Biology subjects

Dhingra, S.

Publications and source records attributed to Dhingra, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Macrophage PHOX Restricts Aspergillus fumigatus Germination and Hyphal Development Through ROS-Dependent Immunity

Aspergillus fumigatus is a fungal pathogen that causes invasive disease in immunocompromised individuals. Innate immune cells, including macrophages and neutrophils, are essential for controlling A. fumigatus, but the mechanisms by which specific phagocytes control distinct stages of infection remain incompletely understood. In particular, the contribution of macrophage-derived reactive oxygen species (ROS) to conidial clearance and control of fungal germination and hyphae remains unclear. Here, we used larval zebrafish with mutation of p22, an essential subunit of the phagocyte NADPH oxidase (PHOX) complex, and neutrophil-deficiency, to define the role of macrophage-derived ROS during A. fumigatus infection. Mutation of p22 or pharmacological ROS scavenging increased host susceptibility, supporting a role for ROS in macrophage-mediated defense. However, macrophage ROS restricts fungal developmental progression rather than promoting clearance of conidia. Using {Delta}pksP conidia, we confirmed that fungal melanin limits the localization of p22 to phagosomes, but found no difference in conidial killing of {Delta}pksP conidia, supporting the finding that PHOX-generated ROS are not required for macrophage conidial killing. However, {Delta}pksP conidia have defective virulence only in ROS-producing larvae, as melanin inhibits ROS-mediated control of germination and hyphal growth. These findings identify macrophage PHOX activity as a stage-specific regulator of A. fumigatus infection that primarily restricts fungal developmental progression, while PksP-produced melanin promotes immune evasion by limiting macrophage ROS-dependent antifungal activity. These findings provide insight into how macrophages inhibit the transition from conidia to invasive fungal growth and how A. fumigatus evades this defense, with implications for understanding determinants of disease progression during invasive aspergillosis.

immunology↗

bioRxiv2026

Spatially resolved multimodal hallmarks of response to neoadjuvant immunotherapies in the melanoma ecosystem in 2D and 3D

Neoadjuvant immunotherapy has transformed cancer treatment, yet the spatial molecular architecture governing response and resistance across distinct immune checkpoint blockade (ICB) regimens remains poorly defined. We assembled the largest neoadjuvant ICB (NICB) spatial multi-omics cohort to date, profiling over 112 million cells at single-cell resolution across three melanoma ICB regimens using MERFISH spatial transcriptomics, multiplexed immunofluorescence, and scRNA-sequencing. These analyses revealed the full multicellular spatial architecture of the NICB tumor microenvironment, including mature TLS with germinal centers, TCF7+ stem-like T cells, myeloid cells organized into spatially distinct cellular neighborhoods with unique intercellular signaling circuits, and CCL19/CCL21-expressing fibroblasts as a previously unrecognized stromal scaffold sustaining these immune hubs. We developed three purpose-built computational tools that together enabled comprehensive quantification of this microenvironment for the first time: SCIRA for whole-slide single-cell receptor-ligand quantification, GCSCAN for molecularly grounded TLS and germinal center structural delineation, and PathNet-TLS for automated TLS detection on H&E images. Applying these tools across the cohort, we defined the immune and stromal composition and cellular neighborhood organization distinguishing responders from non-responders. We also quantified cell-cell interactions and regimen-specific immune architectures, including a markedly stronger mature TLS/germinal center response with IPI-NIVO than NIVO-RELA. Importantly, GCSCAN-quantified TLS and germinal center density each stratified disease-free survival, with responders that lack germinal centers having an elevated risk of relapse. Open-top light-sheet imaging and CODA-based 3D reconstruction further uncovered interconnected germinal center-TLS tunnels invisible to standard 2D histopathology. These findings establish a discovery-to-tool paradigm linking single-cell tumor microenvironment interrogation to clinically deployable computational pathology for biomarker-driven NICB assessment across cancer types.

cancer biology↗

bioRxiv2026

Plasma-derived extracellular vesicles as potential biomarkers and mediators of functional alterations in MELAS

