Biology subjects

Cohen, S.

Publications and source records attributed to Cohen, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

PRDM6 promotes medulloblastoma by repressing chromatin accessibility and altering gene expression

SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

cancer biology↗

bioRxiv2023

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection

The gammaherpesviruses establish a lifelong infection, with the cellular outcome of infection intimately regulated by cell type. Though myeloid cells are an early infection target, infection results in widely divergent outcomes without a clear explanation. Here we use murine gammaherpesvirus 68 to demonstrate that macrophages are readily infectable, resulting in three divergent infection outcomes dictated by viral and host factors. Infection in vivo and in a cell culture model results in a high frequency of viable cells characterized by restricted viral transcription and unexpected transcription of the ORF75 locus, with rare cells initiating but failing to complete lytic replication. Restricted infection can be fully converted to productive lytic replication by pre-treatment with the host cytokine IL-4. Finally, infection with a high viral dose also results in productive infection, but triggers increased cell death. These studies demonstrate stepwise regulation of virus infection and identify virus and host factors that dictate divergent outcomes in a single cell type.

microbiology↗

bioRxiv2023

APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

The E4 variant of APOE strongly predisposes individuals to late-onset Alzheimers disease. We demonstrate that in response to neutral lipid synthesis, apolipoprotein E (APOE) in astrocytes can avoid translocation into the ER lumen and traffic to lipid droplets (LDs) via membrane bridges at ER-LD contacts. APOE knockdown promotes fewer, larger LDs containing more unsaturated triglyceride. This LD size distribution phenotype was rescued by chimeric APOE that targets only LDs. APOE4-expressing astrocytes also form a small number of large LDs enriched in unsaturated triglyceride. Additionally, the larger LDs in APOE4 cells exhibit impaired turnover and increased sensitivity to lipid peroxidation. Our data indicate that APOE plays a previously unrecognized role as an LD surface protein that regulates LD size and composition. APOE4 is a toxic gain of function variant that causes aberrant LD composition and morphology. We propose that APOE4 astrocytes with large, unsaturated LDs are sensitized to lipid peroxidation or lipotoxicity, which could contribute to Alzheimers disease risk. SummaryWindham et al. discover that APOE in astrocytes can traffic to lipid droplets (LDs), where it modulates LD composition and size. Astrocytes expressing the Alzheimers risk variant APOE4 form large LDs with impaired turnover and increased peroxidation sensitivity.

cell biology↗

bioRxiv2023

Proteasome gene expression is controlled by the coordinated functions of multiple transcription factors

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 d after denervation) it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and -PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and -PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g. type-2 diabetes, cancer).

cell biology↗

bioRxiv2023

Graph-pMHC: Graph Neural Network Approach to MHC Class II Peptide Presentation and Antibody Immunogenicity

Antigen presentation on MHC Class II (pMHCII presentation) plays an essential role in the adaptive immune response to extracellular pathogens and cancerous cells. But it can also reduce the efficacy of large-molecule drugs by triggering an anti-drug response. Significant progress has been made in pMHCII presentation modeling due to the collection of large-scale pMHC mass spectrometry datasets (ligandomes) and advances in deep machine learning. Here, we develop graph-pMHC, a graph neural network approach to predict pMHCII presentation. We derive adjacency matrices for pMHCII using Alphafold2-multimer, and address the peptide-MHC binding groove alignment problem with a simple graph enumeration strategy. We demonstrate that graph-pMHC dramatically outperforms methods with suboptimal inductive biases, such as the multilayer-perceptron-based NetMHCIIan-4.0 (+22.84% average precision). Finally, we create an antibody drug immunogenicity dataset from clinical trial data, and develop a method for measuring anti-antibody immunogenicity risk using pMHCII presentation models. In comparison with BioPhis Sapiens score, a deep learning based measure of the humanness of an antibody drug, our strategy achieves a 7.14% ROC AUC improvement in predicting antibody drug immunogenicity.

immunology↗

bioRxiv2022

Learning orientation-invariant representations enables accurate and robust morphologic profiling of cells and organelles

Cell and organelle morphology are driven by diverse genetic and environmental factors and thus accurate quantification of cellular phenotypes is essential to experimental cell biology. Representation learning methods for phenotypic profiling map images to feature vectors that form an embedding space of morphological variation useful for clustering, dimensionality reduction, outlier detection, and supervised learning problems. Morphology properties do not change with orientation, and thus we argue that representation learning methods should encode this orientation invariance. We show that prior methods are sensitive to orientation, which can lead to suboptimal clustering. To address this issue, we develop O2-VAE, an unsupervised learning method that learns robust, orientation-invariant representations. We use O2-VAE to discover novel morphology subgroups in segmented cells and mitochondria, detect outlier cells, and rapidly characterise cellular shape and texture in large datasets, including in a newly generated synthetic benchmark.

