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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Synthetic β-sheets mimicking fibrillar and oligomeric structures for evaluation of spectral X-ray scattering technique for biomarker quantification

Archetypical cross-β spines sharpen the boundary between functional and pathological proteins including β-amyloid, tau, α-synuclein and transthyretin are linked to many debilitating human neurodegenerative and non-neurodegenerative amyloidoses. An increased focus on development of pathogenic β-sheet specific fluid and imaging structural biomarkers and conformation-specific monoclonal antibodies in targeted therapies has been recently observed. Identification and quantification of pathogenic oligomers remain challenging for existing neuroimaging modalities. We propose two artificial β-sheets which can mimic the nanoscopic structural characteristics of pathogenic oligomers and fibrils for evaluating the performance of a label free, X-ray based biomarker detection and quantification technique. Highly similar structure with elliptical cross-section and parallel cross-β motif is observed among recombinant α-synuclein fibril, Aβ-42 fibril and artificial β-sheet fibrils. We then use these β-sheet models to assess the performance of spectral small angle X-ray scattering (sSAXS) technique for detecting β-sheet structures. sSAXS showed quantitatively accurate detection of antiparallel, cross-β artificial oligomers from a tissue mimicking environment and significant distinction between different oligomer packing densities such as diffuse and dense packings. The proposed synthetic β-sheet models mimicked the nanoscopic structural characteristics of β-sheets of fibrillar and oligomeric states of Aβ and α-synuclein based on the ATR-FTIR and SAXS data. The tunability of β-sheet proportions and shapes of structural motifs, and the low-cost of these β-sheet models can become useful test materials for evaluating β-sheet or amyloid specific biomarkers in a wide range of neurological diseases. By using the proposed synthetic β-sheet models, our study indicates that the sSAXS has potential to evaluate different stages of β-sheet-enriched structures including oligomers of pathogenic proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Phylogenetic Analyses of Shigella and Enteroinvasive Escherichia coli for the Identification of Molecular Epidemiological Markers: Whole-Genome Comparative Analysis Does Not Support Distinct Genera Designation

As a leading cause of bacterial dysentery, Shigella represents a significant threat to public health and food safety. Related, but often overlooked, enteroinvasive Escherichia coli (EIEC) can also cause dysentery. Current typing methods have limited ability to identify and differentiate between these pathogens despite the need for rapid and accurate identification of pathogens for clinical treatment and outbreak response. We present a comprehensive phylogeny of Shigella and EIEC using whole genome sequencing of 169 samples, constituting unparalleled strain diversity, and observe a lack of monophyly between Shigella and EIEC and among Shigella taxonomic groups. The evolutionary relationships in the phylogeny are supported by analyses of population structure and hierarchical clustering patterns of translated gene homolog abundance. Lastly, we identified a panel of 404 single nucleotide polymorphism (SNP) markers specific to each phylogenetic cluster for more accurate identification of Shigella and EIEC. Our findings show that Shigella and EIEC are not distinct evolutionary groups within the E. coli genus and, thus, EIEC as a group is not the ancestor to Shigella. The multiple analyses presented provide evidence for reconsidering the taxonomic placement of Shigella. The SNP markers offer more discriminatory power to molecular epidemiological typing methods involving these bacterial pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genetic diversity, population structure and anthracnose resistance response in a novel sweet sorghum diversity panel

Sweet sorghum is an attractive feedstock for the production of renewable chemicals and fuels due to the readily available fermentable sugars that can be extracted from the juice, and the additional stream of fermentable sugars that can be obtained from the cell wall polysaccharides in the bagasse. An important selection criterion for new sweet sorghum germplasm is resistance to anthracnose, a disease caused by the fungal pathogen Colletotrichum sublineolum. The identification of novel anthracnose-resistance sources present in sweet sorghum germplasm offers a fast track towards the development of new resistant sweet sorghum germplasm. We established a sweet sorghum diversity panel (SWDP) of 272 accessions from the USDA-ARS National Plant Germplasm (NPGS) collection that includes landraces from 22 countries and advanced breeding material, and that represents ~15% of the NPGS sweet sorghum collection. Genomic characterization of the SWDP identified 171,954 single nucleotide polymorphisms (SNPs) with an average of one SNP per 4,071 kb. Population structure analysis revealed that the SWDP could be stratified into four populations and one admixed group, and that this population structure could be aligned to sorghum’s racial classification. Results from a two-year replicated trial of the SWDP for anthracnose resistance response in Texas, Georgia, Florida, and Puerto Rico showed 27 accessions to be resistant across locations, while 145 accessions showed variable resistance response against local pathotypes. A genome-wide association study identified 16 novel genomic regions associated with anthracnose resistance. Four resistance loci on chromosomes 3, 6, 8 and 9 were identified against pathotypes from Puerto Rico, and two resistance loci on chromosomes 3 and 8 against pathotypes from Texas. In Georgia and Florida, three resistance loci were detected on chromosomes 4, 5, 6 and four on chromosomes 4, 5 (two loci) and 7, respectively. One resistance locus on chromosome 2 was effective against pathotypes from Texas and Puerto Rico and a genomic region of 41.6 kb at the tip of chromosome 8 was associated with resistance response observed in Georgia, Texas, and Puerto Rico. This publicly available SWDP and the extensive evaluation of anthracnose resistance represent a valuable genomic resource for the improvement of sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira

