Mapping disease regulatory circuits at cell-type resolution from single-cell multiomics data.

Resolving chromatin-remodeling-linked gene expression changes at cell-type resolution is important for understanding disease states. Here we describe MAGICAL (Multiome Accessibility Gene Integration Calling and Looping), a hierarchical Bayesian approach that leverages paired single-cell RNA sequencing and single-cell transposase-accessible chromatin sequencing from different conditions to map disease-associated transcription factors, chromatin sites, and genes as regulatory circuits. By simultaneously modeling signal variation across cells and conditions in both omics data types, MAGICAL achieved high accuracy on circuit inference. We applied MAGICAL to study Staphylococcus aureus sepsis from peripheral blood mononuclear single-cell data that we generated from subjects with bloodstream infection and uninfected controls. MAGICAL identified sepsis-associated regulatory circuits predominantly in CD14 monocytes, known to be activated by bacterial sepsis. We addressed the challenging problem of distinguishing host regulatory circuit responses to methicillin-resistant and methicillin-susceptible S. aureus infections. Although differential expression analysis failed to show predictive value, MAGICAL identified epigenetic circuit biomarkers that distinguished methicillin-resistant from methicillin-susceptible S. aureus infections.

Ohio Coronavirus Wastewater Monitoring Network: Implementation of Statewide Monitoring for Protecting Public Health.

CONTEXT: Prior to the COVID-19 pandemic, wastewater influent monitoring for tracking disease burden in sewered communities was not performed in Ohio, and this field was only on the periphery of the state academic research community. PROGRAM: Because of the urgency of the pandemic and extensive state-level support for this new technology to detect levels of community infection to aid in public health response, the Ohio Water Resources Center established relationships and support of various stakeholders. This enabled Ohio to develop a statewide wastewater SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) monitoring network in 2 months starting in July 2020. IMPLEMENTATION: The current Ohio Coronavirus Wastewater Monitoring Network (OCWMN) monitors more than 70 unique locations twice per week, and publicly available data are updated weekly on the public dashboard. EVALUATION: This article describes the process and decisions that were made during network initiation, the network progression, and data applications, which can inform ongoing and future pandemic response and wastewater monitoring. DISCUSSION: Overall, the OCWMN established wastewater monitoring infrastructure and provided a useful tool for public health professionals responding to the pandemic.

Human Immune Cell Epigenomic Signatures in Response to Infectious Diseases and Chemical Exposures.

Variations in DNA methylation patterns in human tissues have been linked to various environmental exposures and infections. Here, we identified the DNA methylation signatures associated with multiple exposures in nine major immune cell types derived from peripheral blood mononuclear cells (PBMCs) at single-cell resolution. We performed methylome sequencing on 111,180 immune cells obtained from 112 individuals who were exposed to different viruses, bacteria, or chemicals. Our analysis revealed 790,662 differentially methylated regions (DMRs) associated with these exposures, which are mostly individual CpG sites. Additionally, we integrated methylation and ATAC-seq data from same samples and found strong correlations between the two modalities. However, the epigenomic remodeling in these two modalities are complementary. Finally, we identified the minimum set of DMRs that can predict exposures. Overall, our study provides the first comprehensive dataset of single immune cell methylation profiles, along with unique methylation biomarkers for various biological and chemical exposures.

Analysis of metatranscriptomic methods to enable wastewater-based biosurveillance of all infectious diseases.

Introduction: Wastewater-based surveillance emerged during the COVID-19 pandemic as an efficient way to quickly screen large populations, monitor infectious disease transmission over time, and identify whether more virulent strains are becoming more prevalent in the region without burdening the health care system with individualized testing. Ohio was one of the first states to implement wastewater monitoring through its Ohio Coronavirus Wastewater Monitoring Network (OCWMN), originally tracking the prevalence of COVID-19 by quantitative qPCR from over 67 sites across the state. The OCWMN evolved along with the pandemic to include sequencing the SARS-CoV-2 genome to assess variants of concern circulating within the population. As the pandemic wanes, networks such as OCWMN can be expanded to monitor other infectious diseases and outbreaks of interest to the health department to reduce the burden of communicable diseases. However, most surveillance still utilizes qPCR based diagnostic tests for individual pathogens, which is hard to scale for surveillance of multiple pathogens. Methods: Here we have tested several genomic methods, both targeted and untargeted, for wastewater-based biosurveillance to find the most efficient procedure to detect and track trends in reportable infectious diseases and outbreaks of known pathogens as well as potentially novel pathogens or variants on the rise in our communities. RNA extracts from the OCWMN were provided weekly from 10 sites for 6 weeks. Total RNA was sequenced from the samples on the Illumina NextSeq and on the MinION to identify pathogens present. The MinION long read platform was also used to sequence SARS-CoV-2 with the goal of reducing the complexity of variant calling in mixed populations as occurs with short Illumina reads. Finally, a targeted hybridization approach was tested for compatibility with wastewater RNA samples. Results and discussion: The data analyzed here provides a baseline assessment that demonstrates that wastewater is a rich resource for infectious disease epidemiology and identifies technology gaps and potential solutions to enable this resource to be used by public health laboratories to monitor the infectious disease landscape of the regions they serve.

