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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A conserved viral RNA fold enables nuclease resistance across kingdoms of life

Abstract Viral exoribonuclease-resistant RNA (xrRNA) structures block cellular nucleases to produce subgenomic viral RNAs during infection. High sequence variability among xrRNAs from distantly related viruses raises questions about the shared molecular features that enable these RNAs to withstand the strong unwinding forces of exoribonucleases. Here, we present the first structure of a plant-virus xrRNA in its active conformation and uncover universal principles of xrRNA folding. Comparison with the structure of a human-pathogenic flavivirus xrRNA reveals that both share a core structural motif—a protective ring encircling the RNA’s 5′ end—despite lacking sequence similarity. Disrupting this core motif through targeted mutagenesis eliminates exoribonuclease-resistance and attenuates viral infection. We identify hundreds of related structures across multiple virus families, supporting the conservation of this mechanism. Our study demonstrates how distantly related RNA viruses have converged on a common structural strategy to inhibit cellular nucleases, with a universal ring topology as the defining feature of viral xrRNAs.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Topologically associating domain boundaries are required for normal genome function

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1,2,3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11–80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The ChiS-Family DNA-Binding Domain Contains a Cryptic Helix-Turn-Helix Variant

Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2005

Microbial assessment of cabin air quality on commercial airliners

The microbial burdens of 69 cabin air samples collected from commercial airliners were assessed via conventional culture-dependent, and molecular-based microbial enumeration assays. Cabin air samples from each of four separate flights aboard two different carriers were collected via air-impingement. Microbial enumeration techniques targeting DNA, ATP, and endotoxin were employed to estimate total microbial burden. The total viable microbial population ranged from 0 to 3.6 x10 4 cells per 100 liters of air, as assessed by the ATP-assay. When these same samples were plated on R2A minimal medium, anywhere from 2% to 80% of these viable populations were cultivable. Five of the 29 samples examined exhibited higher cultivable counts than ATP derived viable counts, perhaps a consequence of the dormant nature (and thus lower concentration of intracellular ATP) of cells inhabiting these air cabin samples. Ribosomal RNA gene sequence analysis showed these samples to consist of a moderately diverse group of bacteria, including human pathogens. Enumeration of ribosomal genes via quantitative-PCR indicated that population densities ranged from 5 x 10 1 ' to IO 7 cells per 100 liters of air. Each of the aforementioned strategies for assessing overall microbial burden has its strengths and weaknesses; this publication serves as a testament to the power of their use in concert.

microbe↗
DOE OSTI2020

CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data

Abstract CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays—even isolated arrays—from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.

Podlevsky, Joshua D.↗
DOE OSTI2025

Holo-Omics disentangle drought response and biotic interactions among plant, endophyte and pathogen

Holo-omics provide a novel opportunity to study the interactions among fungi from different functional guilds in host plants in field conditions. We address the entangled responses of plant pathogenic and endophytic fungi associated with sorghum when droughted through the assembly of the most abundant fungal, endophyte genome from rhizospheric metagenomic sequences followed by a comparison of its metatranscriptome with the host plant metabolome and transcriptome. The rise in relative abundance of endophytic Acremonium persicinum (operational taxonomic unit 5 (OTU5)) in drought co-occurs with a rise in fungal membrane dynamics and plant metabolites, led by ethanolamine, a key phospholipid membrane component. The negative association between endophytic A. persicinum (OTU5) and plant pathogenic fungi co-occurs with a rise in expression of the endophyte's biosynthetic gene clusters coding for secondary compounds. Endophytic A. persicinum (OTU5) and plant pathogenic fungi are negatively associated under preflowering drought but not under postflowering drought, likely a consequence of variation in fungal fitness responses to changes in the availability of water and niche space caused by plant maturation over the growing season. Our findings suggest that the dynamic biotic interactions among host, beneficial and harmful microbiota in a changing environment can be disentangled by a blending of field observation, laboratory validation, holo-omics and ecological modelling.

