Biology subjects

Smith, D.

Publications and source records attributed to Smith, D..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

cell biology↗

bioRxiv2024

Lipid A double bond position determination using ozone and laser-induced dissociation

Ozone coupled with tandem MS is a traditional tool to assign double bond position in lipids. However, few studies were reported on the use of ozone for double bond determination of bacterial lipids. Most MS-based double bond localization methods used in bacterial lipidomics focused on low molecular weight phospholipids, but never tested for assigning double bond position in complex glycolipids such as Lipid A. Here, we present a simple approach to identify unsaturated lipid A double bond position using on-MALDI plate ozone treatment of intact Lipid A.

microbiology↗

bioRxiv2023

Short-term social isolation acts on hypothalamic neurons to promote social behavior in a sex- and context-dependent manner

Social animals, including both humans and mice, are highly motivated to engage in social interactions. Short-term social isolation promotes social behavior, but the neural circuits through which it does so remain incompletely understood. Here, we sought to identify neurons that promote social behavior in single-housed female mice, which exhibit increased rates of social investigation, social ultrasonic vocalizations (USVs), and mounting during same-sex interactions that follow a period of short-term (3-day) isolation. We first used immunostaining for the immediate early gene Fos to identify a population of neurons in the preoptic hypothalamus (POA) that increase their activity in single-housed females following same-sex interactions (POAsocial neurons) but not in single-housed females that did not engage in social interactions. TRAP2-mediated chemogenetic silencing of POAsocial neurons in single-housed females significantly attenuates the effects of short-term isolation on social investigation, USV production, and mounting. In contrast, caspase-mediated ablation of POAsocial neurons in single-housed females robustly attenuates mounting but does not decrease social investigation or USV production. Optogenetic activation of POAsocial neurons in group-housed females promotes social investigation and USV production but does not recapitulate the effects of short-term isolation on mounting. To understand whether a similar population of POAsocial neurons promotes social behavior in single-housed males, we performed Fos immunostaining in single-housed males following either same-sex or opposite-sex social interactions. These experiments revealed a population of POA neurons that increase Fos expression in single-housed males following opposite-sex, but not same-sex, interactions. Chemogenetic silencing of POAsocial neurons in single-housed males during interactions with females reduces mounting but does not affect social investigation or USV production. These experiments identify a population of hypothalamic neurons that promote social behavior following short-term isolation in a sex- and social context-dependent manner.

neuroscience↗

bioRxiv2023

Identification and functional characterisation of a locus for target site integration in Fusarium graminearum

BackgroundFusarium Head Blight is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists in identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration. ResultsHere, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region the TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1. ConclusionThe TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work can be an alternative to southern blotting, the gold standard technique to identify ectopic integration and tandem insertions in fungal transformation.

genetics↗

bioRxiv2023

Fitting a square beam in a square camera: novel condensor apertures for low-dose transmission electron microscopy

In transmission electron microscopy (TEM) cameras are square or rectangular but beams are round. With a beam size chosen to fill the camera at a given image magnification, the circular lobes of the beam will extend beyond the cameras field of view and irradiate areas that are not acquired on the camera, damaging and precluding them from future acquisitions if the sample is beam sensitive. In this paper we present development of condenser aperture plates for TEM that have square and rectangular apertures which improve the efficiency of sample area usage by 44% or greater in low dose TEM applications. We demonstrate that the use of these apertures is compatible with high-resolution cryogenic (cryo) TEM by reconstructing sub 2 [A] apo-ferritin models from a datasets recorded with both square and rectangular apertures. Moreover the design of our aperture plates should improve the flexibility of 2 condenser systems for cryo-TEM acquisitions with multiple shots per hole by tailored matching of beam sizes to camera sizes at each magnification.

biophysics↗

bioRxiv2023

Impact of angiotensin-receptor blockers on intrarenal renin-angiotensin system activity in hypertension: A PK/PD modelling study

