Search

Serine hydrolases play important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human protease dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

BackgroundBelize is a middle-income Caribbean country with poorly described cancer epidemiology and no comprehensive cancer care capacity. In 2018, GO, Inc., a US-based NGO, partnered with the Ministry of Health and the national hospital in Belize City to create the first public oncology clinic in the country. Here, we report demographics from the clinic and describe time intervals to care milestones to allow for public health targeting of gaps.Patients and MethodsUsing paper charts and a mobile health platform, we performed a retrospective chart review at the Karl Heusner Memorial Hospital (KHMH) clinic from 2018 to 2022.ResultsDuring this time period, 465 patients with cancer presented to the clinic. Breast cancer (28%) and cervical cancer (12%) were most common. Most patients (68%) presented with stage 3 or 4 disease and were uninsured (78%) and unemployed (79%). Only 21% of patients ever started curative intent treatment. Median time from patient-reported symptoms to a biopsy or treatment was 130 and 189 days. For the most common cancer, breast, similar times were seen at 140 and 178 days. Time intervals at the clinic: ConclusionThis study reports the first clinic-based cancer statistics for Belize. Many patients have months between symptom onset and treatment. In this setting, the clinic has built infrastructure allowing for minimal delays in care despite an underserved population. This further affirms the need for infrastructure investment and early detection programs to improve outcomes in Belize.

Bacterial populations that originate from a single bacterium are not strictly clonal. Often they contain subgroups that have distinct phenotypes. One way that bacteria generate this heterogeneity is through phase variation: enzyme-mediated, reversible inversion of specific intergenic regions of genomic DNA. These DNA inversions can flip the orientation of promoters within otherwise isogenic populations, impacting fitness and survival. We developed and applied bioinformatic approaches that enabled the discovery of thousands of previously undescribed phase-variable regions in prokaryotes using long-read datasets. We identified ‘intragenic invertons’, a surprising new class of invertible elements found entirely within genes, across the prokaryotic tree of life. Intragenic invertons allow a single gene to encode two or more versions of a protein by flipping a DNA sequence within the gene, thereby increasing coding capacity without increasing genome size. We experimentally characterize specific intragenic invertons in the gut commensalBacteroides thetaiotaomicron, presenting a ‘roadmap’ for investigating this gene-diversifying phenomenon.One-Sentence SummaryIntragenic DNA inversions, identified using long-read sequencing datasets, are found across the prokaryotic tree of life.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker.ImportanceHuman exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell–derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal β-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal β-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Ribosome profiling enables sequencing of ribosome-bound fragments of RNA, revealing which transcripts are being translated as well as the position of ribosomes along mRNAs. Although ribosome profiling has been applied to cultured bacterial isolates, its application to uncultured, mixed communities has been challenging. We present MetaRibo-Seq, a protocol that enables the application of ribosome profiling directly to the human fecal microbiome. MetaRibo-Seq is a benchmarked method that includes several modifications to existing ribosome profiling protocols, specifically addressing challenges involving fecal sample storage, purity and input requirements. We also provide a computational workflow to quality control and trim reads, de novo assemble a reference metagenome with metagenomic reads, align MetaRibo-Seq reads to the reference, and assess MetaRibo-Seq library quality ( https://github.com/bhattlab/bhattlab_workflows/tree/master/metariboseq ). This MetaRibo-Seq protocol enables researchers in standard molecular biology laboratories to study translation in the fecal microbiome in ~5 d.

