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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 199 records · Page 11
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

The catalytic domains of Streptococcus mutans glucosyltransferases: a structural analysis

Streptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Muscone suppresses inflammatory responses and neuronal damage in a rat model of cervical spondylotic myelopathy by regulating Drp1‐dependent mitochondrial fission

Abstract Cervical spondylotic myelopathy (CSM) is a common cause of disability with few treatments. Aberrant mitochondrial dynamics play a crucial role in the pathogenesis of various neurodegenerative diseases. Thus, regulation of mitochondrial dynamics may offer therapeutic benefit for the treatment of CSM. Muscone, the active ingredient of an odoriferous animal product, exhibits anti‐inflammatory and neuroprotective effects for which the underlying mechanisms remain obscure. We hypothesized that muscone might ameliorate inflammatory responses and neuronal damage by regulating mitochondrial dynamics. To this end, the effects of muscone on a rat model of chronic cervical cord compression, as well as activated BV2 cells and injured neurons, were assessed. The results showed that muscone intervention improved motor function compared with vehicle‐treated rats. Indeed, muscone attenuated pro‐inflammatory cytokine expression, neuronal‐apoptosis indicators in the lesion area, and activation of the nod‐like receptor family pyrin domain‐containing 3 inflammasome, nuclear transcription factor‐κB, and dynamin‐related protein 1 in Iba1‐ and βIII‐tubulin‐labeled cells. Compared with vehicle‐treated rats, compression sites of muscone‐treated animals exhibited elongated mitochondrial morphologies in individual cell types and reduced reactive oxygen species. In vitro results indicated that muscone suppressed microglial activation and neuronal damage by regulating related‐inflammatory or apoptotic molecules. Moreover, muscone inhibited dynamin‐related protein 1 activation in activated BV2 cells and injured neurons, whereby it rescued mitochondrial fragmentation and reactive oxygen species production, which regulate a wide range of inflammatory and apoptotic molecules. Our findings reveal that muscone attenuates neuroinflammation and neuronal damage in rats with chronic cervical cord compression by regulating mitochondrial fission events, suggesting its promise for CSM therapy. image

Zhou, Long‐yun↗
DOE OSTI2021

ChAdOx1 interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome

Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Exploration of structure-activity relationships for the SARS-CoV-2 macrodomain from shape-based fragment linking and active learning

The macrodomain of severe acute respiratory syndrome coronavirus 2 nonstructural protein 3 is required for viral pathogenesis and is an emerging antiviral target. We previously performed an x-ray crystallography–based fragment screen and found submicromolar inhibitors by fragment linking. However, these compounds had poor membrane permeability and liabilities that complicated optimization. Here, we developed a shape-based virtual screening pipeline—FrankenROCS. We screened the Enamine high-throughput collection of 2.1 million compounds, selecting 39 compounds for testing, with the most potent binding with a 130 μM median inhibitory concentration (IC 50 ). We then paired FrankenROCS with an active learning algorithm (Thompson sampling) to efficiently search the Enamine REAL database of 22 billion molecules, testing 32 compounds with the most potent binding with a 220 μM IC 50 . Further optimization led to analogs with IC 50 values better than 10 μM. This lead series has improved membrane permeability and is poised for optimization. FrankenROCS is a scalable method for fragment linking to exploit synthesis-on-demand libraries.

Science & Technology - Other Topics↗
DOE OSTI2022

NF-κB perturbation reveals unique immunomodulatory functions in Prx1 + fibroblasts that promote development of atopic dermatitis