bioinformatics↗

bioRxiv2022

Insulin receptor turnover in fasting is dependent on NAGLU-mediated beta-dystroglycan deglycosylation

Fasting exerts various physiological effects, most notably, reduced signaling through the insulin receptor. We showed that insulin receptor activity requires association with Dystrophin Glycoprotein Complex (DGC). Here, we demonstrate that insulin receptor turnover by lysosomes during fasting is dependent on deglycosylation of the principal DGC component, {beta}-dystroglycan. We show that the lysosomal enzymes HexA and Man2b1, which specifically remove N-linked glycans, mediate {beta}-dystroglycan deglycosylation and consequently insulin receptor-DGC loss. Surprisingly, the lysosomal enzyme NAGLU, which cannot process N-linked glycosylation, also facilitated {beta}-dystroglycan deglycosylation and insulin receptor loss. NAGLU enhances the activity of the transcriptional complex PPAR-{gamma}/RXR-, which in turn promotes Man2b1 and HexA induction and the resulting {beta}-dystroglycan deglycosylation. Accordingly, downregulation of HexA, Man2b1, NAGLU or RXR- during fasting blocked {beta}-dystroglycan deglycosylation, and caused accumulation of insulin receptor-DGC assemblies on the membrane. Thus, NAGLU mediates physiological adaptation to fasting by promoting indirectly {beta}-dystroglycan deglycosylation.

cell biology↗

bioRxiv2022

Combined drought resistance strategies and the hydraulic limit in co-existing Mediterranean woody species

O_LIWoody species employ various strategies to cope with drought stress. We investigated similarities and differences in response to chronic drought to understand resistance strategies in co-occurring Mediterranean species. C_LIO_LIWe studied five predominant Mediterranean species; Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus lycioides, and Phillyrea latifolia over two summers at three sites with different aridities. We measured key hydraulic and osmotic traits related to drought resistance, including resistance to embolism ({Psi}50), carbon isotope signature ({delta}13C), pre-dawn ({Psi}PD) and mid-day ({Psi}MD) water potentials, and native ({Psi}s) and full turgor ([Pcy]0) osmotic potentials. C_LIO_LISignificant differences among species appeared in resistance to embolism. The species also showed differences in the water potential plastic response over the dry season. This interspecific variation increased at the end of the dry season and resulted in very narrow hydraulic safety margins (HSM). Consequently, predicted loss of hydraulic conductivity revealed species with significant native embolism. Two of the species also had seasonal changes in osmotic adjustment. C_LIO_LIOur detailed analysis indicates that co-existing Mediterranean woody species combine various drought resistance strategies to minimize mortality risk. However, all of them risk mortality as they approach their hydraulic limit near the dry margin of their distribution. C_LI

plant biology↗

bioRxiv2022

Improved And Optimized Drug Repurposing For The SARS-CoV-2 Pandemic

The active global SARS-CoV-2 pandemic caused more than 426 million cases and 5.8 million deaths worldwide. The development of completely new drugs for such a novel disease is a challenging, time intensive process. Despite researchers around the world working on this task, no effective treatments have been developed yet. This emphasizes the importance of drug repurposing, where treatments are found among existing drugs that are meant for different diseases. A common approach to this is based on knowledge graphs, that condense relationships between entities like drugs, diseases and genes. Graph neural networks (GNNs) can then be used for the task at hand by predicting links in such knowledge graphs. Expanding on state-of-the-art GNN research, Doshi et al. recently developed the Dr-COVID model. We further extend their work using additional output interpretation strategies. The best aggregation strategy derives a top-100 ranking of 8,070 candidate drugs, 32 of which are currently being tested in COVID-19-related clinical trials. Moreover, we present an alternative application for the model, the generation of additional candidates based on a given pre-selection of drug candidates using collaborative filtering. In addition, we improved the implementation of the Dr-COVID model by significantly shortening the inference and pre-processing time by exploiting data-parallelism. As drug repurposing is a task that requires high computation and memory resources, we further accelerate the post-processing phase using a new emerging hardware -- we propose a new approach to leverage the use of high-capacity Non-Volatile Memory for aggregate drug ranking.

bioinformatics↗

bioRxiv2022

Quantitative Proteomic Profiling of Murine Embryonic Heart Development Reveals a Role for the Mevalonate Pathway in Cardiomyocyte Proliferation

Defining the molecular mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the cardiac proteome at eight critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 identified proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and established a functional role for the mevalonate pathway in the regulation of embryonic cardiomyocyte proliferation and cell signaling. Overall, our proteomic datasets are an invaluable resource for studying molecular events that regulate embryonic heart development and contribute to congenital heart disease.

developmental biology↗

bioRxiv2022

Perilipin 5 interacts with Fatp4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport

Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (Plin5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and {beta}-oxidation during starvation of myoblasts requires both phosphorylation of Plin5 and an intact Plin5 mitochondrial tethering domain. We further identified the acyl-CoA synthetase, Fatp4 (ACSVL4) as a novel mitochondrial interactor of Plin5. The C-terminal domains of Plin5 and Fatp4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of Plin5, lipolysis, and subsequent channeling of FAs from LDs to Fatp4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.

cell biology↗

bioRxiv2022

Discovery of novel therapeutic targets in cancer using patient-specific gene regulatory networks.

