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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae , an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant Δ VdTrx1 that exhibited significantly higher sensitivity to ROS stress, H 2 O 2 , and tert -butyl hydroperoxide ( t -BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The Δ VdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum , and model plants, Arabidopsis thaliana and Nicotiana benthamiana . Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae .

Tian, Li↗
DOE OSTI2026

High-quality Acinetobacter genomes recovered from combat wounds via metagenomic sequencing resemble cultured isolate genomes

The ability to accurately characterize wound pathogens is critical to informing clinical decisions for wound infections with complex treatment requirements. Acinetobacter baumannii is an impactful nosocomial pathogen in combat wounds and civilian hospital-acquired infections. An informed understanding of the phylogenetics and epidemiology of A. baumannii infections in military and civilian environments could guide approaches that improve antibiotic treatment regimens for both military and civilian patients. Whole-genome data for bacterial strains can be difficult to obtain due to challenges in culturing isolates from preserved military specimens. Metagenomic sequencing and assembly create opportunities for genomic analysis of pathogens directly from clinical specimens. The ability to perform comparative analyses between metagenome-derived genomes and culture-derived genomes would support a range of comparative bacterial genomic studies. Wound tissue biopsy and effluent samples from combat injuries were subjected to metagenomic sequencing and assembly. In total, 42 microbial metagenome-assembled genomes (MAGs) were obtained directly from metagenomic sequence data, 36 of which were designated “high” quality. Thirty of these genomes corresponded to Acinetobacter, with 29 mapping specifically to A. baumannii. Other observed genera included Bordetella, Citrobacter, Escherichia, and Pseudomonas. Single-copy and multi-copy orthologs were identified across Acinetobacter MAGs and publicly available isolate genomes derived from military and civilian sources. Both MAG and military isolate genomes were annotated with antimicrobial resistance data, and MAG genomes were statistically comparable to genomes obtained from isolates. Our results highlight the potential of de novo metagenome assembly for enabling high-resolution characterization directly from clinical specimens, thereby improving diagnostic precision, guiding antimicrobial stewardship, and enhancing understanding of pathogen evolution across diverse healthcare and battlefield environments.

Acinetobacter baumannii↗
DOE OSTI2023

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

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 OSTI2023

Application of quasimetagenomics methods to define microbial diversity and subtype Listeria monocytogenes in dairy and seafood production facilities

Microorganisms frequently colonize surfaces within food production facilities. Detection of Listeria monocytogenes in this setting relies on culture-dependent methods, but the complex dynamics of bacterial interactions within these environments and their impact on pathogen detection remain largely unexplored. To address this challenge, we applied both 16S rRNA and shotgun quasimetagenomic (enriched microbiome) sequencing of swab culture enrichments from five seafood and seven dairy production environments. Utilizing 16S rRNA amplicon sequencing, we observed variability between 355 samples taken from these 12 production facilities and a distinctive microbiome for each environment. With shotgun quasimetagenomic sequencing, we were able to assemble L. monocytogenes metagenome-assembled genomes (MAGs) from 28 of the 32 culture-positive samples. We compared these MAGs to their corresponding whole-genome sequencing assemblies, which resulted in two polyphyletic clades consisting of L. monocytogenes lineages I and II with 13,195 and 25,556 single-nucleotide polymorphism sites, respectively. The remaining four MAGs did not produce sufficient genome coverage. To understand and establish limits for pathogen detection and subtyping using shotgun quasimetagenomics, these same data sets were downsampled in slilico to produce a titration series of abundances of L. monocytogenes and analyzed. Pathogen detection was achieved for all downsampled data sets, even those with only 3× genome coverage. This study contributes to the understanding of microbial diversity within food production environments and presents insights into the level of genome coverage needed in a metagenome sequencing data set to detect, subtype, and source track a foodborne pathogen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genomic and phenotypic comparison of two variants of multidrug-resistant Salmonella enterica serovar Heidelberg isolated during the 2015–2017 multi-state outbreak in cattle

