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At least 901 records · Page 50
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site

Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (M pro ) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on M pro inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Whole-Genome Resequencing to Evaluate Life History Variation in Anadromous Migration of Oncorhynchus mykiss

Anadromous fish experience physiological modifications necessary to migrate between vastly different freshwater and marine environments, but some species such as Oncorhynchus mykiss demonstrate variation in life history strategies with some individuals remaining exclusively resident in freshwater, whereas others undergo anadromous migration. Because there is limited understanding of genes involved in this life history variation across populations of this species, we evaluated the genomic difference between known anadromous ( n = 39) and resident ( n = 78) Oncorhynchus mykiss collected from the Klickitat River, WA, USA, with whole-genome resequencing methods. Sequencing of these collections yielded 5.64 million single-nucleotide polymorphisms that were tested for significant differences between resident and anadromous groups along with previously identified candidate gene regions. Although a few regions of the genome were marginally significant, there was one region on chromosome Omy12 that provided the most consistent signal of association with anadromy near two annotated genes in the reference assembly: COP9 signalosome complex subunit 6 (CSN6) and NACHT, LRR, and PYD domain–containing protein 3 (NLRP3). Previously identified candidate genes for anadromy within the inversion region of chromosome Omy05 in coastal steelhead and rainbow trout were not informative for this population as shown in previous studies. Results indicate that the significant region on chromosome Omy12 may represent a minor effect gene for male anadromy and suggests that this life history variation in Oncorhynchus mykiss is more strongly driven by other mechanisms related to environmental rearing such as epigenetic modification, gene expression, and phenotypic plasticity. Further studies into regulatory mechanisms of this trait are needed to understand drivers of anadromy in populations of this protected species.

Collins, Erin E.↗
DOE OSTI2026

Engineering and Application of a Thermostable MHETase for PET Depolymerization

Enzymatic hydrolysis of poly(ethylene terephthalate) (PET) releases mono(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T m,app ) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043–5 was identified with a comparable thermostability (T m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077 LFK , which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

A deep learning approach to programmable RNA switches

Engineered RNA elements are programmable tools capable of detecting small molecules, proteins, and nucleic acids. Predicting the behavior of these synthetic biology components remains a challenge, a situation that could be addressed through enhanced pattern recognition from deep learning. Here, we investigate Deep Neural Networks (DNN) to predict toehold switch function as a canonical riboswitch model in synthetic biology. To facilitate DNN training, we synthesize and characterize in vivo a dataset of 91,534 toehold switches spanning 23 viral genomes and 906 human transcription factors. DNNs trained on nucleotide sequences outperform (R 2 = 0.43–0.70) previous state-of-the-art thermodynamic and kinetic models (R 2 = 0.04–0.15) and allow for human-understandable attention-visualizations (VIS4Map) to identify success and failure modes. This work shows that deep learning approaches can be used for functionality predictions and insight generation in RNA synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

De Novo Design of a Highly Stable Ovoid TIM Barrel: Unlocking Pocket Shape towards Functional Design

The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels.

Chu, Alexander E.↗
DOE OSTI2022

Improvement of eukaryotic protein predictions from soil metagenomes

During the last decades, metagenomics has highlighted the diversity of microorganisms from environmental or host-associated samples. Most metagenomics public repositories use annotation pipelines tailored for prokaryotes regardless of the taxonomic origin of contigs. Consequently, eukaryotic contigs with intrinsically different gene features, are not optimally annotated. Using a bioinformatics pipeline, we have filtered 7.9 billion contigs from 6,872 soil metagenomes in the JGI’s IMG/M database to identify eukaryotic contigs. We have re-annotated genes using eukaryote-tailored methods, yielding 8 million eukaryotic proteins and over 300,000 orphan proteins lacking homology in public databases. Comparing the gene predictions we made with initial JGI ones on the same contigs, we confirmed our pipeline improves eukaryotic proteins completeness and contiguity in soil metagenomes. The improved quality of eukaryotic proteins combined with a more comprehensive assignment method yielded more reliable taxonomic annotation. This dataset of eukaryotic soil proteins with improved completeness, quality and taxonomic annotation reliability is of interest for any scientist aiming at studying the composition, biological functions and gene flux in soil communities involving eukaryotes.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Prevalence of gp160 polymorphisms known to be related to decreased susceptibility to temsavir in different subtypes of HIV-1 in the Los Alamos National Laboratory HIV Sequence Database