The renin-angiotensin system (RAS) is a primary regulator of volume homeostasis and blood pressure, whose over-activation is commonly associated with hypertension. Indeed, medications that target the RAS are generally effective in reducing blood pressure. However, more can be learned about how these medications influence the intrarenal RAS. Angiotensin-receptor blockers (ARBs) in particular have been shown to exert different effects on the intrarenal and systemic RASs in various experimental models of hypertension. In rats chronically infused with angiotensin II (Ang II), ARBs consistently prevent intrarenal, but not systemic Ang II levels from rising. The former effect is sufficient in preventing the development of hypertension. The regulation of intrarenal RAS, independently of the systemic RAS, by ARBs has been hypothesized to be mediated by the inhibition of all positive feedback loops inherent to the intrarenal RAS, also known as the "key point breakdown effect." To investigate the validity of this hypothesis, we developed a PK/PD model of the ARB Losartan that considers the kidney, and applied the model to study how this class of medication influences intrarenal RAS activity and consequently blood pressure regulation in male rats. Simulations indicate that ARBs more effectively inhibit the activation of the intrarenal RAS because, unlike in the plasma, this process relies on the accumulation of cell-associated Ang II. We hypothesize that it is by blocking this intracellular uptake pathway, and restricting Ang II to extracellular regions of the kidney where the peptide cannot initiate downstream signalling, that Losartan normalizes blood pressure. While the key point break down effect assists in this response, it alone is not sufficient. Our results highlight the intrarenal RAS as the key pharmacological target of ARB treatment and emphasize the importance of this local tissue RAS in the development of hypertension.

physiology↗

bioRxiv2023

Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots

Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally heterogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families; i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 179 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

biochemistry↗

bioRxiv2023

Improved gene annotation of the fungal wheat pathogen Zymoseptoria tritici based on combined Iso-Seq and RNA-Seq evidence

Despite large omics datasets, the establishment of a reliable gene annotation is still challenging for eukaryotic genomes. Here, we used the reference genome of the major fungal wheat pathogen Zymoseptoria tritici (isolate IPO323) as a case study to develop methods to improve eukaryotic gene prediction. Four previous IPO323 annotations identified 10,933 to 13,260 gene models, but only one third of these coding sequences (CDS) have identical structures. To resolve these discrepancies and improve gene models, we generated full-length transcripts using long-read sequencing. This dataset was used together with other evidence (RNA-Seq transcripts and protein sequences) to generate novel ab initio gene models. The selection of the best structure among novel and existing gene models was performed according to transcript and protein evidence using InGenAnnot, a novel bioinformatics suite. Overall, 13,414 re-annotated gene models (RGMs) were predicted, including 671 new genes among which 53 encoded effector candidates. This process corrected many of the errors (15%) observed in previous gene models (coding sequence fusions, false introns, missing exons). While fungal genomes have poor annotations of untranslated regions (UTRs), our Iso-Seq long-read sequences outlined 5 and 3UTRs for 73% of the RGMs. Alternative transcripts were identified for 13% of RGMs, mostly due to intron retention (75%), likely corresponding to unprocessed pre-mRNAs. A total of 353 genes displayed alternative transcripts with combinations of previously predicted or novel exons. Long non-coding transcripts (lncRNAs) and double-stranded RNAs from two fungal viruses were also identified. Most lncRNAs corresponded to antisense transcripts of genes (52%). lncRNAs that were up or down regulated during infection were enriched in antisense transcripts (70%), suggesting their involvement in the control of gene expression. Our results showed that combining different ab initio gene predictions and evidence-driven curation using InGenAnnot improved the quality of gene annotations of a compact eukaryotic genome. Our analysis also provided new insights into the transcriptional landscape of Z. tritici, helping develop an increasingly complex picture of its biology.

genomics↗

bioRxiv2023

Ready-to-use nanopore platform for ethanolamine quantification using an aptamer-based strand displacement assay.

In recent decades, nanopores have become a promising diagnostic tool. Protein and solid-state nanopores are increasingly used for both RNA/DNA sequencing and small molecule detection. The latter is of great importance because small molecules are difficult or expensive to detect using available methods such as HPLC or LC-MS. Moreover, DNA aptamers are an excellent detection element for sensitive and specific detection of small molecules. Here, we describe a method for the quantification of ethanolamine using Oxford Nanopores ready-to-use sequencing platform. To this end, we have developed a strand displacement assay using a binding ethanolamine aptamer and magnetic beads. The displaced aptamer can be detected using the MinION(R) nanopores and analysed/quantified using our in-house developed analysis software.