Salmonella enterica subspecies enterica serovar Heidelberg (Salmonella Heidelberg) has caused several multistate foodborne outbreaks in the United States, largely associated with the consumption of poultry. However, a 2015–2017 multidrug-resistant (MDR) Salmonella Heidelberg outbreak was linked to contact with dairy beef calves. Traceback investigations revealed calves infected with outbreak strains of Salmonella Heidelberg exhibited symptoms of disease frequently followed by death from septicemia. To investigate virulence characteristics of Salmonella Heidelberg as a pathogen in bovine, two variants with distinct pulse-field gel electrophoresis (PFGE) patterns that differed in morbidity and mortality during the multistate outbreak were genotypically and phenotypically characterized and compared. Strain SX 245 with PFGE pattern JF6X01.0523 was identified as a dominant and highly pathogenic variant causing high morbidity and mortality in affected calves, whereas strain SX 244 with PFGE pattern JF6X01.0590 was classified as a low pathogenic variant causing less morbidity and mortality. Comparison of whole-genome sequences determined that SX 245 lacked ~200 genes present in SX 244, including genes associated with the IncI1 plasmid and phages; SX 244 lacked eight genes present in SX 245 including a second YdiV Anti-FlhC(2)FlhD(4) factor, a lysin motif domain containing protein, and a pentapeptide repeat protein. RNA-sequencing revealed fimbriae-related, flagella-related, and chemotaxis genes had increased expression in SX 245 compared to SX 244. Furthermore, SX 245 displayed higher invasion of human and bovine epithelial cells than SX 244. These data suggest that the presence and up-regulation of genes involved in type 1 fimbriae production, flagellar regulation and biogenesis, and chemotaxis may play a role in the increased pathogenicity and host range expansion of the Salmonella Heidelberg isolates involved in the bovine-related outbreak.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Development of LpxH Inhibitors Chelating the Active Site Dimanganese Metal Cluster of LpxH

Abstract Despite the widespread emergence of multidrug‐resistant nosocomial Gram‐negative bacterial infections and the major public health threat it brings, no new class of antibiotics for Gram‐negative pathogens has been approved over the past five decades. Therefore, there is an urgent medical need for developing effective novel antibiotics against multidrug‐resistant Gram‐negative pathogens by targeting previously unexploited pathways in these bacteria. To fulfill this crucial need, we have been investigating a series of sulfonyl piperazine compounds targeting LpxH, a dimanganese‐containing UDP‐2,3‐diacylglucosamine hydrolase in the lipid A biosynthetic pathway, as novel antibiotics against clinically important Gram‐negative pathogens. Inspired by a detailed structural analysis of our previous LpxH inhibitors in complex with K. pneumoniae LpxH ( Kp LpxH), here we report the development and structural validation of the first‐in‐class sulfonyl piperazine LpxH inhibitors, JH‐LPH‐45 ( 8 ) and JH‐LPH‐50 ( 13 ), that achieve chelation of the active site dimanganese cluster of Kp LpxH. The chelation of the dimanganese cluster significantly improves the potency of JH‐LPH‐45 ( 8 ) and JH‐LPH‐50 ( 13 ). We expect that further optimization of these proof‐of‐concept dimanganese‐chelating LpxH inhibitors will ultimately lead to the development of more potent LpxH inhibitors for targeting multidrug‐resistant Gram‐negative pathogens.

Pharmacology & Pharmacy↗
DOE OSTI2023

Single-cell profiling of Arabidopsis leaves to Pseudomonas syringae infection

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We expose Arabidopsis to Pseudomonas syringae or mock treatment and profile >11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from both treatments identifies distinct pathogen-responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection reveals a continuum of disease progression from an immune to a susceptible state. Confocal imaging of promoter-reporter lines for transcripts enriched in immune cell clusters shows expression surrounding substomatal cavities colonized or in close proximity to bacterial colonies, suggesting that cells within immune clusters represent sites of early pathogen invasion. Susceptibility clusters exhibit more general localization and are highly induced at later stages of infection. Overall, our work shows cellular heterogeneity within an infected leaf and provides insight into plant differential response to infection at a single-cell level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Fortifying the frontier: cell wall modifications during plant immunity

The plant cell wall (CW) was long thought to be a rigid barrier encasing the plant cell and protecting it against biotic and abiotic stressors. Different CW polysaccharides interact with each other, and modifications of either the components or organization of these polysaccharides result in impaired growth or immunity. Emerging evidence suggests that the CW is dynamically modified and reorganized based on internal and external cues. Thus, the CW is both the first barrier that pathogens encounter and the critical final step in defense signaling that leads to fortification of the CW. Here, in this work, we review recent findings on how CW components are remodeled to fortify the CW upon pathogen attack and propose a novel concept: layered CW remodeling as an immune strategy. Within this framework, we categorize three interconnected layers of CW remodeling upon pathogen attack: (i) rapid and reversible CW depositions that provide immediate but transient protection; (ii) flexible modifications with plausible signaling functions that integrate defense and surveillance; and (iii) irreversible fortifications that encase pathogen, delimiting infected cells from uninfected cells. This layered framework provides a cohesive view of how different CW modifications are integrated into, and contribute to, plant defense. We also discuss the challenges in studying CW modifications during biotic stresses and highlight important questions that remain unanswered.