Robust benchmarking studies have highlighted how measured relative microbial abundances can vary dramatically depending on how DNA is extracted, made into libraries, sequenced, and analyzed. To build upon prior research, we investigated how sample preservation and storage choices impact observed absolute microbial load and relative metagenomic and metatranscriptomic measurements. Specifically, we studied how two common stool preservatives (OMNIgene GUT OMR200 and Zymo DNA/RNA PowerShield) perform across a range of storage temperatures (−80°C, 23°C and 40°C). For immediately frozen samples with no preservatives, we observed a mean colonic load of ∼100 trillion (1.2 × 1014) prokaryotes across ten donors, revising the gut prokaryote:human cell ratio of ∼1:1 to ∼4:1. We found that both preservatives introduce significant bias in the metagenomics results; and, while OMNIgene results were robust to storage temperature, samples stored in Zymo preservative had further bias with increasing storage temperatures. In terms of measured composition, we observed a ∼1.9x and ∼1.5x difference in the metagenomic Bacteroidetes:Firmicutes ratio in OMNIgene and Zymo preservatives, respectively. Absolute abundance measurements revealed that these differences are driven by higher measured Bacteroidetes in OMNIgene-preserved samples and lower measured Firmicutes in Zymo-preserved samples. For metatranscriptomic measurements, we also found that both preservatives introduced bias, but that RNA likely degraded in samples stored in OMNIgene preservative at high temperature. In summary, we recommend the OMNIgene preservative for studies that include significant field components. For metatranscriptomics studies, we recommend kits rated for RNA preservation such as the Zymo kit; however, existing samples collected in non-RNA rated kits might also be viable for limited metatranscriptomic studies. This study demonstrates how sample collection and storage choices can affect measured microbiome research outcomes, makes additional concrete suggestions for sample handling best practices, and demonstrates the importance of including absolute abundance measurements in microbiome studies.

SummaryProtists in the gut microbiota influence intestinal immunity and epithelial composition, yet the mechanisms used by these protists to shape the gut environment remain poorly understood. Here, we identify a new protist species, Tritrichomonas casperi, and show that metabolic differences between closely related commensal protists modulate their effects on host immunity and determine their ecological niche within the murine microbiota. Genomic and metabolomic analysis of commensal tritrichomonads reveal species-level differences in excretion of the tuft cell activating metabolite succinate, leading to differential induction of Th1, Th2, and Th17 cells in the small intestine. Using defined diets and in vitro growth assays, we show that different tritrichomonad species preferentially rely on dietary polysaccharides or mucus glycans, which leads to trans-kingdom competition with specific bacteria in the microbiota. Our findings elucidate differences in commensal tritrichomonad metabolism and suggest how dietary interventions could regulate the impact of these protists on gut health.

The human gut microbiome is a diverse ecosystem that encompasses multiple domains of life and plays a vital role in human health. Due to technical limitations, most microbiome studies have focused on gut prokaryotes, overlooking bacteriophages and other gut viruses. The most common method to profile viruses is to assemble shotgun metagenomic reads - often from virus-enriched samples - and identify viral genomes de novo. While valuable, this resource-intensive and reference-independent method has limited sensitivity. To overcome these drawbacks, we developed Phanta, which profiles human gut metagenomes in a virus-inclusive manner directly from short reads utilizing recently published catalogs of gut viral genomes. Phanta incorporates k-mer based classification tools and was developed with virus-specific properties in mind. Specifically, it includes optimizations considering viruses’ small genome size, sequence homology with prokaryotes, and interactions with other members of the gut microbial community. Based on simulations, the workflow is fast and accurate with respect to both prokaryotes and viruses, minimizing false positive species identification using a novel genome coverage-based strategy. When applied to metagenomes from healthy adults, Phanta identified ~200 viral species per sample, ~5x more than the standard assembly-based methods. Notably, we observed a 2:1 ratio between gut viruses and bacteria, with higher interindividual variability of the gut virome compared to the gut bacteriome. Phanta performs equally well on bulk vs. virus-enriched metagenomes, making it possible to study prokaryotes and viruses in a single experiment, with a single analysis. Phanta can tandemly profile gut viruses and prokaryotes in existing and novel datasets, and can therefore identify cross-domain interactions with likely relevance to human health. We expect that Phanta will reduce the barrier to virus-inclusive studies of the human gut microbiome, thus making it standard practice.