UltraSEQ, a Universal Bioinformatic Platform for Information-Based Clinical Metagenomics and Beyond.

Applied metagenomics is a powerful emerging capability enabling the untargeted detection of pathogens, and its application in clinical diagnostics promises to alleviate the limitations of current targeted assays. While metagenomics offers a hypothesis-free approach to identify any pathogen, including unculturable and potentially novel pathogens, its application in clinical diagnostics has so far been limited by workflow-specific requirements, computational constraints, and lengthy expert review requirements. To address these challenges, we developed UltraSEQ, a first-of-its-kind accurate and scalable metagenomic bioinformatic tool for potential clinical diagnostics and biosurveillance utility. Here, we present the results of the evaluation of our novel UltraSEQ pipeline using an in silico-synthesized metagenome, mock microbial community data sets, and publicly available clinical data sets from samples of different infection types, including both short-read and long-read sequencing data. Our results show that UltraSEQ successfully detected all expected species across the tree of life in the in silico sample and detected all 10 bacterial and fungal species in the mock microbial community data set. For clinical data sets, even without requiring data set-specific configuration setting changes, background sample subtraction, or prior sample information, UltraSEQ achieved an overall accuracy of 91%. Furthermore, as an initial demonstration with a limited patient sample set, we show UltraSEQ's ability to provide antibiotic resistance and virulence factor genotypes that are consistent with phenotypic results. Taken together, the above-described results demonstrate that the UltraSEQ platform offers a transformative approach for microbial and metagenomic sample characterization, employing a biologically informed detection logic, deep metadata, and a flexible system architecture for the classification and characterization of taxonomic origin, gene function, and user-defined functions, including disease-causing infections. IMPORTANCE Traditional clinical microbiology-based diagnostic tests rely on targeted methods that can detect only one to a few preselected organisms or slow, culture-based methods. Although widely used today, these methods have several limitations, resulting in rates of cases of an unknown etiology of infection of >50% for several disease types. Massive developments in sequencing technologies have made it possible to apply metagenomic methods to clinical diagnostics, but current offerings are limited to a specific disease type or sequencer workflow and/or require laboratory-specific controls. The limitations associated with current clinical metagenomic offerings result from the fact that the backend bioinformatic pipelines are optimized for the specific parameters described above, resulting in an excess of unmaintained, redundant, and niche tools that lack standardization and explainable outputs. In this paper, we demonstrate that UltraSEQ uses a novel, information-based approach that enables accurate, evidence-based predictions for diagnosis as well as the functional characterization of a sample.

Feasibility of neighborhood and building scale wastewater-based genomic epidemiology for pathogen surveillance.

The benefits of wastewater-based epidemiology (WBE) for tracking the viral load of SARS-CoV-2, the causative agent of COVID-19, have become apparent since the start of the pandemic. However, most sampling occurs at the wastewater treatment plant influent and therefore monitors the entire catchment, encompassing multiple municipalities, and is conducted using quantitative polymerase chain reaction (qPCR), which only quantifies one target. Sequencing methods provide additional strain information and also can identify other pathogens, broadening the applicability of WBE to beyond the COVID-19 pandemic. Here we demonstrate feasibility of sampling at the neighborhood or building complex level using qPCR, targeted sequencing, and untargeted metatranscriptomics (total RNA sequencing) to provide a refined understanding of the local dynamics of SARS-CoV-2 strains and identify other pathogens circulating in the community. We demonstrate feasibility of tracking SARS-CoV-2 at the neighborhood, hospital, and nursing home level with the ability to detect one COVID-19 positive out of 60 nursing home residents. The viral load obtained was correlative with the number of COVID-19 patients being treated in the hospital. Targeted wastewater-based sequencing over time demonstrated that nonsynonymous mutations fluctuate in the viral population. Clades and shifts in mutation profiles within the community were monitored and could be used to determine if vaccine or diagnostics need to be adapted to ensure continued efficacy. Furthermore, untargeted RNA sequencing identified several other pathogens in the samples. Therefore, untargeted RNA sequencing could be used to identify new outbreaks or emerging pathogens beyond the COVID-19 pandemic.