Bhandari, Deepak D. [Michigan State Univ., East La↗
DOE OSTI2023

Large-scale genomic analyses with machine learning uncover predictive patterns associated with fungal phytopathogenic lifestyles and traits

Abstract Invasive plant pathogenic fungi have a global impact, with devastating economic and environmental effects on crops and forests. Biosurveillance, a critical component of threat mitigation, requires risk prediction based on fungal lifestyles and traits. Recent studies have revealed distinct genomic patterns associated with specific groups of plant pathogenic fungi. We sought to establish whether these phytopathogenic genomic patterns hold across diverse taxonomic and ecological groups from the Ascomycota and Basidiomycota, and furthermore, if those patterns can be used in a predictive capacity for biosurveillance. Using a supervised machine learning approach that integrates phylogenetic and genomic data, we analyzed 387 fungal genomes to test a proof-of-concept for the use of genomic signatures in predicting fungal phytopathogenic lifestyles and traits during biosurveillance activities. Our machine learning feature sets were derived from genome annotation data of carbohydrate-active enzymes (CAZymes), peptidases, secondary metabolite clusters (SMCs), transporters, and transcription factors. We found that machine learning could successfully predict fungal lifestyles and traits across taxonomic groups, with the best predictive performance coming from feature sets comprising CAZyme, peptidase, and SMC data. While phylogeny was an important component in most predictions, the inclusion of genomic data improved prediction performance for every lifestyle and trait tested. Plant pathogenicity was one of the best-predicted traits, showing the promise of predictive genomics for biosurveillance applications. Furthermore, our machine learning approach revealed expansions in the number of genes from specific CAZyme and peptidase families in the genomes of plant pathogens compared to non-phytopathogenic genomes (saprotrophs, endo- and ectomycorrhizal fungi). Such genomic feature profiles give insight into the evolution of fungal phytopathogenicity and could be useful to predict the risks of unknown fungi in future biosurveillance activities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Arabidopsis bZIP11 Is a Susceptibility Factor During Pseudomonas syringae Infection

The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Biochemistry & Molecular Biology↗
DOE OSTI2025

Apple Bitter Rot: Biology, Ecology, Omics, Virulence Factors, and Management of Causal Colletotrichum Species

ABSTRACT Apple bitter rot is caused by various Colletotrichum spp. that threaten apple production globally resulting in millions of dollars in damage annually. The fungus causes a decline in fruit quality and yield, eventually rotting the fruit and rendering it inedible. The pathogen is difficult to keep out of orchards because of its broad host range and transmissibility by rain splash and insects. Once the disease manifests, pathogen identification is difficult due to evolving taxonomy and similar morphology between species. Current management strategies are threatened by an increase in fungicide resistance and regulations on many multisite fungicides, leading to a pressing need for new management options for control. This review aims to summarise the most current knowledge regarding the biology, virulence factors, ecology, omics and emerging management strategies for Colletotrichum species that cause apple bitter rot. Taxonomy Colletotrichum species—Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Class Sordariomycetes, Order Glomerellales, Family Glomerellaceae, Genus Colletotrichum . Biology Hemibiotrophic pathogen with a wide host range that establishes a biotrophic interaction where it penetrates host plants using appressoria followed by a switch to necrotrophy causing rot symptoms. Toxins Cercosporin, colletotrichins, colletotric acid, ferricrocin. Host Range The host range varies by species but largely occurs on dicotyledonous plants and is less prevalent on monocots as well as gymnosperms, ferns, mosses and animals (e.g., insects). Disease Symptoms Symptoms often manifest as flat to sunken necrotic areas on fruit. Lesions on leaves and fruit can have concentric rings with abundant pathogen sporulation. Disease Control Colletotrichum spp. are primarily managed by single‐site quinone outside inhibitor (Qol), methyl benzimidazole carbamate (MBC), demethylation inhibitor (DMI) fungicides, and multisite dithiocarbamate and phthalimide fungicides. Susceptibility may vary with species, strain specificity, or geographic region. Other management options include clean stock production, cultural practices, resistance breeding, and biological control through the introduction of protective or competing microorganisms.