Serine hydrolases play important roles in signaling and human metabolism, yet little is known about the functions of these enzymes in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human protease dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Functional studies reveal that BT4193 is a true homolog of hDPP4 while the other is misannotated and is a proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and is inhibited by FDA-approved type 2 diabetes drugs that target hDPP4. Loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. Taken together, our findings suggest that serine hydrolases contribute to gut microbiota dynamics and may be off-targets for existing drugs that could cause unintended impact on the microbiota.

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts., Almost 190,000 draft-quality DNA virus genomes are recovered by mining more than 11,000 deposited human stool metagenomes to improve resources for understanding the human gut virome.

In recognition of the rising incidence and mortality of cancer in low- and middle-resource settings, as well as the increasingly international profile of its membership, ASCO has prioritized efforts to enhance its engagement at a global level. Among the recommendations included in the 2016 Global Oncology Leadership Task Force report to the ASCO Board of Directors was that ASCO should promote the recognition of global oncology as an academic field. The report suggested that ASCO could serve a role in transitioning global oncology from an informal field of largely voluntary activities to a more formal discipline with strong research and well-defined training components. As a result of this recommendation, in 2017, ASCO formed the Academic Global Oncology Task Force (AGOTF) to guide ASCO’s contributions toward formalizing the field of global oncology. The AGOTF was asked to collect and analyze key issues and barriers toward the recognition of global oncology as an academic discipline, with an emphasis on training, research, and career pathways, and produce a set of recommendations for ASCO action. The outcome of the AGOTF was the development of recommendations designed to advance the status of global oncology as an academic discipline.

Tetralogy of Fallot (TOF) is a complex congenital cardiac defect. Surgical correction is well established as the treatment of choice and has resulted in a rapidly growing group of adults living with TOF. We describe potential complications of patients who have undergone TOF repair and were lost to follow-up. (Level of Difficulty: Intermediate.), Graphical abstract, Tetralogy of Fallot (TOF) is a complex congenital cardiac defect. Surgical correction is well established as the treatment of choice and has resulted in…

Purpose Misinformation and lack of information about cancer and its treatment pose significant challenges to delivering cancer care in resource-limited settings and may undermine patient engagement in care. We aimed to investigate patients’ knowledge and attitudes toward cancer and its treatment and to adapt, implement, and evaluate a low-literacy cancer patient education booklet at the Hôpital Universitaire de Mirebalais (HUM) in rural Haiti. Materials and Methods A low-literacy cancer patient education booklet was adapted into Haitian Creole in collaboration with clinicians at HUM. Patients were recruited for structured interviews (n = 20) and two focus groups (n = 13) designed to explore patients’ attitudes toward cancer and its treatment and to assess whether the booklet increased patients’ knowledge via an investigator-designed knowledge test. Results Participants reported a subjective lack of knowledge about cancer and its treatments and described views of cancer as deadly or incurable. Patients of varying education levels valued receiving written materials that set expectations about cancer treatment and expressed a desire to share the booklet with caregivers and others in their community. Participants across all levels of education significantly increased their performance on a knowledge test after counseling using the booklet (p < .001). Conclusion We found that an educational booklet about cancer developed in collaboration with local providers was well received by patients with variable literacy levels and improved their knowledge of cancer and its treatment in a resource-limited setting. Such educational materials have the potential to serve as tools to engage patients with cancer and their families in care. Implications for Practice Misinformation and lack of information pose significant challenges to delivering cancer care in resource-limited settings; however, there are often no culturally and literacy appropriate tools available to aid in patient education. This article shows that written educational materials are well received by patients of variable literacy levels and can be effective tools for increasing patients’ knowledge of cancer and its treatment in a limited-resource setting. Furthermore, the authors have made their educational booklet, Cancer and You, freely available online and welcome the opportunity to connect with readers of The Oncologist interested in implementing this educational booklet in clinical care.