molecular biology↗

bioRxiv2023

The long and short of it: Benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid approaches that combine long and short read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock phage community of previously sequenced phage genomes and sequenced using Illumina, Nanopore, and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies ~4x and 120x higher than found in Illumina only assemblies respectively. While the best PacBio assemblies had SNPs at frequencies ~3.5 x and 12x higher than found in Illumina only assemblies respectively. Despite high read coverage, long-read only assemblies failed to recover a complete genome for any of the 15 phage, down sampling of reads did increase the proportion of a genome that could be assembled into a single contig. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short read only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes. Data SummaryAll reads from virome sequencing were submitted to the ENA under study PRJEB56639. The assemblies are provided via FigShare (https://figshare.com/s/2d9b5121eb421d370455). Author NotesEight Supplementary Tables and nine Supplementary Figures are available with the online version of this article.

microbiology↗

bioRxiv2023

A Single Laccase Acts as a Key Component of Environmental Sensing in a Broad Host Range Fungal Pathogen

Secreted laccases are important enzymes on an ecological scale for their role in mediating plant-fungal interactions, but their function in fungal pathogenesis has yet to be elucidated. Ascomycete laccases have been primarily associated with cell wall melanin deposition, and laccase mutants in ascomycete species often demonstrate reduced pigmentation. In this study, a putatively secreted laccase, Sslac2, was characterized from the broad host-range plant pathogen Sclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Of the seven putative laccases in the S. sclerotiorum genome, Sslac2 was the only one found to be highly upregulated during pathogenesis of soybeans and was additionally found to be induced during growth on solid surfaces. Gene knockouts of Sslac2 demonstrate wide ranging developmental defects, including abolished sclerotial formation, and are functionally non-pathogenic on unwounded tissue. While these mutants demonstrated a clear radial growth defect, enhanced growth was observed in liquid culture, likely due to altered hydrophobicity and thigmotropic responsiveness. Sslac2 mutants were also unable to respond to a host environment, and accordingly unable to differentiate penetration structures, respond appropriately to chemical stress, or produce the key virulence factor oxalic acid. Transmission and scanning electron microscopy of WT and mutant strains show apparent differences in extracellular matrix structure that may explain the inability of the mutant to respond to environmental cues. Targeting Sslac2 using host-induced gene silencing significantly improved resistance to S. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad host-range pathogen S. sclerotiorum and propose a potentially novel role for fungal laccases in modulating environmental sensing.

microbiology↗

bioRxiv2023

How random is the review outcome? A systematic study of the impact of external factors on eLife peer review

The advance of science rests on a robust peer review process. However whether or not a paper is accepted can depend on random external factors--e.g. the timing of the submission, the matching of editors and reviewers--that are beyond the quality of the work. This article systematically investigates the impact of these random factors independent of the papers quality on peer review outcomes in a major biomedical journal, eLife. We analyzed all of the submissions to eLife between 2016 to 2018, with 23,190 total submissions. We examined the effects of random factors at each decision point in the review process, from the gatekeeping senior editors who may desk-reject papers to review editors and reviewers who recommend the final outcome. Our results suggest that the peer-review process in eLife is robust overall and that random external factors have relatively little quantifiable bias.

bioinformatics↗

bioRxiv2022

Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.

genetics↗

bioRxiv2022

A single-administration therapeutic interfering particle reduces SARS-CoV-2 viral shedding and pathogenesis in hamsters

The high transmissibility of SARS-CoV-2 is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, post-exposure TIP dose lowers SARS-CoV-2 nasal shedding and at 5 days post-infection infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Co-housed contact animals exposed to infected, TIP-treated, animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals co-housed with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission. SignificanceCOVID-19 vaccines are exceptionally effective in preventing severe disease and death, but they have mixed efficacy in preventing virus transmission, consistent with established literature that parenteral vaccines for other viruses fail to prevent mucosal virus shedding or transmission. Likewise, small-molecule antivirals, while effective in reducing viral-disease pathogenesis, also appear to have inconsistent efficacy in preventing respiratory virus transmission including for SARS-CoV-2. Recently, we reported the discovery of a single-administration antiviral Therapeutic Interfering Particle (TIP) against SARS-CoV-2 that prevents severe disease in hamsters and exhibits a high genetic barrier to the evolution of resistance. Here, we report that TIP intervention also reduces SARS-CoV-2 transmission between hamsters.

