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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 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Lessons learned from studying Histoplasma capsulatum extracellular vesicles

Histoplasma capsulatum is a major endemic mycosis. Our laboratories have demonstrated that H. capsulatum produces extracellular vesicles (EV) that are loaded with diverse compounds that influence virulence. We have further shown that H. capsulatum dynamically regulates the loading and release of fungal EV in response to stimuli and growth conditions. This chapter details the current knowledge of EV biology in H. capsulatum and the impact of this information on our understanding of this important process that is closely linked to pathogenesis.

Zamith-Miranda, Daniel↗
DOE OSTI2025

Bleach Rescues Nannochloropsis from an Obligate Parasite and Alters Microbial and Metabolite Signatures of Outdoor Cultures

Chemical agents are commonly used to protect algal crops. Yet, few studies have characterized the effects of these agents on associated microbial communities to understand effects on microbial functions relevant to algal crop production and protection. Here, we used shotgun metagenomic sequencing and untargeted exometabolite profiling to link the application of bleach, a -cidal agent used to protect algae from pests, to changes in community composition, metabolic pathways, and exometabolies - at a whole community level. Bleach protected the algal crop from crashing but altered bacterial diversity. Analysis of metagenome-assembled genomes (MAGs) revealed a classic predator-prey cycle between Oligoflexus and our target alga Nannochloropsis. Olifoflexus genomes from our study were notably similar to a previously identified BALO (Bdellovibrio and like organism), FD111, known to kill Nannochloropsis cultures, providing strong evidence that an FD111-like organism was responsible for the crash. Metabolic pathway composition differed between bleached and unbleached ponds, with abundance of twelve pathways related to stress tolerance, including the superpathway of methylglyoxal degradation, lipid IVA biosynthesis, and ectoine biosynthesis, greater in bleached ponds compared to unbleached ponds. Virulence factors related to adherence, biofilm formation, motility, and pathogenicity increased dramatically in bleached ponds with time, although this increase was not coupled with an increase in pathogens - algal or otherwise - or a decline in algal health. Our study highlights the importance of coupling 16S rRNA gene sequencing with whole genome data and other -omics tools to sketch a larger picture of community structure and function in crop systems. Moreover, our results highlight that continued long-term bleaching may lead to negative effects to crop health or downstream adverse health effects to humans or animals, depending on the algal product (i.e. human supplements or animal feedstocks). Future work on alternative treatment methods that would reduce resistance is necessary in the field.

09 BIOMASS FUELS↗
DOE OSTI2025

Evaluating size exclusion chromatography for nucleic acid removal in Klebsiella pneumoniae cell surface polysaccharide purification

Cell surface-associated polysaccharides in Klebsiella are major virulence determinants and crucial targets for developing vaccines. Traditionally, the purification of these cell surface polysaccharides from Klebsiella pneumoniae involves a multi-step process comprising phenol extraction, nuclease digestion, ultracentrifugation, and repeated ethanol extractions. In this study, we evaluated size exclusion chromatography for effectively eliminating nucleic acid contamination while purifying high molecular weight cell surface-associated polysaccharides. Post-initial extraction, the nucleic acid content remains significantly elevated, and kinetic analysis reveals that DNase I and RNase A digestion is neither economically viable nor effective for removing these contaminants. Employing an appropriate size exclusion resin removes over 99 % of nucleic acid contamination, as confirmed by nucleic acid content analysis and agarose gel electrophoresis. Purity and structural analysis using 1H 1D-NMR and 2D-NMR demonstrate that the cell surface-associated polysaccharide purified with this study is highly homogeneous and identified as antigenic O-polysaccharide. This approach streamlines the purification process by removing the need for nuclease digestion and additional ethanol precipitation steps.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Formin nanoclustering-mediated actin assembly during plant flagellin and DSF signaling