Chen, Peilin↗
DOE OSTI2025

Visualizing and analyzing 3D biomolecular structures using Mol* at RCSB.org: Influenza A H5N1 virus proteome case study

The easiest and often most useful way to work with experimentally determined or computationally predicted structures of biomolecules is by viewing their three-dimensional (3D) shapes using a molecular visualization tool. Mol* was collaboratively developed by RCSB Protein Data Bank (RCSB PDB, RCSB.org) and Protein Data Bank in Europe (PDBe, PDBe.org) as an open-source, web-based, 3D visualization software suite for examination and analyses of biostructures. It is capable of displaying atomic coordinates and related experimental data of biomolecular structures together with a variety of annotations, facilitating basic and applied research, training, education, and information dissemination. Across RCSB.org, the RCSB PDB research-focused web portal, Mol* has been implemented to support single-mouse-click atomic-level visualization of biomolecules (e.g., proteins, nucleic acids, carbohydrates) with bound cofactors, small-molecule ligands, ions, water molecules, or other macromolecules. RCSB.org Mol* can seamlessly display 3D structures from various sources, allowing structure interrogation, superimposition, and comparison. Using influenza A H5N1 virus as a topical case study of an important pathogen, we exemplify how Mol* has been embedded within various RCSB.org tools—allowing users to view polymer sequence and structure-based annotations integrated from trusted bioinformatics data resources, assess patterns and trends in groups of structures, and view structures of any size and compositional complexity. In addition to being linked to every experimentally determined biostructure and Computed Structure Model made available at RCSB.org, Standalone Mol* is freely available for visualizing any atomic-level or multi-scale biostructure at rcsb.org/3d-view.

3D biostructure↗
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 OSTI2024

The activity of antimicrobial peptoids against multidrug-resistant ocular pathogens

Background: Ocular infections caused by antibiotic-resistant pathogens can result in partial or complete vision loss. The development of pan-resistant microbial strains poses a significant challenge for clinicians as there are limited antimicrobial options available. Synthetic peptoids, which are sequence-specific oligo-N-substituted glycines, offer potential as alternative antimicrobial agents to target multidrug-resistant bacteria. Methods: The antimicrobial activity of synthesised peptoids against multidrug-resistant (MDR) ocular pathogens was evaluated using the microbroth dilution method. Hemolytic propensity was assessed using mammalian erythrocytes. Peptoids were also incubated with proteolytic enzymes, after which their minimum inhibitory activity against bacteria was re-evaluated. Results: Several alkylated and brominated peptoids showed good inhibitory activity against multidrug-resistant Pseudomonas aeruginosa strains at concentrations of ≤15 μg mL -1 (≤12 µM). Similarly, most brominated compounds inhibited the growth of methicillin-resistant Staphylococcus aureus at 1.9 to 15 μg mL -1 (12 µM). The N-terminally alkylated peptoids caused less toxicity to erythrocytes. The peptoid denoted as TM5 had a high therapeutic index, being non-toxic to either erythrocytes or corneal epithelial cells, even at 15 to 22 times its MIC. Additionally, the peptoids were resistant to protease activity. Conclusions: Peptoids studied here demonstrated potent activity against various multidrug-resistant ocular pathogens. Their properties make them promising candidates for controlling vision-related morbidity associated with eye infections by antibiotic-resistant strains.

60 APPLIED LIFE SCIENCES↗
NASA NTRSDate not supplied

Development of Computational Environmental Microbiome Workflows for the Laboratory and the International Space Station

Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.