Boeckman, Nathanial J. [Plant Pathology Laboratory↗
DOE OSTI2024

Detection of anaerobic and aerobic bacteria from commercial tattoo and permanent makeup inks

ABSTRACT Tattooing and use of permanent makeup (PMU) have dramatically increased over the last decade, with a concomitant increase in ink-related infections. Studies have shown evidence that commercial tattoo and PMU inks are frequently contaminated with pathogenic microorganisms. Considering that tattoo inks are placed into the dermal layer of the skin where anaerobic bacteria can thrive and cause infections in low-oxygen environments, the prevalence of anaerobic and aerobic bacteria should be assessed in tattoo and PMU inks. In this study, we tested 75 tattoo and PMU inks using the analytical methods described in the FDA Bacteriological Analytical Manual Chapter 23 for the detection of both aerobic and anaerobic bacterial contamination, followed by 16S rRNA gene sequencing for microbial identification. Of 75 ink samples, we found 26 contaminated samples with 34 bacterial isolates taxonomically classified into 14 genera and 22 species. Among the 34 bacterial isolates, 19 were identified as possibly pathogenic bacterial strains. Two species, namelyCutibacterium acnes(four strains) andStaphylococcus epidermidis(two strains) were isolated under anaerobic conditions. Two possibly pathogenic bacterial strains,Staphylococcus saprophyticusandC. acnes, were isolated together from the same ink samples (n= 2), indicating that tattoo and PMU inks can contain both aerobic (S. saprophyticus) and anaerobic bacteria (C. acnes). No significant association was found between sterility claims on the ink label and the absence of bacterial contamination. The results indicate that tattoo and PMU inks can also contain anaerobic bacteria. IMPORTANCE The rising popularity of tattooing and permanent makeup (PMU) has led to increased reports of ink-related infections. This study is the first to investigate the presence of both aerobic and anaerobic bacteria in commercial tattoo and PMU inks under aerobic and anaerobic conditions. Our findings reveal that unopened and sealed tattoo inks can harbor anaerobic bacteria, known to thrive in low-oxygen environments, such as the dermal layer of the skin, alongside aerobic bacteria. This suggests that contaminated tattoo inks could be a source of infection from both types of bacteria. The results emphasize the importance of monitoring these products for both aerobic and anaerobic bacteria, including possibly pathogenic microorganisms.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Acinetobacter baumannii Carbapenemase OXA-23 by Impeding Deacylation

Acinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to β-lactam antibiotics by producing β-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of b-lactam antibiotics have been the drugs of choice for treating A. baumannii infections. However, their effectiveness has been significantly diminished due to the pathogen’s production of carbapenem-hydrolyzing class D β-lactamases (CHDLs); thus, new antibiotics and inhibitors of these enzymes are urgently needed. Here, we describe a new carbapenem antibiotic, MA-1-206, in which the canonical C6 hydroxyethyl group has been replaced with hydroxymethyl. The antimicrobial susceptibility studies presented here demonstrated that this compound is more potent than meropenem and imipenem against A. baumannii producing OXA-23, the most prevalent CHDL of this pathogen, and also against strains producing the CHDL OXA-24/40 and the class B metallo-β-lactamase VIM-2. Our kinetic and mass spectrometry studies revealed that this drug is a reversible inhibitor of OXA-23, where inhibition takes place through a branched pathway. X-ray crystallographic studies, molecular docking, and molecular dynamics simulations of the OXA-23-MA-1-206 complex show that the C6 hydroxymethyl group forms a hydrogen bond with the carboxylated catalytic lysine of OXA-23, effectively preventing deacylation. These results provide a promising strategy for designing a new generation of CHDL-resistant carbapenems to restore their efficacy against deadly A. baumannii infections. Carbapenem antibiotics are the drugs of choice for treatment of deadly infections caused by Gram-negative bacteria. However, their efficacy is severely compromised by the wide spread of carbapenem-hydrolyzing class D β-lactamases (CHDLs). The importance of this research is the discovery that substitution of the canonical hydroxyethyl group of carbapenems by a hydroxymethyl significantly enhances stability against inactivation by the major CHDL of Acinetobacter baumannii, OXA-23. These results provide a novel strategy for designing next-generation, carbapenemase-stable carbapenems to fight multidrug-resistant infections caused by Gram-negative pathogens

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

SARS-CoV-2 wastewater variant surveillance: pandemic response leveraging FDA’s GenomeTrakr network