Skin is composed of diverse cell populations that cooperatively maintain homeostasis. Up-regulation of the nuclear factor κB (NF-κB) pathway may lead to the development of chronic inflammatory disorders of the skin, but its role during the early events remains unclear. Here, through analysis of single-cell RNA sequencing data via iterative random forest leave one out prediction, an explainable artificial intelligence method, we identified an immunoregulatory role for a unique paired related homeobox-1 (Prx1) + fibroblast subpopulation. Disruption of Ikkb–NF-κB under homeostatic conditions in these fibroblasts paradoxically induced skin inflammation due to the overexpression of C-C motif chemokine ligand 11 (CCL11; or eotaxin-1) characterized by eosinophil infiltration and a subsequent T H 2 immune response. Because the inflammatory phenotype resembled that seen in human atopic dermatitis (AD), we examined human AD skin samples and found that human AD fibroblasts also overexpressed CCL11 and that perturbation of Ikkb–NF-κB in primary human dermal fibroblasts up-regulated CCL11. Monoclonal antibody treatment against CCL11 was effective in reducing the eosinophilia and T H 2 inflammation in a mouse model. Together, the murine model and human AD specimens point to dysregulated Prx1 + fibroblasts as a previously unrecognized etiologic factor that may contribute to the pathogenesis of AD and suggest that targeting CCL11 may be a way to treat AD-like skin lesions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Clostridioides difficile utilizes siderophores as an iron source and FhuDBGC contributes to ferrichrome uptake

ABSTRACT Clostridioides difficileremains a public health threat commonly observed following antibiotic use. Due to the importance of iron in many cell processes, most bacteria, includingC. difficile, have multiple mechanisms of acquiring iron. Previous studies have examined ferrous iron uptake inC. difficile; here we focus on the role of siderophores. In a growth assay, we show thatC. difficilecan use a variety of siderophores as the sole iron source. InC. difficile,two ABC transporters induced under low iron conditions are predicted siderophore importers: FhuDBGC and YclNOPQ. We hypothesized that these transporters are responsible for the uptake of the siderophores we tested. To investigate the specificity of these transporters, we purified the substrate binding proteins and examined siderophore binding using thermal shift. We demonstrate increased stability between one siderophore binding protein, FhuD, and the siderophore ferrichrome, suggesting a binding interaction. This specificity correlates with the inability of anΔfhuDBGCmutant to grow efficiently under iron limiting conditions in the presence of ferrichrome. WhileC. difficileused additional siderophores in our growth experiments, we did not observe increased thermal stability between the receptor proteins and any of the other siderophores tested, suggesting these siderophores do not bind these receptors and other siderophore import mechanisms remain to be elucidated. Redundancy in iron acquisition is a microbial survival adaptation to cope with the constant battle for iron within a host. Greater knowledge about howC. difficileacquires iron will provide insight about howC. difficilecolonizes and persists in the colon. IMPORTANCE This study is the first example ofC. difficilegrowing with siderophores as the sole iron source and describes the characterization of the ferric hydroxamate uptake ABC transporter (FhuDBGC). This transporter shows specificity to the siderophore ferrichrome. While not required for pathogenesis, this transporter highlights the redundancy in iron acquisition mechanisms thatC. difficileuses to compete for iron during an infection.

Microbiology↗
DOE OSTI2023

Structural Basis for Binding of Neutralizing Antibodies to Clostridioides difficile Binary Toxin

Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb—the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol—showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Tailoring a Global Iron Regulon to a Uropathogen

Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O 2 -dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O 2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Physiological roles of an Acinetobacter -specific σ factor

ABSTRACT The Gram-negative pathogen Acinetobacter baumannii is considered an “urgent threat” to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF “SigAb”; however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE-like ECFs. We combine promoter mutagenesis, motif scanning, and chromatin immunoprecipitation-sequencing (ChIP-seq) to define the direct SigAb regulon, which consists of genes encoding SigAb itself, the stringent response mediator, RelA, and the uncharacterized small RNA, “SabS.” However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper-induced SigAb-dependent transcription. Furthermore, we found that two uncharacterized genes in the sigAb operon, “ aabA ” and “ aabB ,” have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb , aabA , and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii . IMPORTANCE Acinetobacter baumannii is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how A. baumannii senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the Acinetobacter -specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically relevant contexts.