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is a genetic disorder characterized by progressive neuromuscular and multisystem symptoms. MELAS typically manifests during childhood, can be difficult to diagnose, and has no cure. Extracellular vesicles (EVs) are lipid-enclosed nanoparticles secreted from cells that contain biological cargo and have demonstrated potential as biomarkers. We investigated the potential of plasma-derived EVs as diagnostic biomarkers of MELAS and examined their functional effects on mitochondrial respiration in treated skeletal muscle myotubes. Plasma-derived EVs were isolated from MELAS patients and age- and sex-matched control individuals, and biophysical characteristics and cargo of EVs analyzed. A Mito Stress Test was performed to assess oxygen consumption rate (OCR) in healthy myotubes treated with Control- or MELAS-EVs to determine the functional effects of circulatory EVs. Nine MELAS patients from two families were studied, and the results were categorized by age, sex and mtDNA heteroplasmy level. EV size and zeta potential remained unchanged. However, total EV concentration was higher in MELAS patients, particularly for small-EVs (<200 nm) and in younger patients (<25 years old). Relative protein yield per EV was lower in the MELAS group, especially among female and younger individuals. EV double-stranded DNA (dsDNA) concentration did not differ between MELAS- and Control-EVs overall, but was higher in male MELAS patients. Protein markers typically enriched in small-EVs showed altered expression in MELAS EVs: TSG101 and CD63 were lower, while flotillin-1 was higher compared to Control-EVs. A decrease in basal OCR was shown in cells treated with MELAS-EVs, with a similar response noted in the group treated with EVs from female MELAS patients. Post-treatment analysis showed no differences in oxidative phosphorylation (OXPHOS) subunit levels between cells treated with MELAS- and Control-EVs. In conclusion, plasma-derived EVs show promise as potential biomarkers for MELAS, and circulating EVs in this patient population may contribute to systemic metabolic dysfunction.

Cell Biology↗

bioRxiv2026

A novel mRNA Lipid Nanoparticle Therapy Improves Heart Failure Phenotype and Suppresses Endothelial-Mesenchymal Transition In Vitro

ObjectivesTo evaluate the therapeutic potential of BMP-7 mRNA-lipid nanoparticle formulation in attenuating cardiac fibrosis and improving function in non-ischemic heart failure, and to assess its impact on endothelial phenotype and function under pro endothelial-to-mesenchymal transition (EndMT) conditions. BackgroundDespite advances in neurohormonal blockade, heart failure (HF) progression remains driven in part by fibrotic remodeling. Endothelial-to-mesenchymal transition (EndMT) has emerged as a contributor to myocardial fibrosis, while recent work suggests endothelial phenotypic plasticity may also participate in myocardial recovery. Bone morphogenetic protein-7 (BMP-7) is a known anti-fibrotic regulator, but effective therapeutic delivery strategies remain limited. MethodsA patent pending, custom-designed BMP-7 mRNA formulated in lipid nanoparticles (AET-1978) was administered subcutaneously in a murine model of non-ischemic HF induced by L-NAME and angiotensin II. Cardiac function and fibrosis were assessed by echocardiography and histology. In an invitro EndMT model, human umbilical vascular endothelial cells (HUVECs) were treated with BMP-7 mRNA and endothelial and mesenchymal morphology, and markers were assessed along with endothelial functional tests. ResultsAET-1978 therapy significantly improved left ventricular systolic and diastolic function and reduced myocardial fibrosis compared with untreated HF mice, without evidence of renal or hepatic toxicity. In vitro, BMP-7 mRNA delivery restored endothelial morphology, suppressed EndMT-associated mesenchymal and profibrotic marker expression while preserving nitric oxide production, lipoprotein uptake, and angiogenic capacity of the HUVECs. ConclusionsA novel formulation of BMP-7-mRNA-LNP called AET-1978 represents a novel, transient, non-integrating strategy to attenuate fibrotic remodeling and improve cardiac function in heart failure, with supportive evidence of being anti endothelial to mesenchymal transition. HighlightsO_LIA novel BMP-7 mRNA-lipid nanoparticle formulation delivered as a subcutaneous injection attenuated myocardial fibrosis and improved systolic and diastolic function in a murine model of non-ischemic heart failure. C_LIO_LIBMP-7 mRNA therapy preserved endothelial phenotype and suppressed endothelial-to-mesenchymal transition in an in vitro platform of human umbilical vascular endothelial cells. C_LIO_LIBMP-7 mRNA therapy preserved endothelial function including restoration of nitric oxide production, lipoprotein uptake, and angiogenic capacity in vitro. C_LIO_LIThis study introduces AET-1978, a transient, non-integrating mRNA therapeutic platform, as a novel approach to target residual fibrotic pathways in heart failure using a clinically scalable delivery route. C_LI

bioengineering↗

bioRxiv2026

Thermal adaptation crosstalk with azole response through lncRNA in Aspergillus fumigatus

