Biology subjects

Gnirke, A.

Publications and source records attributed to Gnirke, A..

9 recordsLinked to original sources

bioRxiv2025

An Anaerobic Pathogen Rewires Host Metabolism to Fuel Oxidative Growth in the Inflamed Gut

To colonize their host and cause disease, enteric pathogens must deploy their virulence factors to establish distinct nutrient niches. How obligate anaerobic pathogens construct nutrient niches in the densely populated large intestine remains poorly understood. Enterotoxigenic Bacteroides fragilis (ETBF) is considered an obligate anaerobic bacterium and has been implicated in inflammation-associated diseases, including colitis and colorectal cancer. Here we show that ETBF uses its virulence factor, Bacteroides fragilis toxin, to reprogram colonic epithelial cell metabolism to colonize the inflamed gut. Bacteroides fragilis toxin activates colonic epithelial signaling and hijacks the host bile acid recycling pathway, inducing a metabolic shift in the epithelium from oxidative phosphorylation to glycolysis. This shift increases local concentrations of lactate and oxygen, nutrients that support an oxidative metabolism in ETBF. These findings reveal an unexpected strategy by which a pathogenic organism, previously considered to be an obligate anaerobic bacterium, generates and exploits an oxidative niche in the inflamed gut.

microbiology↗

bioRxiv2024

Distinct Escherichia coli transcriptional profiles in the guts of recurrent UTI sufferers revealed by pan-genome hybrid selection

Low-abundance members of microbial communities are difficult to study in their native habitats. This includes Escherichia coli, a minor, but common inhabitant of the gastrointestinal tract and opportunistic pathogen, including of the urinary tract, where it is the primary pathogen. While multi-omic analyses have detailed critical interactions between uropathogenic Escherichia coli (UPEC) and the bladder that mediate UTI outcome, comparatively little is known about UPEC in its pre-infection reservoir, partly due to its low abundance there (<1% relative abundance). To accurately and sensitively explore the genomes and transcriptomes of diverse E. coli in gastrointestinal communities, we developed E. coli PanSelect which uses a set of probes designed to specifically recognize and capture E. colis broad pangenome from sequencing libraries. We demonstrated the ability of E. coli PanSelect to enrich, by orders of magnitude, sequencing data from diverse E. coli using a mock community and a set of human stool samples collected as part of a cohort study investigating drivers of recurrent urinary tract infections (rUTI). Comparisons of genomes and transcriptomes between E. coli residing in the gastrointestinal tracts of women with and without a history of rUTI suggest that rUTI gut E. coli are responding to increased levels of oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of native in vivo biology of E. coli in other environments where it is at low relative abundance, and the framework described here has broad applicability to other highly diverse, low abundance organisms.

microbiology↗

bioRxiv2023

Comparative genomics of Cryptococcus and Kwoniella reveals pathogenesis evolution and contrasting modes of karyotype evolution via chromosome fusion or intercentromeric recombination

A large-scale comparative genomic analysis was conducted for the global human fungal pathogens within the Cryptococcus genus, compared to non-pathogenic Cryptococcus species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species of both genera, resulting in a dataset encompassing virtually all of their known diversity. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at pre-adaptive pathogenic potential, our analysis found evidence in pathogenic Cryptococcus species of specific examples of gene gain (via horizontal gene transfer) and gene loss, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the two genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5 or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes, underwent chromosome reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Taken together, our findings advance our understanding of genomic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

genomics↗

bioRxiv2023

Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample

Ancient DNA (aDNA) sequencing analysis typically involves alignment to a modern reference genome assembly from a related species. Since aDNA molecules are fragmentary, these alignments yield information about small-scale differences, but provide no information about larger features such as the chromosome structure of ancient species. We report the genome assembly of a female Late Pleistocene woolly mammoth (Mammuthus primigenius) with twenty-eight chromosome-length scaffolds, generated using mammoth skin preserved in permafrost for roughly 52,000 years. We began by creating a modified Hi-C protocol, dubbed PaleoHi-C, optimized for ancient samples, and using it to map chromatin contacts in a woolly mammoth. Next, we developed "reference-assisted 3D genome assembly," which begins with a reference genome assembly from a related species, and uses Hi-C and DNA-Seq data from a target species to split, order, orient, and correct sequences on the basis of their 3D proximity, yielding accurate chromosome-length scaffolds for the target species. By means of this reference-assisted 3D genome assembly, PaleoHi-C data reveals the 3D architecture of a woolly mammoth genome, including chromosome territories, compartments, domains, and loops. The active (A) and inactive (B) genome compartments in mammoth skin more closely resemble those observed in Asian elephant skin than the compartmentalization patterns seen in other Asian elephant tissues. Differences in compartmentalization between these skin samples reveal sequences whose transcription was potentially altered in mammoth. We observe a tetradic structure for the inactive X chromosome in mammoth, distinct from the bipartite architecture seen in human and mouse. Generating chromosome-length genome assemblies for two other elephantids (Asian and African elephant), we find that the overall karyotype, and this tetradic Xi structure, are conserved throughout the clade. These results illustrate that cell-type specific epigenetic information can be preserved in ancient samples, in the form of DNA geometry, and that it may be feasible to perform de novo genome assembly of some extinct species.