ABSTRACT Wastewater surveillance has emerged as a crucial public health tool for population-level pathogen surveillance. Supported by funding from the American Rescue Plan Act of 2021, the FDA‘s genomic epidemiology program, GenomeTrakr, was leveraged to sequence SARS-CoV-2 from wastewater sites across the United States. This initiative required the evaluation, optimization, development, and publication of new methods and analytical tools spanning sample collection through variant analyses. Version-controlled protocols for each step of the process were developed and published on protocols.io. A custom data analysis tool and a publicly accessible dashboard were built to facilitate real-time visualization of the collected data, focusing on the relative abundance of SARS-CoV-2 variants and sub-lineages across different samples and sites throughout the project. From September 2021 through June 2023, a total of 3,389 wastewater samples were collected, with 2,517 undergoing sequencing and submission to NCBI under the umbrella BioProject,PRJNA757291. Sequence data were released with explicit quality control (QC) tags on all sequence records, communicating our confidence in the quality of data. Variant analysis revealed wide circulation of Delta in the fall of 2021 and captured the sweep of Omicron and subsequent diversification of this lineage through the end of the sampling period. This project successfully achieved two important goals for the FDA’s GenomeTrakr program: first, contributing timely genomic data for the SARS-CoV-2 pandemic response, and second, establishing both capacity and best practices for culture-independent, population-level environmental surveillance for other pathogens of interest to the FDA. IMPORTANCE This paper serves two primary objectives. First, it summarizes the genomic and contextual data collected during a Covid-19 pandemic response project, which utilized the FDA’s laboratory network, traditionally employed for sequencing foodborne pathogens, for sequencing SARS-CoV-2 from wastewater samples. Second, it outlines best practices for gathering and organizing population-level next generation sequencing (NGS) data collected for culture-free, surveillance of pathogens sourced from environmental samples.

Microbiology↗
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 OSTI2022

Host genotype controls ecological change in the leaf fungal microbiome

Leaf fungal microbiomes can be fundamental drivers of host plant success, as they contain pathogens that devastate crop plants and taxa that enhance nutrient uptake, discourage herbivory, and antagonize pathogens. We measured leaf fungal diversity with amplicon sequencing across an entire growing season in a diversity panel of switchgrass ( Panicum virgatum ). We also sampled a replicated subset of genotypes across 3 additional sites to compare the importance of time, space, ecology, and genetics. We found a strong successional pattern in the microbiome shaped both by host genetics and environmental factors. Further, we used genome-wide association (GWA) mapping and RNA sequencing to show that 3 cysteine-rich receptor-like kinases (crRLKs) were linked to a genetic locus associated with microbiome structure. We confirmed GWAS results in an independent set of genotypes for both the internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA markers. Fungal pathogens were central to microbial covariance networks, and genotypes susceptible to pathogens differed in their expression of the 3 crRLKs, suggesting that host immune genes are a principal means of controlling the entire leaf microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparing variability in diagnosis of upper respiratory tract infections in patients using syndromic, next generation sequencing, and PCR-based methods

Early and accurate diagnosis of respiratory pathogens and associated outbreaks can allow for the control of spread, epidemiological modeling, targeted treatment, and decision making–as is evident with the current COVID-19 pandemic. Many respiratory infections share common symptoms, making them difficult to diagnose using only syndromic presentation. Yet, with delays in getting reference laboratory tests and limited availability and poor sensitivity of point-of-care tests, syndromic diagnosis is the most-relied upon method in clinical practice today. Here, we examine the variability in diagnostic identification of respiratory infections during the annual infection cycle in northern New Mexico, by comparing syndromic diagnostics with polymerase chain reaction (PCR) and sequencing-based methods, with the goal of assessing gaps in our current ability to identify respiratory pathogens. Of 97 individuals that presented with symptoms of respiratory infection, only 23 were positive for at least one RNA virus, as confirmed by sequencing. Whereas influenza virus (n = 7) was expected during this infection cycle, we also observed coronavirus (n = 7), respiratory syncytial virus (n = 8), parainfluenza virus (n = 4), and human metapneumovirus (n = 1) in individuals with respiratory infection symptoms. Four patients were coinfected with two viruses. In 21 individuals that tested positive using PCR, RNA sequencing completely matched in only 12 (57%) of these individuals. Few individuals (37.1%) were diagnosed to have an upper respiratory tract infection or viral syndrome by syndromic diagnostics, and the type of virus could only be distinguished in one patient. Thus, current syndromic diagnostic approaches fail to accurately identify respiratory pathogens associated with infection and are not suited to capture emerging threats in an accurate fashion. We conclude there is a critical and urgent need for layered agnostic diagnostics to track known and unknown pathogens at the point of care to control future outbreaks.

60 APPLIED LIFE SCIENCES↗