Plants respond to bacterial infection acutely with actin remodeling during plant immune responses. The mechanisms by which bacterial virulence factors (VFs) modulate plant actin polymerization remain enigmatic. Here, we show that plant-type-I formin serves as the molecular sensor for actin remodeling in response to two bacterial VFs: Xanthomonas campestris pv. campestris (Xcc) diffusible signal factor (DSF), and pathogen-associated molecular pattern (PAMP) flagellin in pattern-triggered immunity (PTI). Both VFs regulate actin assembly by tuning the clustering and nucleation activity of formin on the plasma membrane (PM) at the nano-sized scale. By being integrated within the cell-wall-PM-actin cytoskeleton (CW-PM-AC) continuum, the dynamic behavior and function of formins are highly dependent on each scaffold layer’s composition within the CW-PM-AC continuum during both DSF and PTI signaling. Our results reveal a central mechanism for rapid actin remodeling during plant-bacteria interactions, in which bacterial signaling molecules fine tune plant formin nanoclustering in a host mechanical-structure-dependent manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Genetics & Heredity↗
DOE OSTI2023

From Tg O/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat’s oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with “Rosetta stone”-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite’s capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification and characterization of mono- and bifunctional galactan synthases in the pediatric pathogen Kingella kingae

The emerging pediatric pathogen Kingella kingae elaborates a lipopolysaccharide (LPS) that is extended with a galactofuranose homopolymer called galactan, which is a key virulence determinant that contributes to resistance to complement-mediated and neutrophil-mediated killing. Previous work has demonstrated that the pamABCDE locus is required for galactan synthesis. In this study, mutational studies suggested that the pamC gene product is a UDP-galactofuranose (Galf) transferase and is the galactan synthase. Analysis of genome sequence data revealed two distinct pamC alleles designated pamC1 and pamC2, which correlate with the two galactan structures in K. kingae. Examination of isogenic mutants expressing either pamC1 or pamC2 demonstrated that the pamC alleles are the determinants of galactan structure. Experiments with recombinant PamC1 and PamC2 in vitro established that these proteins are galactan synthases capable of extending synthetic Galf disaccharide acceptors in the presence of UDP-Galf. Homology analysis identified critical amino acids that are essential for PamC1 and PamC2 enzymatic activity both in vitro and in K. kingae. Structural analysis of the in vitro-modified synthetic acceptors implicated PamC1 as a monofunctional enzyme capable of generating a β-(1 → 5) Galf linkage and PamC2 as a bifunctional enzyme capable of generating β-(1 → 3) and β-(1 → 6) Galf linkages. This study advances our understanding of the GT2 family of UDP-galactofuranosyltransferases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Highly tail-asymmetric lipids interdigitate and cause bidirectional ordering

Phospholipids form structurally and compositionally diverse membranes. A less studied type of compositional diversity involves phospholipid tail variety. Some phospholipids contain two acyl tails which differ in length. These tail-asymmetric lipids are shown to contribute to temperature sensitivity, oxygen adaptability, and membrane fluidity. Membranes of a highly virulent intracellular bacterium, Francisella tularensis, contain highly tail-asymmetric 1-lignoceroyl-2-decanoyl-sn-glycero-3-phosphatidylethanolamine (XJPE) lipids which were previously shown to inhibit inflammatory responses in host cells. XJPE tails have unusually high asymmetry, and how they contribute to membrane properties on a molecular level is unknown. Here, we use small angle X-ray scattering and molecular dynamics simulations to investigate how varying XJPE ratios alters properties of simple membranes. Our results demonstrate that at high concentration they promote liquid-to-gel transition in otherwise liquid membranes, while at low concentration they are tolerated well, minimally altering membrane properties. In liquid membranes, XJPE lipids dynamically adopt two main conformations; with the long tail extended into the opposing leaflet or bent-back residing in its own leaflet. When added to both leaflets XJPE primarily adopts an extended confirmation, while asymmetric addition results in more bent-back orientations. The former increases tail ordering and the latter decreases it. XJPE tails adopt different conformations that induce composition- and leaflet-dependent bidirectional effect on membrane fluidity and this suggests that Francisella tularensis could use tail asymmetry to facilitate vesicle fusion and destabilize host cells. The effect of tail-asymmetric lipids on complex membranes should be further investigated to reveal the regulatory roles of high tail asymmetry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Predicting metal-binding proteins and structures through integration of evolutionary-scale and physics-based modeling