Fungal pathogens are adapting to increased temperatures altering host-pathogen interactions, disease patterns, and response to the antimicrobial drugs. Here, we show that thermal adaptation to 42{degrees}C leads to reversible changes in fungal colony size and azole drug response in the human pathogenic fungus Aspergillus fumigatus. Importantly, this adaptation is mediated by the lncRNA, afu-182, whose RNA levels negatively correlate with temperature. Growth at a lower temperature or ectopic upregulation of afu-182 RNA levels reverses the temperature adaptation. Previously, we have shown that {Delta}afu-182 strains produce worse disease outcomes in a murine model of invasive pulmonary aspergillosis (IPA). Here, more importantly, we show that the overexpression of afu-182 in clinically azole-resistant isolates increased survival in a murine model of IPA. Taken together, fungal adaptation to increased temperature leads to a decrease in afu-182 RNA levels that is associated with worse disease outcomes upon azole treatment. This provides a framework to take temperature into account when analyzing the rise in azole MIC in environmental and clinical isolates.

microbiology↗

bioRxiv2026

Autophagy Cholesterol Axis Remodeling Supports Malignant Progression and Chemoresistance in Glioma

Glioma progression and resistance to temozolomide (TMZ) remain major clinical challenges. Here, we investigated whether dysregulated autophagy and cholesterol metabolism are coordinately remodeled during glioma progression and TMZ resistance. Tissue microarray analysis of astrocytoma and glioblastoma specimens revealed progressive autophagosome accumulation, reflected by increased LC3{beta} puncta, coupled with impaired autophagic flux compared with adjacent normal brain tissue. These alterations intensified with tumor grade and were associated with upregulation of farnesyl diphosphate synthase (FDPS), linking malignant progression to cholesterol pathway remodeling. TMZ-resistant (R) glioblastoma cells exhibited epithelial-to-mesenchymal transition, mitotic quiescence, and mitochondrial remodeling consistent with a therapy-tolerant phenotype. Bioenergetic profiling demonstrated reduced respiratory reserve, diminished ATP-linked respiration, and elevated proton leak, indicating constrained metabolic flexibility. In parallel, impaired autophagy flux was associated with suppression of de novo cholesterol synthesis and transcriptional downregulation of SREBP-2 and LDL-R. Comprehensive lipidomic profiling revealed marked cholesterol metabolic reprogramming in R cells, characterized by accumulation of specific cholesteryl esters, including CE 22:5, CE 22:6, CE 22:4, and CE 20:4, despite reduced cholesterol biosynthesis. Pharmacologic inhibition of the mevalonate pathway with simvastatin significantly altered cholesteryl ester profiles but failed to restore autophagy flux or sensitize R cells to TMZ-induced apoptosis, even under combined TMZ-simvastatin treatment. Lay AbstractAs gliomas progress from astrocytoma to glioblastoma, autophagy becomes dysregulated and cholesterol metabolism is rewired. This coordinated remodeling supports tumor survival, metabolic plasticity, and resistance to temozolomide therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/697885v2_ufig1.gif" ALT="Figure 1000"> View larger version (79K): org.highwire.dtl.DTLVardef@1183dd2org.highwire.dtl.DTLVardef@82e20dorg.highwire.dtl.DTLVardef@c6c8dforg.highwire.dtl.DTLVardef@adb427_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsAutophagy flux blockade intensifies during progression from astrocytoma to glioblastoma Dysregulated autophagy is coupled to altered cholesterol metabolism in malignant gliomas TMZ-resistant glioblastoma cells undergo epithelial-to-mesenchymal transition and mitotic quiescence Resistant cells exhibit constrained bioenergetic capacity and mitochondrial remodeling Impaired autophagy suppresses de novo cholesterol synthesis and lipid recycling Lipidomics reveals accumulation of long-chain cholesteryl esters in TMZ-resistant cells Statin-based cholesterol inhibition fails to resensitize glioblastoma cells to temozolomide

cancer biology↗

bioRxiv2026

A long non-coding RNA, afu-254, is required for the oxidative stress response, cell wall stress response, azole susceptibility and virulence in Aspergillus fumigatus