The draft genome of the hyperthermophilic archaeon Pyrodictium delaneyi strain hulk, an iron and nitrate reducer, reveals the capacity for sulfate reduction.

Pyrodictium delaneyi strain Hulk is a newly sequenced strain isolated from chimney samples collected from the Hulk sulfide mound on the main Endeavour Segment of the Juan de Fuca Ridge (47.9501 latitude, -129.0970 longitude, depth 2200 m) in the Northeast Pacific Ocean. The draft genome of strain Hulk shared 99.77% similarity with the complete genome of the type strain Su06T, which shares with strain Hulk the ability to reduce iron and nitrate for respiration. The annotation of the genome of strain Hulk identified genes for the reduction of several sulfur-containing electron acceptors, an unsuspected respiratory capability in this species that was experimentally confirmed for strain Hulk. This makes P. delaneyi strain Hulk the first hyperthermophilic archaeon known to gain energy for growth by reduction of iron, nitrate, and sulfur-containing electron acceptors. Here we present the most notable features of the genome of P. delaneyi strain Hulk and identify genes encoding proteins critical to its respiratory versatility at high temperatures. The description presented here corresponds to a draft genome sequence containing 2,042,801 bp in 9 contigs, 2019 protein-coding genes, 53 RNA genes, and 1365 hypothetical genes.

Therapeutic Development of Mesenchymal Stem Cells or Their Extracellular Vesicles to Inhibit Autoimmune-Mediated Inflammatory Processes in Systemic Lupus Erythematosus.

Since being discovered over half a century ago, mesenchymal stem cells (MSCs) have been investigated extensively to characterize their cellular and physiological influences. MSCs have been shown to possess immunosuppressive capacity through inhibiting lymphocyte activation/proliferation and proinflammatory cytokine secretion while simultaneously demonstrating limited allogenic reactivity, which subsequently led to the evaluation of therapeutic feasibility to treat inflammatory diseases. Although regulatory constraints have restricted MSC development pharmacologically, limited clinical studies have shown encouraging results using MSC infusions to treat systemic lupus erythematosus (SLE); but, more trials will have to be performed to conclusively determine the clinical efficacy of MSCs to treat SLE. Moreover, there are some data to suggest that MSCs possess tumorigenic potential and that the immunosuppressive influence can be dramatically affected by both donor variability and ex vivo expansion. Given that recent studies have found that the immunosuppressive effects of MSCs are a result, at least in part, to extracellular vesicle (EV) secretion, the use of MSC-derived EVs has been suggested as a cell-free therapeutic alternative. Despite the positive data observed using EVs isolated from human MSCs to suppress inflammatory responses in vitro and in inhibiting autoimmune disease pathogenesis in preclinical work, there are no studies to date examining EVs from MSCs to treat SLE in humans or animal models. Considering that EVs are not subject to the strict regulatory constraints of stem cell-based pharmacological development and are more readily standardized with regard to industrial-scale production and storage, this review outlines the anti-inflammatory biology of MSCs and the scientific evidence supporting the potential use of EVs derived from human MSCs to treat patients with SLE.

Host Characteristics and Bacterial Traits Predict Experimental Virulence for Escherichia coli Bloodstream Isolates From Patients With Urosepsis.