microbiology↗

bioRxiv2022

A unified peptide array platform for antibody epitope binning, mapping, specificity and predictive off-target binding

Therapeutic antibody efficacy is largely determined by the target epitope. In addition, off-target binding can result in unanticipated side-effects. Therefore, characterization of the epitope and binding specificity are critical in antibody discovery. Epitope binning provides low-resolution of an antibody epitope and is typically performed as a cross-blocking assay to group antibodies into overlapping or non-overlapping bins. Epitope mapping identifies the epitope with high resolution but requires low throughput methods. In addition to binning and mapping, there is a need for a scalable and predictive approach to reveal off-target binding early in antibody discovery to reduce the risk of in vivo side effects. Peptide microarrays are an information-rich platform for antibody characterization. However, the potential of peptide microarrays in early-stage antibody discovery has not been realized because they are not produced at the scale, quality and format needed for reliable high-throughput antibody characterization. A unified, peptide library platform for high-resolution antibody epitope binning, mapping and predictive off-target binding characterization is described here. This platform uses highly scalable array synthesis and photolithography to synthesize more than 3 million addressable peptides. These arrays conform to a microplate format and each synthesis is qualified with mass spectrometry. Using this platform, a scalable approach to early-stage epitope and specificity characterization, with prediction of off-target interaction(s), is demonstrated using a panel of anti-HER2 monoclonal antibodies. This study highlights the prospect of this platform to improve antibody discovery productivity by generating epitope and specificity information much earlier with potentially hundreds of antibody clones.

bioengineering↗

bioRxiv2022

Hunter-gatherer oral microbiomes are shaped by contact network structure

Ancestral humans evolved a complex social structure still observed in extant hunter-gatherers. Here we investigate the effects of extensive sociality and mobility on the oral microbiome of 138 Agta hunter-gatherers from the Philippines. Comparisons of microbiome composition showed that the Agta are more similar to Central African Bayaka hunter-gatherers than to neighboring farmers. We also defined the Agta social microbiome as a set of 137 oral bacteria (only 7% of 1980 amplicon sequence variants) significantly influenced by social contact (quantified through wireless sensors of short-range interactions). We show that interaction networks covering large areas, and their strong links between close kin, spouses, and even unrelated friends, can significantly predict bacterial transmission networks across Agta camps. Finally, more central individuals to social networks are also bacterial supersharers. We conclude that hunter-gatherer social microbiomes, which are predominantly pathogenic, were shaped by evolutionary tradeoffs between extensive sociality and disease spread.

microbiology↗

bioRxiv2022

The making of the oral microbiome in Agta hunter-gatherers

Ecological and genetic factors have influenced the composition of the human microbiome during our evolutionary history. We analyzed the oral microbiota of the Agta, a hunter-gatherer population where part of its members is adopting an agricultural diet. We show that age is the strongest factor modulating the microbiome, likely through immunosenescence as there is an increase of pathogenicity with age. Biological and cultural processes generate sexual dimorphism in the oral microbiome. A small subset of oral bacteria is influenced by the host genome, linking host collagen genes to bacterial biofilm formation. Our data also suggests that shifting from a fish/meat to a rice-rich diet transforms their microbiome, mirroring the Neolithic transition. All these factors have implications in the epidemiology of oral diseases. Thus, the human oral microbiome is multifactorial, and shaped by various ecological and social factors that modify the oral environment.

microbiology↗

bioRxiv2022

Human and murine Cryptococcus neoformans infection selects for common genomic changes in an environmental isolate.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from the cerebral spinal fluid (CSF) of an immunocompromised patient with cryptococcosis based on the molecular analyses available in 2000. Here we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MAT strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including 8 nonsynonymous changes involving 7 genes. To ascertain whether changes in these genes are selected during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frame-shift mutation in one of the seven genes altered in the human sample, a gene predicted to encode a SWI-SNF chromatin-remodeling complex protein. Both cockatoo and patient strains as well as mouse passaged isolates obtained from brain tissue had a premature stop codon in a homolog of ZFC3, a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and appears to have reverted to a full-length protein in the mouse passaged isolates obtained from lung tissue. The patient strain and mouse passaged isolates show variability in the expression of virulence factors, with differences in capsule size, melanization, and rates on non-lytic expulsion from macrophages observed. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

genomics↗