Most cancer types lack effective targeted therapeutic options and in cancers where first-line targeted therapies are available, treatment resistance is a huge challenge. Recent technological advances enable the use of ATAC-seq and RNA-seq on patient biopsies in a high-throughput manner. Here we present a computational approach that leverages these datasets to identify novel drug targets based on tumor lineage. We constructed patient-specific gene regulatory networks for 371 patients of 22 cancer types using machine learning approaches trained using three-dimensional genomic data for enhancer to promoter contacts. Next, we identify the key transcription factors (TFs) in these networks, which are used to identify therapeutic vulnerabilities either by direct targeting of TFs or proteins that they co-operate with. We validate four novel candidates identified for neuroendocrine, liver and renal cancers, which have a dismal prognosis with current therapeutic options. We present a novel approach to use the increasing amounts of functional genomics data from patient biospecimens for identification of novel drug targets.

systems biology↗

bioRxiv2022

The AAA-ATPase Atad1 and its partners promote degradation of desmin intermediate filaments in muscle

Maintenance of desmin intermediate filaments (IF) is vital for muscle plasticity and function, and their perturbed integrity due to accelerated loss or aggregation causes atrophy and myopathies. Calpain-1-mediated disassembly of ubiquitinated desmin IF is a prerequisite for desmin loss, myofibril breakdown and atrophy. Because calpain-1 does not harbor a bona fide ubiquitin binding domain, the precise mechanism for desmin IF disassembly remains unknown. Here, we demonstrate that the AAA-ATPase, ATAD1, is required to facilitate disassembly and turnover of ubiquitinated desmin IF. We identified PLAA and UBXN4 as ATAD1s interacting partners, and show they are required for desmin filaments turnover because their downregulation attenuated desmin loss upon denervation. The ATAD1-PLAA-UBXN4 complex binds desmin filaments and promotes a release of phosphorylated and ubiquitinated species into the cytosol, presenting ATAD1 as the only known AAA-ATPase that preferentially acts on phosphorylated substrates. Desmin filaments disassembly was accelerated by the coordinated functions of Atad1 and calpain-1, which interact in muscle. Thus, by extracting ubiquitinated desmin from the insoluble filament, ATAD1 may expose calpain-1 cleavage sites on desmin, consequently enhancing desmin solubilization and degradation in the cytosol.

cell biology↗

bioRxiv2021

Recapitulation of patient-specific 3D chromatin conformation using machine learning and validation of identified enhancer-gene targets

Regulatory networks containing enhancer to gene edges define cellular state and their rewiring is a hallmark of cancer. While efforts, such as ENCODE, have revealed these networks for reference tissues and cell-lines by integrating multi-omics data, the same methods cannot be applied for large patient cohorts due to the constraints on generating ChIP-seq and three-dimensional data from limited material in patient biopsies. We trained a supervised machine learning model using genomic 3D signatures of physical enhancer-gene connections that can predict accurate connections using data from ATAC-seq and RNA-seq assays only, which can be easily generated from patient biopsies. Our method overcomes the major limitations of correlation-based approaches that cannot distinguish between distinct target genes of given enhancers in different samples, which is a hallmark of network rewiring in cancer. Our model achieved an AUROC (area under receiver operating characteristic curve) of 0.91 and, importantly, can distinguish between active regulatory elements with connections to target genes and poised elements with no connections to target genes. Our predicted regulatory elements are validated by multi-omics data, including histone modification marks from ENCODE, with an average specificity of 0.92. Application of our model on chromatin accessibility and transcriptomic data from 400 cancer patients across 22 cancer types revealed novel cancer-type and subtype-specific enhancer-gene connections for known cancer genes. In one example, we identified two enhancers that regulate the expression of ESR1 in only ER+ breast cancer (BRCA) samples but not in ER-samples. These enhancers are predicted to contribute to the high expression of ESR1 in 93% of ER+ BRCA samples. Functional validation using CRISPRi confirms that inhibition of these enhancers decreases the expression of ESR1 in ER+ samples.

cancer biology↗

bioRxiv2021

A novel and highly divergent Canine Distemper Virus lineage causing distemper in ferrets in Australia