No method exists to measure large-scale translation of genes in uncultured organisms in microbiomes. To overcome this limitation, we develop MetaRibo-Seq, a method for simultaneous ribosome profiling of tens to hundreds of organisms in microbiome samples. MetaRibo-Seq was benchmarked against gold-standard Ribo-Seq in a mock microbial community and applied to five different human fecal samples. Unlike RNA-Seq, Ribo-Seq signal of a predicted gene suggests it encodes a translated protein. We demonstrate two applications of this technique: First, MetaRibo-Seq identifies small genes, whose identification until now has been challenging. For example, MetaRibo-Seq identifies 2,091 translated, previously unannotated small protein families from five fecal samples, more than doubling the number of small proteins predicted to exist in this niche. Second, the combined application of RNA-Seq and MetaRibo-Seq identifies differences in the translation of transcripts. In summary, MetaRibo-Seq enables comprehensive translational profiling in microbiomes and identifies previously unannotated small proteins., Defining the functions of individual organisms or communities within microbiomes is a challenging task. Here, the authors develop MetaRibo-Seq, a method for simultaneous high-throughput ribosome profiling of organisms in uncultured microbiome samples.

Background Populations of closely related microbial strains can be simultaneously present in bacterial communities such as the human gut microbiome. We recently developed a de novo genome assembly approach that uses read cloud sequencing to provide more complete microbial genome drafts, enabling precise differentiation and tracking of strain-level dynamics across metagenomic samples. In this case study, we present a proof-of-concept using read cloud sequencing to describe bacterial strain diversity in the gut microbiome of one hematopoietic cell transplantation patient over a 2-month time course and highlight temporal strain variation of gut microbes during therapy. The treatment was accompanied by diet changes and administration of multiple immunosuppressants and antimicrobials. Methods We conducted short-read and read cloud metagenomic sequencing of DNA extracted from four longitudinal stool samples collected during the course of treatment of one hematopoietic cell transplantation (HCT) patient. After applying read cloud metagenomic assembly to discover strain-level sequence variants in these complex microbiome samples, we performed metatranscriptomic analysis to investigate differential expression of antibiotic resistance genes. Finally, we validated predictions from the genomic and metatranscriptomic findings through in vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples. Results During the 56-day longitudinal time course that was studied, the patient’s microbiome was profoundly disrupted and eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allowed us to identify differences in substrain populations over time. Based on this, we predicted that particular mobile element integrations likely resulted in increased antibiotic resistance, which we further supported using in vitro antibiotic susceptibility testing. Conclusions We find read cloud assembly to be useful in identifying key structural genomic strain variants within a metagenomic sample. These strains have fluctuating relative abundance over relatively short time periods in human microbiomes. We also find specific structural genomic variations that are associated with increased antibiotic resistance over the course of clinical treatment.

Microbial genomes can be assembled from short-read sequencing data, but the assembly contiguity of these metagenome-assembled genomes is constrained by repeat elements. Correct assignment of genomic positions of repeats is crucial for understanding the effect of genome structure on genome function. We applied nanopore sequencing and our workflow, named Lathe, which incorporates long-read assembly and short-read error correction, to assemble closed bacterial genomes from complex microbiomes. We validated our approach with a synthetic mixture of 12 bacterial species. Seven genomes were completely assembled into single contigs and three genomes were assembled into four or fewer contigs. Next, we used our methods to analyze metagenomics data from 13 human stool samples. We assembled 20 circular genomes, including genomes of Prevotella copri and a candidate Cibiobacter sp. Despite the decreased nucleotide accuracy compared with alternative sequencing and assembly approaches, our methods improved assembly contiguity, allowing for investigation of the role of repeat elements in microbial function and adaptation., Nanopore-metagenomics workflow enables assembly of complete, closed bacterial genomes from human stool samples.

Small genes (40,000 small-gene families in ~2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.