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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 919 records · Page 51
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Roles of Rack1 Proteins in Fungal Pathogenesis

Pathogenic fungi cause diseases on various organisms. Despite their differences in life cycles, fungal pathogens use well-conserved proteins and pathways to regulate developmental and infection processes. In this review, we focus on Rack1, a multifaceted scaffolding protein involved in various biological processes. Rack1 is well conserved in eukaryotes and plays important roles in fungi, though limited studies have been conducted. To accelerate the study of Rack1 proteins in fungi, we review the functions of Rack1 proteins in model and pathogenic fungi and summarize recent progress on how Rack1 proteins are involved in fungal pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Existence of a Nonzero Worst‐Case ACH for Short‐Term Exposure in Ventilated Indoor Spaces

A well‐ventilated room is essential to reduce the risk of airborne transmission. As such, the scientific community sets minimum limits on ventilation with the idea that increased ventilation reduces pathogen concentration and thus reduces the risk of transmission. In contrast, the upper limit on ventilation is usually determined by human comfort and the need to reduce energy consumption. While average pathogen concentration decreases with increased ventilation, local concentration depends on multiple factors and may not follow the same trend, especially within short exposure times over large separation distances. Here, we show through experiments and high‐fidelity simulations the existence of a worst‐case ventilation where local pathogen concentration increases near the receiving host. This occurs during the type of meetings that were recommended during the pandemic (and in some cases solely authorized) with reduced occupancy adhering to social distancing and short exposure times below 20 minutes. We maintain that for cases of high occupancy and long exposure time, increased ventilation remains necessary.

Construction & Building Technology↗
DOE OSTI2014

Genomic Island Identification Software v 1.0

Genomic islands are key mobile DNA elements in bacterial evolution, that can distinguish pathogenic strains from each other, or distinguish pathogenic strains from non-pathogenic strains. Their detection in genomes is a challenging problem. We present 3 main software components that attack the island detection problem on two different bases: 1) the preference of islands to insert in chromosomal tRNA or tmRNA genes (islander.pl), and 2) islands' sporadic occurrence among closely related strains. The latter principle is employed in both an algorithm (learnedPhyloblocks.pl) and a visualization method (panGenome.pl). Component islander.pl finds islands based on their preference for a particular target gene type. We annotate each tRNA and tmRNA gene, find fragments of each such gene as candidates for the distal ends of islands, and filter candidates to remove false positives. Component learnedPhyloblocks.pl uses islands found by islander.pl and other methods as a training set to find new islands. Reference genomes are aligned using mugsy, then the "phylotypes" or patterns of occurrence in the reference set are determined for each position in the target genome, and those phylotypes most enriched in the training set of islands are followed to detect yet more islands. Component panGenome.pl produces a big-data visualization of the chromosomally-ordered "pan-genome", that includes every gene of every reference genome (x-axis, pan-genome order; y-axis, reference genomes; color-coding, gene presence/absence etc.), islands appearing as dark patches.

Williams, KellyPorter↗
DOE OSTI2015

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses

Background: The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. Results: Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. Conclusions: Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

PRIME: An evaluation framework for protein representation inference and generalization in viral mutation space

Background Protein language models (PLMs) have revolutionized protein fitness prediction, yet their application to rapidly evolving viral pathogens is often confounded by extreme sequence homology. This homology leads to “data leakage” in standard random validation splits, yielding inflated performance metrics that fail to translate into real-world biosurveillance utility. Results We present Protein Representation Inference for Mutation Evaluation (PRIME), a framework that integrates domain-specific fine-tuning with a rigorous position-stratified validation protocol to evaluate viral threats. Using a dataset of 347,432 SARS-CoV-2 receptor binding domain (RBD) sequences, we demonstrate that while random training data split yields deceptive R 2 values (> 0.90), they fail to generalize to novel mutational sites. By benchmarking models up to 650 M parameters, we show that domain-specific fine-tuning of the ESM-C 600 M model with correctly stratified data provides an initial demonstration of predictive signal for binding affinity and expression at unseen mutational sites of binding affinity and expression on unseen sites (R 2 ~0.23), a significant advancement over base foundation models which exhibit no predictive power (R 2 <0). PRIME’s embedding-based clustering identified 3.03% of bat coronavirus sequences as candidates for further experimental prioritization based on their functional similarity to human-infective strains in embedding space, offering a perspective complementary to traditional phylogenetic methods. Conclusion PRIME establishes a new benchmark for the application of PLMs in pathogen surveillance. Our findings demonstrate that state-of-the-art models and fine-tuning, when paired with stratified validation, provide biologically meaningful insights into pathogen evolution and zoonotic risk.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing

