Biology subjects

Wagner, A. S.

Publications and source records attributed to Wagner, A. S..

8 recordsLinked to original sources

bioRxiv2024

A conserved in vivo burn wound infection model for diverse pathogenic fungi.

Secondary fungal infections represent a major complication following thermal injuries. However, the mechanisms of fungal colonization of burn tissue and how the host subsequently responds to fungi within this niche remain unclear. We have previously reported a zebrafish model of thermal injury that recapitulates many of the features of human burn wounds. Here, we characterize host-fungal interaction dynamics within the burn wound niche using two of the most common fungal pathogens isolated from burn injuries, Aspergillus fumigatus and Candida albicans. Both A. fumigatus and C. albicans colonize burned tissue in zebrafish larvae and induce a largely conserved innate immune response following colonization. Using drug-induced cell depletion strategies and transgenic zebrafish lines with impaired innate immune function, we found that macrophages control fungal burden while neutrophils primarily control invasive hyphal growth at the early stages of infection. However, we also found that loss of either immune cell can be compensated by the other at the later stages of infection, and that fish with both macrophage and neutrophil deficiencies show more invasive hyphal growth that is sustained throughout the infection process, suggesting redundancy in their antifungal activities. Finally, we demonstrate that C. albicans strains with increased {beta}(1,3)-glucan exposure are cleared faster from the burn wound, demonstrating a need for shielding this immunogenic cell wall epitope for successful fungal colonization of burn tissue. Together, our findings support the use of zebrafish larvae as a model to study host-fungal interaction dynamics within burn wounds. ImportanceSecondary fungal infections within burn wound injuries are a significant problem that delay wound healing and increase the risk of patient mortality. Currently, little is known about how fungi colonize and infect burn tissue or how the host responds to pathogen presence. In this report, we expand upon an existing thermal injury model using zebrafish larvae to begin to elucidate both the host immune response to fungal burn colonization and fungal mechanisms for persistence within burn tissue. We found that both Aspergillus fumigatus and Candida albicans, common fungal burn wound isolates, successfully colonize burn tissue and are effectively cleared in immunocompetent zebrafish by both macrophages and neutrophils. We also find that C. albicans mutants harboring mutations that impact their ability to evade host immune system recognition are cleared more readily from burn tissue. Collectively, our work highlights the efficacy of using zebrafish to study host-fungal interaction dynamics within burn wounds.

microbiology↗

bioRxiv2024

Isotonic medium treatment limits burn wound microbial colonization and improves tissue repair

Burn injuries undergo a complex healing process in which progressive spreading of epithelial damage can lead to secondary complications such as wound infection, which is a major driver of mortality among burn patients. We recently reported that burning larval zebrafish triggers dysregulated keratinocyte dynamics compared to mechanical injury. Here, we investigate keratinocyte behavior following burn injury and the subsequent potential for microbial colonization of burn wounds over time. Real-time imaging, coupled with tracking of photoconverted cells, revealed that early keratinocyte motility contributes to the spread of epithelial damage beyond the initial site of burn injury and that increased epithelial damage was associated with wound colonization by the fungal pathogen Candida albicans. Modulating osmotic balance by treating larval zebrafish with isotonic medium limited the spread of epithelial damage and reduced microbial colonization of burn wounds. Using cultured human skin, we found that topical treatment with isotonic solution (saline) similarly prevented the spread of epithelial damage over time. These findings indicate that keratinocyte behavior contribute to burn wound progression in larval zebrafish and link keratinocyte dynamics to microbial colonization of burn wounded tissue.

cell biology↗

bioRxiv2024

GATA1-deficient human pluripotent stem cells generate neutrophils with improved antifungal immunity that is mediated by the integrin CD18

Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal {beta}-glucan, including an upregulation of the pentose phosphate pathway (PPP), similar to primary human neutrophils in vitro. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections, and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation, limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus, demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively, these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity.

microbiology↗

bioRxiv2023

Plant inositol transport influences bacterial colonization phenotypes

Plant microbiomes are assembled and modified through a complex milieu of biotic and abiotic factors. Despite dynamic and fluctuating contributing variables, specific host metabolites are consistently identified as important mediators of microbial interactions. We combine information from a large-scale metatranscriptomic dataset from natural poplar trees and experimental genetic manipulation assays in model Arabidopsis seedlings to converge on a conserved role for transport of the plant metabolite myo-inositol in mediating host-microbe interactions. While microbial catabolism of this compound is often linked to increased host colonization, we identify motility phenotypes that occur independently of catabolism, suggesting that inositol may additionally serve as a eukaryotic-derived signaling molecule to modulate microbial activities. Our data suggests host control of this compound and resulting microbial behavior are important mechanisms at play surrounding the host metabolite inositol.