Fostemsavir, a prodrug of the gp120-directed attachment inhibitor temsavir, is indicated for use in heavily treatment-experienced individuals with MDR HIV-1. Reduced susceptibility to temsavir in the clinic maps to discrete changes at amino acid positions in gp160: S375, M426, M434 and M475.To query the Los Alamos National Laboratory (LANL) HIV Sequence Database for the prevalence of polymorphisms at gp160 positions of interest. Full-length gp160 sequences (N = 7560) were queried for amino acid polymorphisms relative to the subtype B consensus at positions of interest; frequencies were reported for all sequences and among subtypes/circulating recombinant forms (CRFs) with ≥10 isolates in the database. Among 239 subtypes in the database, the 5 most prevalent were B (n = 2651, 35.1%), C (n = 1626, 21.5%), CRF01_AE (n = 674, 8.9%), A1 (n = 273, 3.6%) and CRF02_AG (n = 199, 2.6%). Among all 7560 sequences, the most prevalent amino acids at positions of interest (S 375 , 73.5%; M 426 , 82.1%; M 434 , 88.2%; M 475 , 89.9%) were the same as the subtype B consensus. Specific polymorphisms with the potential to decrease temsavir susceptibility (S 375 H/I/M/N/T/Y, M 426 L/P, M 434 I/K and M475I) were found in <10% of isolates of subtypes D, G, A6, BC, F1, CRF07_BC, CRF08_BC, 02A, CRF06_cpx, F2, 02G and 02B. S 375 H and M 475 I were predominant among CRF01_AE (S375H, 99.3%; M 475 I, 76.3%; consistent with previously reported low temsavir susceptibility of this CRF) and 01B (S 375 H, 71.7%; M 475 I, 49.5%). Analysis of the LANL HIV Sequence Database found a low prevalence of gp160 amino acid polymorphisms with the potential to reduce temsavir susceptibility overall and among most of the common subtypes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Clades of huge phages from across Earth’s ecosystems

Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is—to our knowledge—the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR–Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR–Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR–Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth’s ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Regulation of L - and D -Aspartate Transport and Metabolism in Acinetobacter baylyi ADP1

Here, the regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N7-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

National Prevalence of Caprine Prion Protein Genetic Variability at Codons 146, 211, and 222 in Goat Herds in the United States

Scrapie is a neurodegenerative disease that impacts sheep and goats, characterized by gradual and progressive changes in neurological function. Recent research shows that the scrapie incubation period is significantly influenced by specific variations in amino acids within the prion protein gene (PRNP). The objective of this study was to estimate the national prevalence of caprine PRNP genetic variability at codons 146, 211, and 222 in goat populations across the United States. A total of 3052 blood, ear tissue, and brain tissue samples were collected from goats from 50 states. The participating states were categorized into four Veterinary Service (VS) district regions. The samples underwent DNA extraction, and the PRNP variants corresponding to codons 146, 211, and 222 were amplified and sequenced. The analysis of PRNP variants, when compared to the PRNP reference sequence, revealed seven alleles in twelve genotypes. The homozygous 146NN, 211RR, and 222QQ alleles, which have been linked to an increased risk of scrapie, were found to be the most prevalent among all the goats. The heterozygous 222QK, 211RQ, 146SD, 146ND, and 146NS alleles and the homozygous 222KK, 146SS, and 146DD alleles, known to be associated with reduced scrapie susceptibility and a prolonged incubation period after experimental challenge, were found in 1.098% (222QK), 2.33% (211RQ), 0.58% (146SD), 3.13% (146ND), 20.68% (146NS), 0.005% (222KK), 3.31% (146SS), and 0.67% (146DD) of goats, respectively. The 222QK allele was found most frequently in goats tested from the east (VS District 1, 1.59%) and southwest (VS District 4, 1.08%) regions, whereas the 211RQ allele was found most often in goats tested from the Midwest (VS District 2, 8.03%) and east (VS District 1, 6.53%) regions. The 146NS allele was found most frequently in goats tested from the northwest (VS District 3, 29.02%) and southwest (VS District 4, 20.69%) regions. Our results showed that the prevalence of less susceptible genotypes at PRNP codon 146 may be sufficient to use genetic susceptibility testing in some herds. This may reduce the number of goats removed as part of a herd clean-up plan and may promote the selective breeding goats for less susceptible alleles in high-risk herds at the national level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering RuBisCo for food safety