Background: Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvements to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq. Results: Time zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples. Conclusions: Our cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve detection sensitivity for foodborne enteric pathogens

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Epidemiological surveillance of common respiratory viruses in patients with suspected COVID-19 in Southwest China

Abstract Background The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the peak season of common respiratory viral infections. However, the clinical symptoms of most SARS-CoV-2 infected patients are not significantly different from those of common respiratory viral infections. Therefore, knowing the epidemiological patterns of common respiratory viruses may be valuable to improve the diagnostic and therapeutic efficacy of patients with suspected COVID-19, especially in Southwest China (a mild epidemic area). Methods A total of 2188 patients with clinically suspected of COVID-19 in Southwest China were recruited from January 21 to February 29, 2020. Nasopharyngeal swabs, throat swabs and sputum specimens were collected to detect SARS-CoV-2 by using real-time reverse transcription-polymerase chain reaction (RT-PCR) and other 12 viruses via PCR fragment analysis combined with capillary electrophoresis. Clinical characteristics and laboratory test findings were acquired from electronic medical records. All data were analyzed to unravel the epidemiological patterns. Results Only 1.1% (24/2188) patients with suspected COVID-19 were eventually confirmed to have SARS-CoV-2 infection, and the most frequently observed symptoms were fever (75.0%, 18/24) and cough (20.8%, 5/24). The overall detection rate of other respiratory pathogens was 10.3% (226/2188). Among them, human rhinovirus (3.2%, 71/2188), human parainfluenza viruses (1.6%, 35/2188), influenza B virus (1.2%, 26/2188) and mycoplasma pneumonia (1.2%, 26/2188) were the predominantly detected pathogens in this study. Moreover, the co-infection was observed in 22 specimens. Notably, one COVID-19 case had a coexisting infection with human parainfluenza virus (4.2%, 1/24) and bocavirus was the most common virus tending to occur in co-infection with other respiratory pathogens. Conclusions This study reveals the epidemiological features of common respiratory viruses and their clinical impact during the ongoing outbreak of COVID-19 in a mild epidemic area. The findings highlight the importance of understanding the transmission patterns of the common respiratory virus in COVID-19 regions, which can provide information support for the development of appropriate treatment plans and health policies, while eliminating unnecessary fear and tension.

Si, Yanjun↗
DOE OSTI2016

Elevated blood lead and cadmium levels associated with chronic infections among non-smokers in a cross-sectional analysis of NHANES data

Background: Experimental animal studies, in vitro experiments, and clinical assessments have shown that metal toxicity can impair immune responses. We analyzed data from a United States representative National Health and Nutrition Examination Survey (NHANES) to explore associations between chronic infections and elevated blood concentrations of lead and cadmium among non-smoking NHANES participants. Methods: NHANES data from 1999 to 2012 were examined and weighted to represent the United States population. Multivariable logistic regression was used to estimate adjusted odds ratios (AOR) and 95 % confidence intervals (CI) for heavy metal associations with seropositivity for Helicobacter pylori, Toxoplasma gondii, and Hepatitis B virus (HBV) infections. Results: Available 2-year survey cycles for infection seroprevalence varied by pathogen, from 1 to 7 cycles. Available sample size, disease seroprevalence, and participant age range also varied by pathogen of interest. After controlling for demographic characteristics and general health condition, an elevated blood lead level above the survey population median was significantly associated with seropositivity for all three pathogens (AORs = 1.2–1.5). In addition, an elevated blood cadmium level above the median was significantly associated with HBV (AOR = 1.5; 95 % CI = 1.2–2.0) and H. pylori (AOR = 1.5; 95 % CI = 1.2–1.7) seropositivity. Age-specific analyses for H. pylori and T. gondii indicated stronger associations among children under 13 years of age, particularly for lead exposure and H. pylori seropositivity, and weaker associations among those over 35 years of age. Conclusions: The results of this cross-sectional human health survey suggest that the immunological effects of lead and cadmium toxicity may be associated with an increased susceptibility to chronic infections.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