Long non-coding RNAs play an important role in stress response in all forms of life; however, a tight regulation of lncRNAs is required for normal function. Abnormal expression of lncRNA is associated with uncontrolled cell growth in many forms of cancer. Recent studies have highlighted the role of lncRNAs in Aspergillus fumigatus in azole stress response and virulence. Here, using a transcriptome dataset of A. fumigatus response to stress, we identified afu-254 as an 854bp lncRNA that plays a role in modulating oxidative stress, fungal sub-MIC azole response to posaconazole and itraconazole, cell wall stress, macrophage phagocytosis and killing ex vivo, and virulence in an invertebrate model of Aspergillus infection. Importantly, afu-254 does not produce cross-azole susceptible response and plays a role in fungal azole response against posaconazole and itraconazole but not voriconazole. Furthermore, we showed that stochiometric levels of afu-254 are important for its function, and ectopic overexpression of afu-254 in the WT strain leads to an antimorph. This phenotype may stem from a higher ordered structure that is denatured with heat, indicating the presence of a non-functional isoform. In conclusion, we characterized a novel lncRNA, afu-254, that is important for stress response and virulence in the pathogenic fungus A. fumigatus. ImportanceFailure of azole treatment for invasive Aspergillus infection by both drug-resistant and drug-sensitive isolates is an area of concern and global importance. Fungal stress response is multifaceted, and long non-coding RNAs have emerged as important players in mediating it, including regulating responses to azole antifungals. Here, we have identified a long non-coding RNA, afu-254, that plays a role in modulating fungal response to oxidative stress, cell wall stress, azole stress, immune cell stress and virulence in an invertebrate model of invasive Aspergillus infection.

microbiology↗

bioRxiv2025

Mechanisms of ligand recognition and channel opening for P2X2 receptors in lipid nanodiscs

Extracellular ATP activates P2X receptor channels (P2XRs) that serve important roles in the immune and nervous systems. Available structures of P2XRs in detergents reveal that ATP binding to the extracellular domain leads to severing of subunit interfaces within transmembrane regions as the pore opens. Here we report cryo-EM structures of the human P2X2R in lipid nanodiscs in an apo closed state, with ATP4-, Mg-ATP2- and suramin bound. We find that a unique Arg residue coordinates the {gamma}-PO4 of ATP4- in P2X2R and underlies the requirement of this subtype for ATP4-. Channel opening and desensitization occur when ATP4- binds, whereas the channel remains closed when Mg-ATP2- binds. A continuous belt of partially resolved lipids in the outer leaflet stabilizes the closed state, and the presence of lipids prevents the severing of subunit interfaces as the channel opens. These findings establish key mechanistic principles of gating for P2X2R in a membrane-like environment, providing a framework for future mechanistic studies and therapeutic development.

biophysics↗

bioRxiv2025

A long non-coding RNA regulates triazole antifungal susceptibility and virulence in Aspergillus fumigatus

Azole-resistant Aspergillus infections are a source of increasing concern with limited alternative therapeutic options. However, as most infections are still caused by azole-susceptible Aspergillus strains, there is a need to better understand fungal responses to azole antifungals. To this end, we discover that a long non-coding RNA, afu-182, is a major regulator of cyp51-independent sub-MIC azole response. We observe that loss of afu-182 leads to increased surface attached growth and poor treated disease outcomes in a murine model of invasive pulmonary aspergillosis upon azole treatment. In contrast, overexpression of afu-182 significantly reduces fungal burden in animals treated with the azole drug, posaconazole. Importantly, afu-182 levels decrease upon azole exposure and in an azole adaptation experiment, continuous exposure to low dose azole led to MIC increase in an afu-182 dependent manner. Whole transcriptome analyses revealed that azole drug treatment leads to an increase in transcripts of genes encoding 7-transmembrane domain proteins of the RTA1 family, and these proteins are negatively regulated by afu-182. Two RTA1 family genes have individual and combined effects and are sufficient to increase fungal susceptibility to azole drugs in the WT strain. Taken together, our data show a role of the long non-coding RNA afu-182 in regulating Aspergillus fumigatus response to azole drugs both in vitro and in vivo. ImportanceDrug resistance in Aspergillus is a major challenge that is often associated with the agricultural use of azoles in the environment. How drug resistance arises in vivo is still an active area of research. Here, we show that azole exposure results in fungal adaptation by lowering the RNA levels of lncRNA, which upon low dose azole exposure leads to an increase in azole drug MIC.