Background. Extraintestinal Escherichia coli infections are common, costly, and potentially serious. A better understanding of their pathogenesis is needed. Methods. Sixty-seven E coli bloodstream isolates from adults with urosepsis (Seattle, WA; 1980s) underwent extensive molecular characterization and virulence assessment in 2 infection models (murine subcutaneous sepsis and moth larval lethality). Statistical comparisons were made among host characteristics, bacterial traits, and experimental virulence. Results. The 67 source patients were diverse for age, sex, and underlying medical and urological conditions. The corresponding E coli isolates exhibited diverse phylogenetic backgrounds and virulence profiles. Despite the E coli isolates' common bloodstream origin, they exhibited a broad range of experimental virulence in mice and moth larvae, in patterns that (for the murine model only) corresponded significantly with host characteristics and bacterial traits. The most highly mouse-lethal strains were enriched with classic "urovirulence" traits and typically were from younger women with anatomically and functionally normal urinary tracts. The 2 animal models corresponded poorly with one another. Conclusions. Host compromise, including older age and urinary tract abnormalities, allows comparatively low-virulence E coli strains to cause urosepsis. Multiple E coli traits predict both experimental and epidemiological virulence. The larval lethality model cannot be a substitute for the murine sepsis model.

A Moonlighting Enolase from Lactobacillus gasseri does not Require Enzymatic Activity to Inhibit Neisseria gonorrhoeae Adherence to Epithelial Cells.

Enolases are generally thought of as cytoplasmic enzymes involved in glycolysis and gluconeogenesis. However, several bacteria have active forms of enolase associated with the cell surface and these proteins are utilized for functions other than central metabolism. Recently, a surface-associated protein produced by Lactobacillus gasseri ATCC 33323 with homology to enolase was found to inhibit the adherence of the sexually transmitted pathogen, Neisseria gonorrhoeae, to epithelial cells in culture. Here, we show that the protein is an active enolase in vitro. A recombinantly expressed, C-terminal His-tagged version of the protein, His6-Eno3, inhibited gonococcal adherence. Assays utilizing inhibitors of enolase enzymatic activity showed that this inhibitory activity required the substrate-binding site to be in an open conformation; however, the enolase enzymatic activity of the protein was not necessary for inhibition of gonococcal adherence. An L. gasseri strain carrying an insertional mutation in eno3 was viable, indicating that eno3 is not an essential gene in L. gasseri 33323. This observation, along with the results of the enzyme assays, is consistent with reports that this strain encodes more than one enolase. Here we show that the three L. gasseri genes annotated as encoding an enolase are expressed. The L. gasseri eno3 mutant exhibited reduced, but not abolished, inhibition of gonococcal adherence, which supports the hypothesis that L. gasseri inhibition of gonococcal adherence is a multifactorial process.

Genome Sequence of Klebsiella pneumoniae Respiratory Isolate IA565.

Klebsiella pneumoniae is a clinically significant opportunistic bacterial pathogen as well as a normal member of the human microbiota. K. pneumoniae strain IA565 was isolated from a tracheal aspirate at the University of Iowa Hospitals and Clinics. Here, we present the genome sequence of K. pneumoniae IA565.

Genome Sequence of Klebsiella pneumoniae Urinary Tract Isolate Top52.

Klebsiella pneumoniae is a significant cause of nosocomial infections, including ventilator-associated pneumonias and catheter-associated urinary tract infections. K. pneumoniae strain TOP52 #1721 (Top52) was isolated from a woman presenting with acute cystitis and subsequently characterized using various murine models of infection. Here we present the genome sequence of K. pneumoniae Top52.

Crystal structure of an efficacious gonococcal adherence inhibitor: an enolase from Lactobacillus gasseri.

Enolases are highly conserved metalloenzymes ubiquitous to cellular metabolism. While these enzymes share a large degree of sequence and structural similarity, they have been shown to possess a wide range of moonlighting functions. Recent studies showed that an enolase from Lactobacillus gasseri impedes the ability of Neisseria gonorrhoeae to adhere to epithelial cells. We present the crystal structure of this enolase, the first from Lactobacillus, with one of its Mg(2+) cofactors. Determined using molecular replacement to 2.08Å, the structure has a flexible and surface exposed catalytic loop containing lysines, and may play a role in the inhibitory function.

Genome Sequences of Campylobacter jejuni 81-176 Variants with Enhanced Fitness Relative to the Parental Strain in the Chicken Gastrointestinal Tract.

Campylobacter jejuni is a major cause of food-borne infections in the United States due to its ability to asymptomatically colonize the gastrointestinal tracts of chickens. Using competition assays with parental C. jejuni 81-176, variants with consistently improved fitness in chicken ceca relative to the parental strain were identified and sequenced.

Genome-wide detection of fitness genes in uropathogenic Escherichia coli during systemic infection.