genomics↗

bioRxiv2020

A rapid, low cost, and highly sensitive SARS-CoV-2 diagnostic based on whole genome sequencing

Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosing and studying SARS-CoV-2 infection. Dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), this method amplifies the entirety of the SARS-CoV-2 genome. This contrasts with typical RT-PCR-based diagnostic tests, which amplify only a few loci. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. Using this method, we can reliably (>95% accuracy) detect SARS-CoV-2 at a concentration of 84 genome equivalents per milliliter (GE/mL). Almost all diagnostic methods currently authorized for use by the United States Food and Drug Administration with the Coronavirus Disease 2019 (COVID-19) Emergency Use Authorization require larger concentrations of the virus to achieve this degree of accuracy. In addition, we can reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. The genotypic data contained in these genome assemblies enable the more effective analysis of disease spread than is possible with an ordinary binary diagnostic. These data can also help identify vaccine and drug targets. Finally, we show that the diagnoses obtained using POLAR of both positive and negative clinical nasopharyngeal swab samples 100% match the diagnoses obtained in a clinical diagnostic lab using the Center for Disease Controls 2019-Novel Coronavirus test. Using POLAR, a single person can manually process 192 samples over an 8- hour experiment at the cost of [~]$36 per patient (as of December 7th, 2022), enabling a 24-hour turnaround with sequencing and data analysis time. We anticipate that further testing and refinement will allow greater sensitivity in this approach.

genomics↗

bioRxiv2020

Distinct evolutionary paths in chronic lymphocytic leukemia during resistance to graft-versus-leukemia

Resistance to the graft-versus-leukemia (GvL) effect remains the major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive hematologic malignancies. The basis of GvL resistance for advanced lymphoid malignancies remains incompletely understood. We hypothesized that for patients with chronic lymphocytic leukemia (CLL) treated with allo-HSCT, leukemic cell-intrinsic features shape GvL outcomes by directing the evolutionary trajectories of CLL cells. Integrated genetic, transcriptomic and epigenetic analyses of CLL cells from 10 patients revealed that the clinical kinetics of post- HSCT relapse are shaped by distinct molecular dynamics and suggest that the selection pressures of the GvL bottleneck are unlike those imposed by chemotherapy. No selective advantage for HLA loss was observed, even when present in pre-transplant subpopulations. Regardless of post-transplant relapse kinetics, gain of stem cell modules was a common signature associated with leukemia relapse. These data elucidate the biological pathways that underlie GvL resistance and post-transplant relapse. One Sentence SummaryWe find that the clinical kinetics of chronic lymphocytic leukemia relapse after stem cell transplant are underwritten by distinct genetic and epigenetic evolutionary trajectories and suggest that the selection pressures of the post-transplant, immunologic bottleneck are unlike those imposed by chemotherapy.

cancer biology↗

bioRxiv2020

Mechanism of monoallelic expression and allelic rheostat role of DNA methylation

In diploid cells, maternal and paternal copies of genes usually have similar transcriptional activity. Mammalian allele-specific epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. The discovery of mitotically stable monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. However, despite its prevalence, the mechanistic basis of MAE remains unknown. To uncover the mechanism of MAE maintenance, we devised a small-molecule screen for reactivation of silenced alleles across multiple loci using targeted RNA sequencing. Contrary to previous reports, we identified DNA methylation as a key mechanism of MAE mitotic maintenance. In contrast with the binary choice of the active allele in XCI, stringent transcriptome-wide analysis revealed MAE as a regulatory mode with tunable control of allele-specific expression, dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to changes in DNA methylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance, reveal tunability of this mode of gene regulation, and provide the essential platform for probing the biological role of MAE in development and disease.

genomics↗

bioRxiv2018

The Hi-Culfite assay reveals relationships between chromatin contacts and DNA methylation state

Hi-Culfite, a protocol combining Hi-C and whole-genome bisulfite sequencing (WGBS), determines chromatin contacts and DNA methylation simultaneously. Hi-Culfite also reveals relationships that cannot be seen when the two assays are performed separately. For instance, we show that loci associated with open chromatin exhibit context-sensitive methylation: when their spatial neighbors lie in closed chromatin, they are much more likely to be methylated.

genomics↗

bioRxiv2017

Multiple introductions of Zika virus into the United States revealed through genomic epidemiology

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital syndromes1,2. In July 2016, mosquito-borne ZIKV transmission was first reported in the continental United States and since then, hundreds of locally-acquired infections have been reported in Florida3. To gain insights into the timing, source, and likely route(s) of introduction of ZIKV into the continental United States, we tracked the virus from its first detection in Miami, Florida by direct sequencing of ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least four distinct ZIKV introductions contributed to the outbreak in Florida and that local transmission likely started in the spring of 2016 - several months before its initial detection. By analyzing surveillance and genetic data, we discovered that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions are phylogenetically linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. By comparing mosquito abundance and travel flows, we describe the areas of southern Florida that are especially vulnerable to ZIKV introductions. Our study provides a deeper understanding of how ZIKV initiates and sustains transmission in new regions.

epidemiology↗