molecular biology↗

bioRxiv2025

Aperiodicity in mouse CA1 and DG power spectra

Rodent hippocampal power spectra comprise of periodic and aperiodic components. The periodic components (brain rhythms) contain information about the behavioral or cognitive state of the animal. The aperiodic components are rarely studied and their functionality is not well understood, though have shown to be correlated with animals age or the excitation-inhibition ratio of the brain region. To study these components in the mouse hippocampus we modified the existing open-source FOOOF toolbox, which was originally optimized for EEG data. First, using simulated data, we show that our modifications decrease the error in assessment of the periodic components from 3% to 0.1%. Second, using tetrode electrophysiological signals, we compare the aperiodic activity within mice hippocampal sub-regions, CA1 and dentate gyrus (DG). Our optimization of FOOOF improved the aperiodic assessment errors by about 50% and were critical in making the first-ever assessments of the aperiodic components in these brain regions. Our results show that the aperiodic parameters in the DG are multi-fold and significantly larger than in CA1, revealing higher excitation and longer timescales in CA1 than DG. Our work highlights the subtle differences in electrophysiology field potentials between hippocampal sub-regions, and presents the improvements needed in the existing open-source toolbox to be able to see such differences.

neuroscience↗

bioRxiv2024

BCL2L13 Influences Autophagy and Ceramide Metabolism without Affecting Temozolomide Resistance in Glioblastoma

Temozolomide (TMZ) resistance in glioblastoma (GBM) arises through metabolic rewiring that links mitochondrial function, autophagy balance, and sphingolipid metabolism. TMZ resistant (R) U251 cells exhibited suppressed apoptosis and complete blockade of autophagy flux, evidenced by LC3II and p62 accumulation and insensitivity to Bafilomycin A1. BCL2L13, strongly upregulated in R cells, emerged as a dual regulator of mitophagy and ceramide metabolism. BCL2L13 knockdown (KD) produced opposite effects in TMZ sensitive (NR) and resistant cells: in NR cells, KD elevated LC3II, reduced respiratory reserve, and triggered compensatory lipid synthesis; in R cells, KD decreased LC3II without restoring flux or TMZ sensitivity. Lipidomic profiling revealed that BCL2L13 loss reactivated CerS6 in NR cells, increasing C16:0 and mid-chain ceramides, while relieving CerS2 inhibition in R cells, elevating very long chain (C22 to C24) and glycosylated ceramides. These distinct sphingolipid signatures were confirmed by PLS-DA and KEGG enrichment, which highlighted steroid hormone, arachidonic, and linoleic acid metabolism in NR KD cells versus neuroactive ligand-receptor and signaling pathways in R KD cells. Together, these findings position BCL2L13 as a molecular integrator of mitochondrial respiration, autophagy flux, and CerS-dependent lipid remodeling, unveiling a context-specific metabolic mechanism that supports GBM cell survival under chemotherapeutic stress.

cancer biology↗

bioRxiv2024

Code for Collagen Folding Deciphered

Collagen triple helix folds in two steps: nucleation of three polypeptides at the C-termini followed by zip-chain like propagation. The triple helices found in all domains of life as well as viruses contain upto 6000 amino acids in each polypeptide that are also frequently interrupted with non-helical sequences that disrupt folding and reduce stability. Given the length of polypeptide and the disruptive interruptions, compensating mechanisms that stabilize against local unfolding during propagation and offset the entropic cost of folding the long polypeptides are not fully understood. Here, we show that the information for correct folding of collagen triple helices is encoded in their sequence as interchain electrostatic interactions. In case of humans, disrupting these interactions causes severe to lethal diseases. Key ResultCollagen triple helices found in all the three domains of life as well as viruses have converged on similar mechanism to fold correctly.

biochemistry↗

bioRxiv2023

Role of the osaA gene in Aspergillus fumigatus development, secondary metabolism and virulence.

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. Deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for expression of the conidiation regulatory genes brlA, abaA and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both the neutropenic and corticosteroid-immunosuppressed mouse models.

microbiology↗

bioRxiv2023

Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expressions have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have an extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of crucial splice residues that influence receptor properties and their modulation. Furthermore, the cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.

neuroscience↗

bioRxiv2023

Engineered CD47 protects T cells for enhanced antitumor immunity

Adoptively transferred T cells and agents designed to block the CD47/SIRP axis are promising antitumor therapeutics, which activate distinct arms of the immune system. We administered anti-CD47 (CD47) with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which blunted therapeutic benefit. CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRP and provided a "dont-eat-me" signal that was not blocked by CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy resulted in synergistic enhancement in antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests markedly enhanced potency against solid tumors.