Metals are essential elements in all living organisms, binding to approximately 50% of proteins. They serve to stabilize proteins, catalyze reactions, regulate activities, and fulfill various physiological and pathological functions. While there have been many advancements in determining the structures of protein-metal complexes, numerous metal-binding proteins still need to be identified through computational methods and validated through experiments. Here, to address this need, we have developed the ESMBind workflow, which combines evolutionary scale modeling (ESM) for metal-binding prediction and physics-based protein-metal modeling. Our approach utilizes the ESM-2 and ESM-IF models to predict metal-binding probability at the residue level. In addition, we have designed a metal-placement method and energy minimization technique to generate detailed 3D structures of protein-metal complexes. Our workflow outperforms other models in terms of residue and 3D-level predictions. To demonstrate its effectiveness, we applied the workflow to 142 uncharacterized fungal pathogen proteins and predicted metal-binding proteins involved in fungal infection and virulence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bacterial effectors mimicking ubiquitin-proteasome pathway tweak plant immunity

Plant pathogenic Gram-negative bacteria evade the host plant immune system by secreting Type III (T3E) and Type IV effector (T4E) proteins into the plant cytoplasm. Mostly T3Es are secreted into the plant cells to establish pathogenicity by affecting the vital plant process viz. metabolic pathways, signal transduction and hormonal regulation. Ubiquitin-26S proteasome system (UPS) exists as one of the important pathways in plants to control plant immunity and various cellular processes by employing several enzymes and enzyme components. Pathogenic and non-pathogenic bacteria are found to secrete effectors into plants with structural and/or functional similarity to UPS pathway components like ubiquitin E3 ligases, F-box domains, cysteine proteases, inhibitor of host UPS or its components, etc. The bacterial effectors mimic UPS components and target plant resistance proteins for degradation by proteasomes, thereby taking control over the host cellular activities as a strategy to exert virulence. Thus, the bacterial effectors circumvent plant cellular pathways leading to infection and disease development. Finally, this review highlights known bacterial T3E and T4E proteins that function and interfere with the ubiquitination pathway to regulate the immune system of plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of the Extracellular Region of the Bacterial Type VIIb Secretion System Subunit EsaA

Gram-positive bacteria use type VII secretion systems (T7SSs) to export effector proteins that manipulate the physiology of nearby prokaryotic and eukaryotic cells. Several mycobacterial T7SSs have established roles in virulence. By contrast, the genetically distinct T7SSb pathway found in Firmicutes bacteria more often functions to mediate bacterial competition. A lack of structural information on the T7SSb has limited the understanding of effector export by this protein secretion apparatus. Here, we present the 2.4 angstrom crystal structure of the extracellular region of the T7SSb subunit EsaA from Streptococcus gallolyticus. Our structure reveals that homodimeric EsaA is an elongated, arrow-shaped protein with a surface-accessible "tip", which in some species of bacteria serves as a receptor for lytic bacteriophages. Because it is the only T7SSb subunit large enough to traverse the peptidoglycan layer of Firmicutes, we propose that EsaA plays a critical role in transporting effectors across the entirety of the Gram-positive cell envelope.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cross-reactive immunogenicity of group A streptococcal vaccines designed using a recurrent neural network to identify conserved M protein linear epitopes

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). Furthermore, to examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ≥60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1–50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structure-guided design of a broadly cross-reactive multivalent group a streptococcal vaccine