G. Marie Sharp↗
DOE OSTI2018

A novel variant of torque teno virus 7 identified in patients with Kawasaki disease

Kawasaki disease (KD), first identified in 1967, is a pediatric vasculitis of unknown etiology that has an increasing incidence in Japan and many other countries. KD can cause coronary artery aneurysms. Its epidemiological characteristics, such as seasonality and clinical picture of acute systemic inflammation with prodromal intestinal/respiratory symptoms, suggest an infectious etiology for KD. Interestingly, multiple host genotypes have been identified as predisposing factors for KD. To explore experimental methodology for identifying etiological agent(s) for KD and to optimize epidemiological study design (particularly the sample size) for future studies, we conducted a pilot study. For a 1-year period, we prospectively enrolled 11 patients with KD. To each KD patient, we assigned two control individuals (one with diarrhea and the other with respiratory infections), matched for age, sex, and season of diagnosis. During the acute phase of disease, we collected peripheral blood, nasopharyngeal aspirate, and feces. We also determined genotypes, to identify those that confer susceptibility to KD. There was no statistically significant difference in the frequency of the risk genotypes between KD patients and control subjects. We also used unbiased metagenomic sequencing to analyze these samples. Metagenomic sequencing and PCR detected torque teno virus 7 (TTV7) in two patients with KD (18%), but not in control subjects (P = 0.111). Sanger sequencing revealed that the TTV7 found in the two KD patients contained almost identical variants in nucleotide and identical changes in resulting amino acid, relative to the reference sequence. Additionally, we estimated the sample size that would be required to demonstrate a statistical correlation between TTV7 and KD. Future larger scale studies with carefully optimized metagenomic sequencing experiments and adequate sample size are warranted to further examine the association between KD and potential pathogens, including TTV7.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

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

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

Biochemistry & Molecular Biology↗
DOE OSTI2025

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

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

09 BIOMASS FUELS↗
DOE OSTI2022

Comparing western (Megascops kennicottii) and whiskered (M. trichopsis) screech-owl microbiomes in southern Arizona using a novel 16S rRNA sequencing method

Microbiomes are essential to a host’s physiology and health. Despite the overall importance of microbiomes to animal health, they remain understudied in wildlife. Microbiomes function as physical barriers to invading pathogens, and changes in the diversity or composition of microbes within a host may disrupt this barrier. In order to use microbiomes in wildlife ecology, knowledge of the natural variation within and among species is essential. We compare the diversity and composition of two avian species that share the same habitat and niche in our study area, the western screech-owl (Megascops kennicottii) and the whiskered screech-owl (M. trichopsis). We used a targeted 16S sequencing method to improve the taxonomic resolution of microbiomes. We found similar measures of alpha diversity between species and sample types (cloacal samples vs. fecal samples). However, there were significant differences in bacterial species richness among nestlings from different nest boxes, and the composition differed between the two bird species and among nestlings from different nest boxes. Western screech-owls had more variation in alpha diversity and composition and had fewer bacterial species in their core microbiome than whiskered screech-owls. Siblings are likely to yield similar findings for microbiomes; thus, sampling nestlings from different nests may be most informative for monitoring population-level changes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Unveiling a pervasive DNA adenine methylation regulatory network in the early-diverging fungus Rhizopus microsporus

Development of the DNA affinity purification and sequencing (DAP-seq) technique has allowed genome-scale studies of transcription factor (TF)-binding sites with high reproducibility. Here, we apply this technique to the human opportunistic pathogen Rhizopus microsporus, a mucoralean fungus belonging to the understudied group of early-diverging fungi. We characterize genome-wide binding sites of 58 TFs encoded by genes regulated through adenine methylation and representing major TF families. This analysis reveals their binding profiles and recognized sequences, expanding and diversifying the catalog of known fungal motifs. By integrating this data with DNA 6-methyladenine profiling, we uncover the extensive direct and indirect impact of this epigenetic modification on the regulation of gene expression. Furthermore, we use the generated data to identify TFs involved in biologically relevant processes such as zinc metabolism and light response. Our work enhances our understanding of regulatory mechanisms in R. microsporus and provides broader insights into gene regulation across the fungal kingdom.

Lax, Carlos [Universidad de Murcia (Spain)] (ORCID↗
DOE OSTI2020

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2–6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36–49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

59 BASIC BIOLOGICAL SCIENCES↗