Bacon, Emily E. (ORCID:0000000180907689)↗
DOE OSTI2021

Unfolded Protein Response Inhibition Reduces Middle East Respiratory Syndrome Coronavirus-Induced Acute Lung Injury

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe atypical pneumonia in infected individuals, but the underlying mechanisms of pathogenesis remain unknown. While much has been learned from the few reported autopsy cases, an in-depth understanding of the cells targeted by MERS-CoV in the human lung and their relative contribution to disease outcomes is needed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Biophysical and Biochemical Characterization of TP0037, a D-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism’s robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum. Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein’s propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum’s d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Brachypodium Phenylalanine Ammonia Lyase (PAL) Promotes Antiviral Defenses against Panicum mosaic virus and Its Satellites

Brachypodium distachyon has recently emerged as a premier model plant for monocot biology, akin to Arabidopsis thaliana. We previously reported genome-wide transcriptomic and alternative splicing changes occurring in Brachypodium during compatible infections with Panicum mosaic virus (PMV) and its satellite virus (SPMV). Here, we dissected the role of Brachypodium phenylalanine ammonia lyase 1 (PAL1), a key enzyme for phenylpropanoid and salicylic acid (SA) biosynthesis and the induction of plant defenses. Targeted metabolomics profiling of PMV-infected and PMV- plus SPMV-infected (PMV/SPMV) Brachypodium plants revealed enhanced levels of multiple defense-related hormones and metabolites such as cinnamic acid, SA, and fatty acids and lignin precursors during disease progression. The virus-induced accumulation of SA and lignin was significantly suppressed upon knockdown of B. distachyon PAL1 (BdPAL1) using RNA interference (RNAi). The compromised SA accumulation in PMV/SPMV-infected BdPAL1 RNAi plants correlated with weaker induction of multiple SA-related defense gene markers (pathogenesis related 1 [PR-1], PR-3, PR-5, and WRKY75) and enhanced susceptibility to PMV/SPMV compared to that of wild-type (WT) plants. Furthermore, exogenous application of SA alleviated the PMV/SPMV necrotic disease phenotypes and delayed plant death caused by single and mixed infections. Together, our results support an antiviral role for BdPAL1 during compatible host-virus interaction, perhaps as a last resort attempt to rescue the infected plant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome sequences of key bacterial symbionts of entomopathogenic nematodes: Xenorhabdus cabanillasii DSM17905, Xenorhabdus ehlersii DSM16337, Xenorhabdus japonica DSM16522, Xenorhabdus koppenhoeferii DSM18168, and Xenorhabdus mauleonii DSM17908

ABSTRACT Xenorhabdus species are bacterial symbionts of entomopathogenic Steinernema nematodes, in which they produce diverse secondary metabolites implicated in pathogenesis. To expand resources for natural product prospecting and exploration of host-symbiont-pathogen relationships, the genomes of Xenorhabdus cabanillasi, Xenorhabdus ehlersii , Xenorhabdus japonica , Xenorhabdus koppenhoeferii , and Xenorhabdus mauleonii were analyzed.

Robertson, Raegan↗
DOE OSTI2020

Metabolic Feedback Inhibition Influences Metabolite Secretion by the Human Gut Symbiont Bacteroides thetaiotaomicron

ABSTRACT Microbial metabolism and trophic interactions between microbes give rise to complex multispecies communities in microbe-host systems. Bacteroides thetaiotaomicron ( B. theta ) is a human gut symbiont thought to play an important role in maintaining host health. Untargeted nuclear magnetic resonance metabolomics revealed B. theta secretes specific organic acids and amino acids in defined minimal medium. Physiological concentrations of acetate and formate found in the human intestinal tract were shown to cause dose-dependent changes in secretion of metabolites known to play roles in host nutrition and pathogenesis. While secretion fluxes varied, biomass yield was unchanged, suggesting feedback inhibition does not affect metabolic bioenergetics but instead redirects carbon and energy to CO 2 and H 2 . Flux balance analysis modeling showed increased flux through CO 2 -producing reactions under glucose-limiting growth conditions. The metabolic dynamics observed for B. theta , a keystone symbiont organism, underscores the need for metabolic modeling to complement genomic predictions of microbial metabolism to infer mechanisms of microbe-microbe and microbe-host interactions. IMPORTANCE Bacteroides is a highly abundant taxon in the human gut, and Bacteroides thetaiotaomicron ( B. theta ) is a ubiquitous human symbiont that colonizes the host early in development and persists throughout its life span. The phenotypic plasticity of keystone organisms such as B. theta is important to understand in order to predict phenotype(s) and metabolic interactions under changing nutrient conditions such as those that occur in complex gut communities. Our study shows B. theta prioritizes energy conservation and suppresses secretion of “overflow metabolites” such as organic acids and amino acids when concentrations of acetate are high. Secreted metabolites, especially amino acids, can be a source of nutrients or signals for the host or other microbes in the community. Our study suggests that when metabolically stressed by acetate, B. theta stops sharing with its ecological partners.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Deciphering the molecular components of the quorum sensing system in the fungus Ophiostoma piceae