bioengineering↗

bioRxiv2023

Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection caused by species in the ubiquitous fungal genus Aspergillus. While leukocyte-generated reactive oxygen species (ROS) are critical for the clearance of fungal conidia from the lung and resistance to IPA, the processes that govern ROS-dependent fungal cell death remain poorly defined. Using a flow cytometric approach that monitors two independent cell death markers, an endogenous histone H2A:mRFP nuclear integrity reporter and Sytox Blue cell impermeable (live/dead) stain, we observed that loss of A. fumigatus cytochrome c (cycA) results in reduced susceptibility to cell death from hydrogen peroxide (H2O2) treatment. Consistent with these observations in vitro, loss of cycA confers resistance to both NADPH-oxidase -dependent and -independent killing by host leukocytes. Fungal ROS resistance is partly mediated in part by Bir1, a homolog to survivin in humans, as Bir1 overexpression results in decreased ROS-induced conidial cell death and reduced killing by innate immune cells in vivo. We further report that overexpression of the Bir1 N-terminal BIR domain in A. fumigatus conidia results in altered expression of metabolic genes that functionally converge on mitochondrial function and cytochrome c (cycA) activity. Together, these studies demonstrate that cycA in A. fumigatus contributes to cell death responses that are induced by exogenous H2O2 and by host leukocytes. ImportanceAspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk of IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with Chronic Granulomatous Disease (CGD). However, treatments for Aspergillus infections remains limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization (WHO) classified A. fumigatus as a critical priority fungal pathogen. Our research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

microbiology↗

bioRxiv2023

Homeobox Transcription Factor HbxA Influences Expression of over One ThousandGenes in the Model Fungus Aspergillus nidulans

In fungi, conserved homeobox-domain (HD) proteins are transcriptional regulators governing development. In Aspergillus species, several HD transcription factor genes have been identified, among them, hbxA/hbx1. For instance, in the opportunistic human pathogen Aspergillus fumigatus, hbxA is involved in conidial production and germination, as well as virulence and secondary metabolism (SM), including production of fumigaclavines, fumiquinazolines, and chaetominine. In the agriculturally important fungus Aspergillus flavus, disruption of hbx1 results in fluffy aconidial colonies unable to produce sclerotia. hbx1 also regulates production of aflatoxins, cyclopiazonic acid and aflatrem. Furthermore, transcriptome studies revealed that hbx1 has a broad effect on the A. flavus genome, including numerous genes involved in SM. These studies underline the importance of the HbxA/Hbx1 regulator, not only in developmental processes but also in the biosynthesis of a broad number of fungal natural products, including potential medical drugs and mycotoxins. To gain further insight into the regulatory scope of HbxA in Aspergilli, we studied its role in the model fungus Aspergillus nidulans. Our present study of the A. nidulans hbxA-dependent transcriptome revealed that more than one thousand genes are differentially expressed when this regulator was not transcribed at wild-type levels, among them numerous transcription factors, including those involved in development as well as in SM regulation. Furthermore, our metabolomics analyses revealed that production of several secondary metabolites, some of them associated with A. nidulans hbxA-dependent gene clusters, was also altered in deletion and overexpression hbxA strains compared to the wild type, including synthesis of nidulanins A, B and D, versicolorin A, sterigmatocystin, austinol, dehydroaustinol, and three unknown novel compounds.

genetics↗

bioRxiv2022

A multi-omics analysis of glioma chemoresistance using a hybrid microphysiological model of glioblastoma

Chemoresistance is a major clinical challenge in the management of glioblastoma (GBM) Temozolomide (TMZ) is the chemotherapeutic drug of choice for GBM; however, the therapeutic effect of TMZ is limited due to the development of resistance. Recapitulating GBM chemoresistance in a controlled environment is thus essential in understanding the mechanism of chemoresistance. Herein, we present a hybrid microphysiological model of chemoresistant GBM-on-a-chip (HGoC) by directly co-culturing TMZ-resistant GBM spheroids with healthy neurons to mimic the microenvironment of both the tumor and the surrounding healthy tissue. We characterized the model with proteomics, lipidomics, and secretome assays. The results showed that our artificial model recapitulated the molecular signatures of recurrent GBM in humans. Both showed alterations in vesicular transport and cholesterol pathways, mitotic quiescence, and a switch in metabolism to oxidative phosphorylation associated with a transition from mesenchymal to amoeboid. This is the first report to unravel the interplay of all these molecular changes as a mechanism of chemoresistance in glioblastoma. Moreover, we have shown that the acquisition of resistance increases invasiveness and the presence of neurons decreases this property.

bioengineering↗