Canine distemper virus (CDV) is a highly contagious systemic viral disease of dogs, that regularly spills-over into other animal species. Despite widespread vaccination, CDV remains endemic in many parts of the world. In this study we report an outbreak of distemper in ferrets in two independent research facilities in Australia. We found that disease severity varied, although most animals had mild to moderate disease signs. Histopathology results of animals with severe disease presented the typical profile of distemper pathology with multi-system virus replication. Through the development of a discriminatory PCR paired with full genome sequencing we revealed that the outbreak at both facilities was caused by a single, novel lineage of CDV. This lineage was highly divergent across the H gene, F signal peptide and full genome and had less than 93% similarity across the H gene to other described lineages, including the vaccine strain. Molecular analysis indicates that this strain belongs to a distinct lineage that diverged from other clades approximately 140 to 400 years ago, and appears to be unique to Australia. Given the differences in key viral proteins of this novel CDV strain, a review of the efficacy of the CDV vaccines currently in use in Australia is warranted to ensure maximum protection of dogs and other vulnerable species. In addition, enhanced surveillance to determine the prevalence of CDV in ferrets, dogs and other at-risk species in Australia would be useful to better understand the diversity of CDV in Australia. ImportanceCanine distemper virus (CDV) is highly contagious and while dogs are the main reservoir, it may spill over into a number of other animal species. In this study we report an outbreak of distemper in ferrets in two research facilities in Australia. Outcomes of pathology and histopathology suggest ferrets have widespread multi-systemic infection, consistent with previously reported distemper infections in ferrets and dogs. Critically, through sequencing and phylogenetic analysis, we revealed that the outbreak at both facilities was caused by a single, novel and highly divergent lineage of CDV. This virus had less than 93% nucleotide similarity to other described lineages and the vaccine strain. This manuscript adds considerably to the epidemiology, ecology and evolution of this virus, and is one of few reports of distemper in Australia in the literature.

microbiology↗

bioRxiv2021

Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems

Alzheimers disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (A{beta}) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that A{beta} is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e. microglia), which culminates in neuronal damage and cognitive decline. In order to test this hypothesis, many in vitro systems have been established to study A{beta}-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale. To address this, we used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic A{beta}. We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic A{beta} does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of A{beta} formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INF{gamma} activate microglia and induce transcriptional changes similar to those observed in disease associated microglia present in the AD brain, suggesting the potential suitability of this model to study AD-related neuroinflammation.

neuroscience↗

bioRxiv2021

Chromosome-level genome assembly of a human fungal pathogen reveals synteny among geographically distinct species

Histoplasma capsulatum, a dimorphic fungal pathogen, is the most common cause of fungal respiratory infections in immunocompetent hosts. Histoplasma is endemic in the Ohio and Mississippi River Valleys in the United States and also distributed worldwide. Previous studies revealed at least eight clades, each specific to a geographic location: North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B), Eurasian, Netherlands, Australian and African, and an additional distinct lineage (H81) comprised of Panamanian isolates. Previously assembled Histoplasma genomes are highly fragmented, with the highly repetitive G217B (NAm 2) strain, which has been used for most whole genome-scale transcriptome studies, assembled into over 250 contigs. In this study, we set out to fully assemble the repeat regions and characterize the large-scale genome architecture of Histoplasma species. We re-sequenced five Histoplasma strains (WU24 (NAm 1), G217B (NAm 2), H88 (African), G186AR (Panama), and G184AR (Panama)) using Oxford Nanopore Technologies long-read sequencing technology. Here we report chromosomal-level assemblies for all five strains, which exhibit extensive synteny among the geographically distant Histoplasma isolates. The new assemblies revealed that RYP2, a major regulator of morphology and virulence, is duplicated in G186AR. In addition, we mapped previously generated transcriptome datasets onto the newly assembled chromosomes. Our analyses revealed that the expression of transposons and transposon-embedded genes are upregulated in yeast phase compared to mycelial phase in G217B and H88 strains. This study provides an important resource for fungal researchers and further highlights the importance of chromosomal-level assemblies in analyzing high-throughput datasets. ImportanceHistoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa and Europe. Phylogenetically distinct species of Histoplasma have been isolated and sequenced. However, for the commonly used strains, genome assemblies have been fragmented, leading to underutilization of genome-scale data. This study provides chromosome-level assemblies of the commonly used Histoplasma strains using long-read sequencing technology. Comparative analysis of these genomes shows largely conserved gene order within the chromosomes. Mapping existing transcriptome data on these new assemblies reveals clustering of transcriptionally co-regulated genes. Results of this study highlight the importance of obtaining chromosome-level assemblies in understanding the biology of human fungal pathogens.

microbiology↗

bioRxiv2021

Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing

Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext(R) Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/l of sample for SWGA and >129 genomic copies/l for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now. ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

genomics↗