Quorum sensing (QS) is a complex cell-cell communication mechanism that coordinates population-level behaviors in microbes. In eukaryotes, this phenomenon has been extensively described in the dimorphic yeast Candida albicans as its main QS molecule, the sesquiterpene alcohol farnesol, is responsible for various phenotypic (i.e., inhibition of yeast-to-hyphae transition, biofilm formation, and, hence, pathogenesis) and metabolic (i.e., induction of oxidative stress and apoptosis) changes. Ophiostoma piceae CECT 20416 is a dimorphic saprotrophic ascomycete with biotechnological interest that also produces farnesol as QS molecule, but in this case, the alcohol promotes the morphological transition to the mycelial form, biofilm formation, enzyme secretion, and melanin production. Here, we characterized the physiological response of Ophiostoma piceae to farnesol, the molecular components of the QS system of this fungus have been investigated using a “multiomics” approach that involved genomic, transcriptomic, and proteomic analyses. Some genes identified in this work are proposed as key factors in farnesol transport and signaling. We have also cataloged the genes undergoing major transcriptional changes triggered by the presence of the autoinducer, such as cell-wall remodeling, reactive oxygen species (ROS) protection, and melanin biosynthesis, using self-organizing maps. This analysis could be useful for applications in the forestry industry, for enzymes production, and for the valorization of residues. Furthermore, it might as well help to investigate the QS mechanisms of clinically relevant fungi phylogenetically related to Ophiostoma.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Sorted-cell proteomics reveals an AT1-associated epithelial cornification phenotype and suggests endothelial redox imbalance in human bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease characterized by inflammation and scarring leading to long-term tissue damage. Previous whole tissue proteomics identified BPD-specific proteome changes and cell type shifts. Little is known about the proteome-level changes within specific cell populations in disease. Here, we sorted epithelial (EPI) and endothelial (ENDO) cell populations based on their differential surface markers from normal and BPD human lungs. Using a low-input compatible sample preparation method (MicroPOT), proteins were extracted and digested into peptides and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteome analysis. Of the 4,970 proteins detected, 293 were modulated in abundance or detection in the EPI population and 422 were modulated in ENDO cells. Modulation of proteins associated with actin-cytoskeletal function, such as SCEL, LMO7, and TBA1B was observed in the BPD EPIs. Using confocal imaging and analysis, we validated the presence of aberrant multilayer-like structures comprising SCEL and LMO7, known to be associated with epidermal cornification, in the human BPD lung. This is the first report of the accumulation of cornification-associated proteins in BPD. Their localization in the alveolar parenchyma, primarily associated with alveolar type 1 (AT1) cells, suggests a role in the BPD postinjury response. In the ENDOs, redox balance and mitochondrial function pathways were modulated. Alternative mRNA splicing and cell proliferative functions were elevated in both populations, suggesting potential dysregulation of cell progenitor fate. This study characterized the proteome of epithelial and endothelial cells from the BPD lung for the first time, identifying population-specific changes in BPD pathogenesis.

BPD↗
DOE OSTI2020

Cryptococcus neoformans Secretes Small Molecules That Inhibit IL-1 β Inflammasome-Dependent Secretion

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501’s conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Endogenous Retroviral Elements Generate Pathologic Neutrophils in Pulmonary Arterial Hypertension

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

60 APPLIED LIFE SCIENCES↗