Uropathogenic Escherichia coli (UPEC) is a leading etiological agent of bacteremia in humans. Virulence mechanisms of UPEC in the context of urinary tract infections have been subjected to extensive research. However, understanding of the fitness mechanisms used by UPEC during bacteremia and systemic infection is limited. A forward genetic screen was utilized to detect transposon insertion mutants with fitness defects during colonization of mouse spleens. An inoculum comprised of 360,000 transposon mutants in the UPEC strain CFT073, cultured from the blood of a patient with pyelonephritis, was used to inoculate mice intravenously. Transposon insertion sites in the inoculum (input) and bacteria colonizing the spleen (output) were identified using high-throughput sequencing of transposon-chromosome junctions. Using frequencies of representation of each insertion mutant in the input and output samples, 242 candidate fitness genes were identified. Co-infection experiments with each of 11 defined mutants and the wild-type strain demonstrated that 82% (9 of 11) of the tested candidate fitness genes were required for optimal fitness in a mouse model of systemic infection. Genes involved in biosynthesis of poly-N-acetyl glucosamine (pgaABCD), major and minor pilin of a type IV pilus (c2394 and c2395), oligopeptide uptake periplasmic-binding protein (oppA), sensitive to antimicrobial peptides (sapABCDF), putative outer membrane receptor (yddB), zinc metallopeptidase (pqqL), a shikimate pathway gene (c1220) and autotransporter serine proteases (pic and vat) were further characterized. Here, we report the first genome-wide identification of genes that contribute to fitness in UPEC during systemic infection in a mammalian host. These fitness factors may represent targets for developing novel therapeutics against UPEC.

The multifunctional protein YdiV represses P fimbria-mediated adherence in uropathogenic Escherichia coli.

YdiV, a degenerate EAL domain protein, represses motility by interacting with FlhD to abolish FlhDC interaction with DNA. Here, we demonstrate that deletion of ydiV dysregulates coordinate control of motility and adherence by increasing adherence of Escherichia coli CFT073 to a bladder epithelial cell line by specifically increasing production of P fimbriae. Interestingly, only one of the two P fimbrial operons, pap_2, present in the genome of E. coli CFT073 was upregulated. This derepression of the pap_2 operon is abolished following deletion of either cya or crp, demonstrating cyclic AMP (cAMP)-dependent activation of the P fimbrial operon. However, the absence of YdiV does not affect the gene expression of cya and crp, and loss of SdiA in the ydiV mutant does not affect the derepression of the pap_2 operon, suggesting that YdiV control of adherence acts in response to cAMP levels. Deletion of ydiV increases motility by increasing expression of fliA, suggesting that in E. coli CFT073, YdiV regulates motility by the same mechanism as that described previously for commensal E. coli strains. Furthermore, analysis of site-directed mutations found two putative Mg(2+)-binding residues of four conserved YdiV residues (E29 and Q219) that were involved in regulation of motility and FliC production, while two conserved c-di-GMP-binding residues (D156 and D165) only affected motility. None of the four conserved YdiV residues appeared to affect regulation of adherence. Therefore, we propose a model in which a degenerate EAL, YdiV, utilizes different domains to regulate motility through interaction with FlhD and adherence to epithelial cells through cAMP-dependent effects on the pap_2 promoter.

Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of Motility or sessility in Escherichia coli CFT073.

Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenic E. coli. IMPORTANCE Uropathogenic Escherichia coli (UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in an E. coli K-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain, E. coli CFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged from E. coli K-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

Escherichia coli isolates that carry vat, fyuA, chuA, and yfcV efficiently colonize the urinary tract.

Extraintestinal Escherichia coli (ExPEC), a heterogeneous group of pathogens, encompasses avian, neonatal meningitis, and uropathogenic E. coli strains. While several virulence factors are associated with ExPEC, there is no core set of virulence factors that can be used to definitively differentiate these pathotypes. Here we describe a multiplex of four virulence factor-encoding genes, yfcV, vat, fyuA, and chuA, highly associated with uropathogenic E. coli strains that can distinguish three groups of E. coli: diarrheagenic and animal-associated E. coli strains, human commensal and avian pathogenic E. coli strains, and uropathogenic and neonatal meningitis E. coli strains. Furthermore, human intestinal isolates that encode all four predictor genes express them during exponential growth in human urine and colonize the bladder in the mouse model of ascending urinary tract infection in higher numbers than human commensal strains that do not encode the four predictor genes (P = 0.02), suggesting that the presence of the predictors correlates with uropathogenic potential.