microbiology↗

bioRxiv2022

Lipid transport by Candida albicans Dnf2 is required for hyphal growth and virulence

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contribute to membrane biogenesis by selecting and transporting specific lipids from the extracellular leaflet to the cytoplasmic leaflet of the bilayer to generate lipid asymmetry. A subset of heterodimeric P4-ATPases, including Dnf1-Lem3 and Dnf2-Lem3 from Saccharomyces cerevisiae, transport phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the sphingolipid glucosylceramide (GlcCer). GlcCer is a critical lipid for Candida albicans polarized growth and virulence, but the role of GlcCer transporters in virulence has not been explored. Here we show that the Candida albicans Dnf2 (CaDnf2) requires association with CaLem3 to form a functional transporter and flip fluorescent derivatives of GlcCer, PC and PE across the plasma membrane. Mutation of conserved substrate-selective residues in the membrane domain strongly abrogates GlcCer transport and partially disrupts PC transport by CaDnf2. Candida strains harboring dnf2 null alleles (dnf2{Delta}{Delta}) or point mutations that disrupt substrate recognition exhibit defects in the yeast to hyphal growth transition, filamentous growth and virulence in systemically infected mice. The influence of CaDNF1 deletion on the morphological phenotypes is negligible although the dnf1{Delta}{Delta} dnf2{Delta}{Delta} strain was less virulent than the dnf2{Delta}{Delta} strain. These results indicate that the transport of GlcCer and/or PC by plasma membrane P4-ATPases is important for pathogenicity of Candida albicans.

microbiology↗

bioRxiv2021

FAIRly big: A framework for computationally reproducible processing of large-scale data

Large-scale datasets present unique opportunities to perform scientific investigations with un-precedented breadth. However, they also pose considerable challenges for the findability, accessibility, interoperability, and reusability (FAIR) of research outcomes due to infrastructure limitations, data usage constraints, or software license restrictions. Here we introduce a DataLad-based, domain-agnostic framework suitable for reproducible data processing in compliance with open science mandates. The framework attempts to minimize platform idiosyncrasies and performance-related complexities. It affords the capture of machine-actionable computational provenance records that can be used to retrace and verify the origins of research outcomes, as well as be re-executed independent of the original computing infrastructure. We demonstrate the frameworks performance using two showcases: one highlighting data sharing and transparency (using the studyforrest.org dataset) and another highlighting scalability (using the largest public brain imaging dataset available: the UK Biobank dataset).

bioinformatics↗

bioRxiv2019

REMoDNaV: Robust Eye Movement Detection for Natural Viewing

Tracking of eye movements is an established measurement for many types of experimental paradigms. More complex and more prolonged visual stimuli have made algorithmic approaches to eye movement event classification the most pragmatic option. A recent analysis revealed that many current algorithms are lackluster when it comes to data from viewing dynamic stimuli such as video sequences. Here we present an event classification algorithm--built on an existing velocity-based approach--that is suitable for both static and dynamic stimulation, and is capable of classifying saccades, post-saccadic oscillations, fixations, and smooth pursuit events. We validated classification performance and robustness on three public datasets: 1) manually annotated, trial-based gaze trajectories for viewing static images, moving dots, and short video sequences, 2) lab-quality gaze recordings for a feature length movie, and 3) gaze recordings acquired under suboptimal lighting conditions inside the bore of a magnetic resonance imaging (MRI) scanner for the same full-length movie. We found that the proposed algorithm performs on par or better compared to state-of-the-art alternatives for static stimulation. Moreover, it yields eye movement events with biologically plausible characteristics on prolonged dynamic recordings. Lastly, algorithm performance is robust on data acquired under suboptimal conditions that exhibit a temporally varying noise level. These results indicate that the proposed algorithm is a robust tool with improved classification accuracy across a range of use cases. The algorithm is cross-platform compatible, implemented using the Python programming language, and readily available as free and open source software from public sources.

bioinformatics↗

bioRxiv2017

Fibril branching dominates self-assembly of mutant huntingtin exon-1 aggregates in vitro

Huntingtin (HTT) fragments with extended polyglutamine (polyQ) tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntingtons disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modelling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

biochemistry↗