ABSTRACT Leptospirosis, caused by pathogenic bacteria from the genusLeptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation ofLeptospirahas been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal toLeptospiracells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3′ end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenicLeptospirafrom multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulateLeptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis. IMPORTANCE Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenicLeptospiraspp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system toLeptospiraenhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique inLeptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in theLeptospira borgpeterseniibackground, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms ofLeptospiraand the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Microbiology↗
DOE OSTI2019

Polysaccharide-based liquid storage and transport media for non-refrigerated preservation of bacterial pathogens

The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F . novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22–40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S . aureus . This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F . novicida and S . aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis

Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producingEscherichia coli(STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.

Biochemistry & Molecular Biology↗
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 OSTI2023

Elucidating Mechanisms of Rust Pathogenesis for Engineering Resistance in Poplar

The overall objective of this research was to elucidate the molecular basis for the virulence of Melampsora larici-populina towards Populus spp. in order to address the formidable challenge of engineering durable resistance in poplar against leaf rust. Our original research plan was built on the technically innovative approach of using dual host systems and known host defense proteins and pathogen avirulent effectors along with genome-wide approaches to identify key pathogenesis effectors of Melampsora larici-populina that target poplar defense and nutrient acquisition. Our efforts focused on developing high-throughput experimental approaches and systems to enable us to address our overarching hypothesis that rust effectors with homology to other fungal effectors will either bypass the poplar immune system and/or suppress effector-triggered immunity through conserved mechanisms, and that an analysis of these interaction networks would provide for new approaches to develop rust-resistant poplar. Three main objectives were originally proposed: (1) mine key rust effectors that suppress host immunity; (2) elucidate interaction networks in poplar targeted by key rust effectors; (3) generate transgenic poplar resources for identification of genes involved in pathogen infection or host defense triggered by rust. Generally, we worked towards all three objectives by developing and applying complementary molecular, genomic, biochemical and plant transformation approaches.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Agnostic capture of pathogens for the detection and diagnostics of emerging threats

The continued emergence of pathogens, whether novel, re-emerging, or engineered, poses a persistent global biosecurity and public health challenge. Recent outbreaks, including COVID-19, Lassa fever, Marburg virus, mpox, and avian influenza, underscore the urgent need for robust systems that enable rapid surveillance, early diagnosis, and timely countermeasures before widespread human transmission occurs. In this article, we focus on early detection technologies and systematically evaluate current diagnostic and sensing modalities. We highlight sequencing and spectroscopy as two complementary approaches capable of providing broad, agnostic detection and rich biological insight. Our analysis emphasizes that scientific innovation alone is insufficient: effective preparedness also requires improved data curation, integration, and sharing to build AI-ready resources that accelerate future responses. We argue for coordinated advances in both technological capabilities and supporting infrastructure to enable the rapid identification and characterization of emerging pathogens and to fully leverage modern science against evolving infectious threats.

Environmental health↗
DOE OSTI2024

A Comparative Metagenomic Analysis of Specified Microorganisms in Groundwater for Non-Sterilized Pharmaceutical Products

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome’s taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436–557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Programmed Cell Death in Neurospora crassa Is Controlled by the Allorecognition Determinant rcd-1

Nonself recognition following cell fusion between genetically distinct individuals of the same species in filamentous fungi often results in a programmed cell death (PCD) reaction, where the heterokaryotic fusion cell is compartmentalized and rapidly killed. The allorecognition process plays a key role as a defense mechanism that restricts genome exploitation, resource plundering, and the spread of deleterious senescence plasmids and mycoviruses. Although a number of incompatibility systems have been described that function in mature hyphae, less is known about the PCD pathways in asexual spores, which represent the main infectious unit in various human and plant fungal pathogens. Here, we report the identification of regulator of cell death-1 (rcd-1), a novel allorecognition gene, controlling PCD in germinating asexual spores of Neurospora crassa; rcd-1 is one of the most polymorphic genes in the genomes of wild N. crassa isolates. The coexpression of two antagonistic rcd-1-1 and rcd-1-2 alleles was necessary and sufficient to trigger cell death in fused germlings and in hyphae. Based on analysis of wild populations of N. crassa and N. discreta, rcd-1 alleles appeared to be under balancing selection and associated with trans-species polymorphisms. We shed light on genomic rearrangements that could have led to the emergence of the incompatibility system in Neurospora and show that rcd-1 belongs to a much larger gene family in fungi. Overall, our work contributes toward a better understanding of allorecognition and PCD in an underexplored developmental stage of filamentous fungi.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2017