Variable genome evolution in fungi after transposon-mediated amplification of a housekeeping gene

Transposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact gene and genome function are largely unknown. Previous analyses revealed that TE-mediated gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis, a histone H3 gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded genome of the banana pathogen, Pseudocercospora fijiensis, was analyzed in greater detail. These analyses identified a housekeeping gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal genera Cercospora, Pseudocercospora and Sphaerulina. These results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified genes, with variable effects on gene and genome evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Detection of Epstein-Barr virus (EBV) in human lymphoma tissue by a novel microbial detection array

Background: Infectious agents are estimated to play a causative role in approximately 20% of cancers worldwide. Viruses, notably the Epstein-Barr virus (EBV), are associated with 10-15% of B-cell lymphomas and are found at a higher frequency in immunosuppressed patients. In this study, we screened human lymphoma tissues using a novel Lawrence Livermore Microbial Detection Array (LLMDA), a comprehensive detection system that contains probes for all sequenced viruses and bacteria. This technology has been applied to identify pathogen-associated diseases. Results: We evaluated samples from 58 cases with various lymphoid tissue disorders using LLMDA. These included 30 B-cell lymphomas (9 indolent and 21 aggressive type), 2 T-cell lymphomas and 2 NK/T cell lymphomas, 4 plasmacytomas as well as 8 specimens of benign lymphoid tissue. Five of 21 high-grade B-cell lymphomas were positive for Epstein-Barr virus-encoded small RNA (EBER+), while all the indolent B-cell lymphomas were EBER-. Similarly, both NK/T cell lymphomas were EBER+, and the benign tissues were EBER-. We also screened 10 cases of post-transplant lymphoproliferative disorder (PTLD). Five of these cases (4 B-cell lymphomas and 1 NK/T cell lymphoma) were EBER+, and the remaining five cases were EBER-. Conclusions: We have confirmed the reliability of the LLMDA methods by detecting EBV in EBV-positive lymphomas while observing no false-positive results in EBV-negative lymphomas. The LLMDA technique provides a sensitive and alternative method for identifying known viral pathogen associated with tumors and may prove useful for future clinical identification of novel cancer-associated viral pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Quantitative differentiation of benign and misfolded glaucoma-causing myocilin variants on the basis of protein thermal stability

Accurate predictions of the pathogenicity of mutations associated with genetic diseases are key to the success of precision medicine. Inherited missense mutations in the myocilin (MYOC) gene, within its olfactomedin (OLF) domain, constitute the strongest genetic link to primary open-angle glaucoma via a toxic gain of function, and thus MYOC is an attractive precision-medicine target. However, not all mutations in MYOC cause glaucoma, and common variants are expected to be neutral polymorphisms. The Genome Aggregation Database (gnomAD) lists ~100 missense variants documented within OLF, all of which are relatively rare (allele frequency <0.001%) and nearly all are of unknown pathogenicity. To distinguish disease-causing OLF variants from benign OLF variants, we first characterized the most prevalent population-based variants using a suite of cellular and biophysical assays, and identified two variants with features of aggregation-prone familial disease variants. Next, we considered all available biochemical and clinical data to demonstrate that pathogenic and benign variants can be differentiated statistically based on a single metric: the thermal stability of OLF. Our results motivate genotyping MYOC in patients for clinical monitoring of this widespread, painless and irreversible ocular disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Functional Metagenomics of Escherichia coli O157:H7 Interactions with Spinach Indigenous Microorganisms during Biofilm Formation