A method of improving a ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) to have a higher protein score is disclosed. The method includes the steps of: making a modified RbcL of the RuBisCo, by, on an RbcL unit of the RuBisCo, either substituting Met for Leu, Phe, Val, or Ile or combinations thereof; substituting Lys for Arg, Thr, or His or combinations thereof; or both of these substitutions. The modified RbcL consequently modifies the RuBisCo and is added to a biomass host where it is stable for homologous recombination. Plastid and nucleus integration was observed. Example RbcL sequences are disclosed with the desirable substitutions. The improved RuBisCo can be used as an improved proteinaceous food source for humans and animals.

Davis, Ryan Wesley↗
DOE OSTI2024

Unveiling the microbial realm with VEBA 2.0: a modular bioinformatics suite for end-to-end genome-resolved prokaryotic, (micro)eukaryotic and viral multi-omics from either short- or long-read sequencing

Abstract The microbiome is a complex community of microorganisms, encompassing prokaryotic (bacterial and archaeal), eukaryotic, and viral entities. This microbial ensemble plays a pivotal role in influencing the health and productivity of diverse ecosystems while shaping the web of life. However, many software suites developed to study microbiomes analyze only the prokaryotic community and provide limited to no support for viruses and microeukaryotes. Previously, we introduced the Viral Eukaryotic Bacterial Archaeal (VEBA) open-source software suite to address this critical gap in microbiome research by extending genome-resolved analysis beyond prokaryotes to encompass the understudied realms of eukaryotes and viruses. Here we present VEBA 2.0 with key updates including a comprehensive clustered microeukaryotic protein database, rapid genome/protein-level clustering, bioprospecting, non-coding/organelle gene modeling, genome-resolved taxonomic/pathway profiling, long-read support, and containerization. We demonstrate VEBA’s versatile application through the analysis of diverse case studies including marine water, Siberian permafrost, and white-tailed deer lung tissues with the latter showcasing how to identify integrated viruses. VEBA represents a crucial advancement in microbiome research, offering a powerful and accessible software suite that bridges the gap between genomics and biotechnological solutions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Hyporheic zone, river, and groundwater metagenome resolved genomes and rpS3 genes in East River Watershed, Colorado USA Summer 2020, 2021

Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from water filter collected across 8 locations along the East River Watershed, CO, and 1 nearby groundwater well. The purpose was to look for connectivity and similarities across the network and to see the impact of the groundwater. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed community composition and strain similarities between the sites and we also compared it to previous metagenomic studies within the watershed looking at floodplain (Matheus Carnevali et al. 2021) and hillslope (Lavy et al. 2019) microbiomes. Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from filters across 8 locations during August 2020 and July 2021. This resulted in 32 samples. The groundwater sample was sequenced at UC Berkley's QB3. The other 31 samples were sequenced at University of Maryland. Metagenomes were assembled using four autobinners and the best bins were selected using dasTool. The genomes were dereplicated at 95% with dRep and the subset of winning genomes were manually curated based on visual inspection of taxonomic profile, GC content, coverage, and a set of 51 bacterial single copy genes (BSCG), and 38 archaeal signal copy genes (ASCG). The dataset includes a zip file of 311 genomes (HZ_River_SW_MAGS_Dereplicated_95.zip). The dataset additionally includes a zipped file of ribosomal protein small subunit 3 (rpS3) proteins from the hyporheic zone and river data (rpS3_Proteins_HZ_River.zip), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a location metadata file (locations.csv). This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

DNA↗
DOE OSTI2020

Desert truffle genomes reveal their reproductive modes and new insights into plant–fungal interaction and ectendomycorrhizal lifestyle

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

ABSTRACT Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeonHaloferax volcanii, withelp3andpat2proposed as a synthetic lethal gene pair. Here, we advance these findings by performing single and double mutagenesis ofelp3with thepat1andpat2lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of Δelp3,Δpat1Δelp3, and Δpat2Δelp3mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence thatelp3andpat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCE Here, we reveal by whole-genome sequencing that the GNAT family gene homologselp3andpat2can be deleted in the sameHaloferax volcaniistrain. Beyond the targeted deletions, minimal differences between the parent and Δelp3Δpat2mutant were observed, suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

Microbiology↗
DOE OSTI2022

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