Microbial Monitoring from the Frontlines to Space: Department of Defense Small Business Innovation Research Technology Aboard the International Space Station

The RAZOR (trademark) EX, a quantitative Polymerase Chain Reaction (qPCR) instrument, is a portable, ruggedized unit that was designed for the Department of Defense (DoD) with its reagent chemistries traceable to a Small Business Innovation Research (SBIR) contract beginning in 2002. The PCR instrument's primary function post 9/11 was to enable frontline soldiers and first responders to detect biological threat agents and bioterrorism activities in remote locations to include field environments. With its success for DoD, the instrument has also been employed by other governmental agencies including Department of Homeland Security (DHS). The RAZOR (Trademark) EX underwent stringent testing by the vendor, as well as through the DoD, and was certified in 2005. In addition, the RAZOR (trademark) EX passed DHS security sponsored Stakeholder Panel on Agent Detection Assays (SPADA) rigorous evaluation in 2011. The identification and quantitation of microbial pathogens is necessary both on the ground as well as during spaceflight to maintain the health of astronauts and to prevent biofouling of equipment. Currently, culture-based monitoring technology has been adequate for short-term spaceflight missions but may not be robust enough to meet the requirements for long-duration missions. During a NASA-sponsored workshop in 2011, it was determined that the more traditional culture-based method should be replaced or supplemented with more robust technologies. NASA scientists began investigating innovative molecular technologies for future space exploration and as a result, PCR was recommended. Shortly after, NASA sponsored market research in 2012 to identify and review current, commercial, cutting edge PCR technologies for potential applicability to spaceflight operations. Scientists identified and extensively evaluated three candidate technologies with the potential to function in microgravity. After a thorough voice-of-the-customer trade study and extensive functional and safety evaluations, the RAZOR (trademark) EX PCR instrument(Bio-Fire Defense, Salt Lake City, UT) was selected as the most promising current technology for spaceflight monitoring applications.

Cherie M Oubre↗
DOE OSTI2020

Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gramnegative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identifed as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1→5)-α-KDOp-(2→partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rapid, antibiotic incubation-free determination of tuberculosis drug resistance using machine learning and Raman spectroscopy

Tuberculosis (TB) is the world’s deadliest infectious disease, with over 1.5 million deaths and 10 million new cases reported anually. The causative organism Mycobacterium tuberculosis (Mtb) can take nearly 40 d to culture, a required step to determine the pathogen’s antibiotic susceptibility. Both rapid identification and rapid antibiotic susceptibility testing of Mtb are essential for effective patient treatment and combating antimicrobial resistance. Here, we demonstrate a rapid, culture-free, and antibiotic incubation-free drug susceptibility test for TB using Raman spectroscopy and machine learning. We collect few-to-single-cell Raman spectra from over 25,000 cells of the Mtb complex strain Bacillus Calmette-Guérin (BCG) resistant to one of the four mainstay anti-TB drugs, isoniazid, rifampicin, moxifloxacin, and amikacin, as well as a pan-susceptible wildtype strain. By training a neural network on this data, we classify the antibiotic resistance profile of each strain, both on dried samples and on patient sputum samples. On dried samples, we achieve >98% resistant versus susceptible classification accuracy across all five BCG strains. In patient sputum samples, we achieve ~79% average classification accuracy. We develop a feature recognition algorithm in order to verify that our machine learning model is using biologically relevant spectral features to assess the resistance profiles of our mycobacterial strains. Finally, we demonstrate how this approach can be deployed in resource-limited settings by developing a low-cost, portable Raman microscope that costs <$5,000. We show how this instrument and our machine learning model enable combined microscopy and spectroscopy for accurate few-to-single-cell drug susceptibility testing of BCG.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. Here, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. Finally, this array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2′,3′-cyclic-PO 4 and 5′-OH ends of tRNA exons into the 3′-OH,2′-PO 4 and 5′-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3′-OH,2′-PO 4 and 5′-PO 4 termini. Here we identify a stand-aloneMucor circinelloidespolynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5′-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales speciesRhizopus azygosporus(RazKIN) andLichtheimia corymbifera(LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg 2+ that illuminates the basis for guanosine nucleotide specificity.

Biochemistry & Molecular Biology↗
DOE OSTI2016

Host-pathogen interactions: A cholera surveillance system

Bacterial pathogen-secreted proteases may play a key role in inhibiting a potentially widespread host-pathogen interaction. Activity-based protein profiling enabled the identification of a major Vibrio cholerae serine protease that limits the ability of a host-derived intestinal lectin to bind to the bacterial pathogen in vivo.

Wright, Aaron T.↗