diabetes (T2DM); thus, our findings have therapeutic implications not only for KCNK16-associated MODY but also for T2DM.

genetics↗

bioRxiv2019

A rare SMAD9 mutation identifies the BMP signalling pathway as a potential osteoanabolic target

To identify targets for novel anabolic medicines for osteoporosis, we recruited a large cohort with unexplained high bone mass (HBM). Exome sequencing identified a rare (minor allele frequency 0.0014) missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in an autosomal dominant family. The same mutation was identified in another two unrelated individuals with HBM. In-silico protein modelling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected individuals have bone mineral density [BMD] Z-Scores +3 to +5, with increased volumetric cortical and trabecular BMD, increased cortical thickness, and low/normal bone turnover. Fractures and nerve compressions are not seen. Both genome-wide, and gene-based association testing of heel estimated-BMD in >362,924 UK-Biobank British subjects showed strong associations with SMAD9 (PGWAS=6x10-16; PGENE =8x10-17). Smad9 is highly expressed in murine osteocytes and zebrafish bone tissue. Our findings support SMAD9 as a novel HBM gene, and a potential novel osteoanabolic target.

genetics↗