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal region of the M protein is variable in sequence, defines the M/emm type, and contains epitopes that elicit opsonic antibodies that protect animals from challenge infections. Although there are >200 M types of Strep A, there is now evidence that structurally related M proteins can be grouped into clusters and that immunity may be cluster-specific in addition to M type-specific. This observation has led to recent studies of structure-based design of multivalent M peptide vaccines to select peptides predicted to cross-react with heterologous M types to improve vaccine coverage. In the current study, we have applied a refined series of peptide structural algorithms to predict immunological cross-reactivity among 117N-terminal M peptides representing the most prevalent M types of Strep A. Based on the results of the structural analyses, in combination with global M type prevalence data, we constructed a 32-valent vaccine containing 19 cross-reactive vaccine candidates predicted to cross-react with 37 heterologous M peptides to which were added 13 type-specific M peptides. Further, the 4-protein recombinant vaccine was immunogenic in rabbits and elicited significant levels of antibodies against 31/32 (97%) vaccine peptides and 28/37 (76%) peptides predicted to cross-react. The vaccine antisera also promoted opsonophagocytic killing of vaccine and cross-reactive M types of Strep A. Based on a recent analysis of M type prevalence of Strep A, the potential global coverage of the 32-valent vaccine is ~90%, ranging from 68% in Africa to 95% in North America. Our results indicate the utility of structure-based design that may be applied to future studies of broadly protective M peptide vaccines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Polyproline peptide targets Klebsiella pneumoniae polysaccharides to collapse biofilms

Hypervirulent Klebsiella pneumoniae is known for its increased extracellular polysaccharide production. Biofilm matrices of hyper- virulent K. pneumoniae have increased polysaccharide abundance and are uniquely susceptible to disruption by peptide bactenecin 7 (bac7 (1–35)). Here, using confocal microscopy, we show that poly- saccharides within the biofilm matrix collapse following bac7 (1–35) treatment. This collapse led to the release of cells from the biofilm, which were then killed by the peptide. Characterization of truncated peptide analogs revealed that their interactions with polysaccharide were responsible for the biofilm matrix changes that accompany bac7 (1–35) treatment. Ultraviolet photodissociation mass spec- trometry with the parental peptide or a truncated analog bac7 (10–35) reveal the important regions for bac7 (1–35) complexing with polysaccharides. Finally, we tested bac7 (1–35) using a murine skin abscess model and observed a significant decrease in the bacte- rial burden. These findings unveil the potential of bac7 (1–35) poly- saccharide interactions to collapse K. pneumoniae biofilms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Enzymatic Biocontrol of Fire Blight ( Erwinia amylovora ) Using an Engineered Glycosyl Hydrolase

Current management of fire blight, caused by Erwinia amylovora , relies heavily on streptomycin a practice that has contributed to the emergence of antibiotic-resistant strains and raised environmental and regulatory concerns. Enzyme-based biocontrol agents offer a promising antibiotic-free approach that combines target specificity with environmental compatibility. This study evaluates CAase, a bacteriophage-derived glycosyl hydrolase, for its ability to disrupt E. amylovora biofilms and reduce disease severity. Biochemical assays and scanning electron microscopy confirmed that CAase efficiently degraded the extracellular polysaccharide (EPS) matrix, releasing cells from biofilms. Gas chromatography–mass spectrometry (GC–MS) linkage analysis of EPS isolated from two Erwinia amylovora strains demonstrated that CAase preferentially cleaves galactan-rich amylovoran produced by strain EA273, while exhibiting only limited activity toward the levan-rich EPS from strain EA1430. Functional assays revealed that CAase reduced bacterial viability by nearly 2 orders of magnitude at higher enzyme concentrations, strongly suppressed surface motility, and induced ultrastructural damage visible by transmission electron microscopy. Importantly, field trials showed that CAase significantly lowered blossom and shoot blight incidence under orchard conditions. These results highlight CAase as a potent enzyme-based strategy for reducing the virulence of E. amylovora and demonstrate its potential as a sustainable alternative to antibiotics in fire blight management.

antibiotic resistance↗
DOE OSTI2022

In Vitro and In Vivo Inhibition of the Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT by Amidinoureas

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure–activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Ca v 1.2 and Na v 1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

SARS-CoV-2 M pro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals

The main protease of SARS-CoV-2 (M pro ) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in M pro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 M pro mutants provided structural insight into their altered functionality. Additionally, we show M pro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting M pro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