The increase in foodborne outbreaks worldwide attributed to fresh fruit and vegetables suggests that produce may serve as an ecological niche for enteric pathogens. Here we examined the interaction of E. coli O157:H7 (EcO157) with spinach leaf indigenous microorganisms during co-colonization and establishment of a mixed biofilm on a stainless steel surface. Stainless steel surface was selected to mimic the surface of produce-processing equipment, where retention of foodborne pathogens such as EcO157 could serve as a potential source for transmission. We observed a positive effect of spinach-associated microbes on the initial attachment of EcO157, but an antagonistic effect on the EcO157 population at the later stage of biofilm formation. Metagenomic analyses of the biofilm community with the GeoChip revealed an extremely diverse community (gene richness, 23409; Shannon-Weiner index H, 9.55). Presence of EcO157 in the mixed biofilm resulted in a significant decrease in the community a-diversity (t test, P,0.05), indicating a putative competition between the pathogen and indigenous spinach microbes. The decrease in the b-diversity of the EcO157-inoculated biofilm at 48 h (ANOVA, P,0.05) suggested a convergent shift in functional composition in response to EcO157 invasion. The success of EcO157 in the mixed biofilm is likely associated with its metabolic potential in utilizing spinach nutrients: the generation time of EcO157 in spinach lysates at 28uC is , 38 min, which is comparable to that in rich broth. The significant decrease in the abundance of many genes involved in carbon, nitrogen, and phosphorus cycling in the EcO157-inoculated biofilms (t test, P,0.05) further support our conclusion that competition for essential macronutrients is likely the primary interaction between the EcO157 and indigenous spinach-biofilm species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

LIK1, A CERK1-Interacting Kinase, Regulates Plant Immune Responses in Arabidopsis

Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1) as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190) and a leucine-rich repeat receptor like kinase (At3g14840), which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1). The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Constraints-based analysis identifies NAD + recycling through metabolic reprogramming in antibiotic resistant Chromobacterium violaceum

In the post genomic era, high throughput data augment stoichiometric flux balance models to compute accurate metabolic flux states, growth and energy phenotypes. Investigating altered metabolism in the context of evolved resistant genotypes potentially provide simple strategies to overcome drug resistance and induce susceptibility to existing antibiotics. A genome-scale metabolic model (GSMM) for Chromobacterium violaceum, an opportunistic human pathogen, was reconstructed using legacy data. Experimental constraints were used to represent antibiotic susceptible and resistant populations. Model predictions were validated using growth and respiration data successfully. Differential flux distribution and metabolic reprogramming were identified as a response to antibiotics, chloramphenicol and streptomycin. Streptomycin resistant populations (StrpR) redirected tricarboxylic acid (TCA) cycle flux through the glyoxylate shunt. Chloramphenicol resistant populations (ChlR) resorted to overflow metabolism producing acetate and formate. This switch to fermentative metabolism is potentially through excess reducing equivalents and increased NADH/NAD ratios. Reduced proton gradients and changed Proton Motive Force (PMF) induced by antibiotics were also predicted and verified experimentally using flow cytometry based membrane potential measurements. Pareto analysis of NADH and ATP maintenance showed the decoupling of electron transfer and ATP synthesis in StrpR. Redox homeostasis and NAD + cycling through rewiring metabolic flux was implicated in re-sensitizing antibiotic resistant C. violaceum. These approaches can be used to probe metabolic vulnerabilities of resistant pathogens. On the verge of a post-antibiotic era, we foresee a critical need for systems level understanding of pathogens and host interaction to extend shelf life of antibiotics and strategize novel therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro . The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Arrayed Guide RNA Libraries for CRISPR-based Gene Knockout Screening

collaboration to develop arrayed formatted genomic screening libraries based on recently developed genome-editing technology termed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-based screening allows for potent knockout of cellular genes with a genome-wide level scalability. With the development of CRISPR knockout libraries, comprehensive analysis of pathogen-host interaction and therapeutic target discovery can be performed against pathogens of biothreat concern and national security interest. Under this work, a CRISPR knockout screen was executed against the mosquito-borne pathogens Zika virus and Rift Valley fever virus. We identified existing as well as novel host genes involved in infection by these viruses. By discovering host genes essential for infection, we can design anti-viral treatments using those identified gene or gene products as targets. The scope of this partnership was within Sandia’s mission area and national security of reducing biological dangers from emerging infections or biodefense threats.

Negrete, Oscar↗