Biology subjects

Golob, J. L.

Publications and source records attributed to Golob, J. L..

10 recordsLinked to original sources

bioRxiv2025

Expanding vaginal microbiome pangenomes via a custom MIDAS database reveals Lactobacillus crispatus accessory genes associated with cervical dysplasia

The vaginal microbiome plays a central role in reproductive health. Vaginal microbiome dysbiosis is associated with many adverse reproductive health outcomes, but most studies have focused on associations at the species level. The potential contribution of intraspecies microbial variation, especially gene content differences across bacterial strains, remains underexplored in reproductive health contexts. The Metagenomic Intra-Species Diversity Analysis (MIDAS) framework enables such analyses but depends on comprehensive reference databases. We constructed a MIDAS-compatible pangenome database from over 18,000 genomes in the Vaginal Microbiome Genome Collection (VMGC). Compared to the Genome Taxonomy Database (GTDB)-derived reference, the VMGC database expanded pangenomes of prevalent vaginal species, better capturing vaginal-specific intraspecies diversity. Applying this database to vaginal samples from a cervical dysplasia cohort, we identified thirteen Lactobacillus crispatus accessory genes significantly associated with cervical dysplasia, including a HicAB toxin-antitoxin system, three transcriptional regulators, and three phage-derived genes. These findings highlight the utility of body site-specific reference resources and shotgun metagenomic sequencing for uncovering intraspecies microbial variation relevant to reproductive health. IMPORTANCEThe vaginal microbiome plays a critical role in reproductive health, and different bacteria from the same species can carry different genes that influence how the strains interact with the host and other microbes. These strain-level differences are often overlooked when microbiomes are analyzed only at the species level. Existing genomic reference databases are heavily biased toward gut and environmental bacteria, leaving the genetic diversity of vaginal microbes understudied. We built a specialized reference database from over 18,000 vaginal bacterial genomes that better reflects this diversity. We then applied this resource to quantify gene-level variation in vaginal samples from a cervical dysplasia cohort. Focusing on Lactobacillus crispatus, a prevalent and often beneficial vaginal species, we identified thirteen genes that were more common in women with cervical dysplasia than in controls. This work demonstrates that body site-specific genomic resources are essential for uncovering strain-level bacterial differences relevant to reproductive health.

microbiology↗

bioRxiv2024

Acarbose Impairs Gut Bacteroides Growth by Targeting Intracellular GH97 Enzymes

Acarbose is a type-2 diabetes medicine that inhibits dietary starch breakdown into glucose by inhibiting host amylase and glucosidase enzymes. Numerous gut species in the Bacteroides genus enzymatically break down starch and change in relative abundance within the gut microbiome in acarbose-treated individuals. To mechanistically explain this observation, we used two model starch-degrading Bacteroides, Bacteroides ovatus (Bo) and Bacteroides thetaiotaomicron (Bt). Bt growth is severely impaired by acarbose whereas Bo growth is not. The Bacteroides use a starch utilization system (Sus) to grow on starch. We hypothesized that Bo and Bt Sus enzymes are differentially inhibited by acarbose. Instead, we discovered that although acarbose primarily targets the Sus periplasmic GH97 enzymes in both organisms, the drug affects starch processing at multiple other points. Acarbose competes for transport through the Sus beta-barrel proteins and binds to the Sus transcriptional regulators. Further, Bo expresses a non-Sus GH97 (BoGH97D) when grown in starch with acarbose. The Bt homolog, BtGH97H, is not expressed in the same conditions, nor can overexpression of BoGH97D complement the Bt growth inhibition in the presence of acarbose. This work informs us about unexpected complexities of Sus function and regulation in Bacteroides, including variation between related species. Further, this indicates that the gut microbiome may be a source of variable response to acarbose treatment for diabetes. ImportanceAcarbose is a type 2 diabetes medication that works primarily by stopping starch breakdown into glucose in the small intestine. This is accomplished by inhibition of host enzymes, leading to better blood sugar control via reduced ability to derive glucose from dietary starches. The drug and undigested starch travel to the large intestine where acarbose interferes with the ability of some bacteria to grow on starch. However, little is known about how gut bacteria interact with acarbose, including microbes that can use starch as a carbon source. Here, we show that two gut species, Bacteroides ovatus (Bo) and Bacteroides thetaiotaomicron (Bt), respond differently to acarbose: Bt growth is inhibited by acarbose while Bo growth is not. We reveal a complex set of mechanisms involving differences in starch import and sensing behind the different Bo and Bt responses. This indicates the gut microbiome may be a source of variable response to acarbose treatment for diabetes via complex mechanisms in common gut microbes.

microbiology↗

bioRxiv2024

Inflammation induced Th17 cells synergize with the inflammation-trained microbiota to mediate host-resiliency against intestinal injury

Background and AimsInflammation can generate pathogenic Th17 cells and cause a inflammatory dysbiosis. In the context of Inflammatory Bowel Disease (IBD) these inflammatory Th17 cells and dysbiotic microbiota may perpetuate injury to intestinal epithelial cells (IECs). However, many models of IBD like T-cell transfer colitis and IL-10-/- mice rely on the absence of regulatory pathways, so it is difficult to tell if inflammationcan also induce protective Th17 cells. MethodsWe subjected C57BL6, RAG1-/- or JH-/- mice to systemic or gastrointestinal (GI) Citrobacter rodentium (Cr). Mice were then subject to 2.5% dextran sodium sulfate to cause epithelial injury. Fecal microbiota transfer was performed by bedding transfer and co-housing. Flow cytometry, qPCR, 16s sequencing and histology were used to assess parameters. ResultsTransient inflammation with GI but not systemic Cr was protective from subsequent intestinal injury. This was replicated with sequential DSS collectively indicating that transient inflammation provides tissue-specific protection. Inflammatory Th17 cells that have a tissue resident memory signature expanded in the intestine. Experiments with reconstituted RAG1-/-, JH-/- mice and cell trafficking inhibitors showed that inflammation induced Th17 cells were required for protection. Fecal microbiota transfer showed that the inflammation-trained microbiota was necessary for protection, likely by maintaining protective Th17 cells in situ. ConclusionInflammation can generate protective Th17 cells which synergize with the inflammation-trained microbiota to provide host resiliency against subsequent injury, indicating that inflammation induced Th17 tissue resident memory T cells are heterogenous and contain protective subsets.

immunology↗

bioRxiv2023

IL-15 promotes inflammatory Th17 cells in the intestine

Ulcerative Colitis (UC) is a chronic gastrointestinal condition with high morbidity. While modern medical therapies have revolutionized the care of UC, 10-25% of patients fail medications and still progress to surgery. Thus, developing new treatments is a core problem in UC. T-cells, especially Th17 cells, are strongly linked with UC and are major targets of medications in UC. Tissue-resident memory T-cells (TRM) are a distinct class of T-cells that are highly enriched in the intestine, closely aligned with the microbiota, and are implicated in the pathogenesis of UC. Unlike circulating T-cells, TRM are difficult to target because they do not recirculate. Thus, we focused on cytokines like IL-15 which act as a tissue danger signal and regulate T-cells in situ. We found that the IL15 axis is upregulated in UC and predicts treatment response. IL-15 was redundant for Th17 differentiation but could activate terminally differentiated Th17 cells to promote intestinal inflammation. Finally, in CD4+ TRM from patients with UC, IL-15 upregulated RORC, the master transcription factor for Th17 cells, via a Janus Kinase (JAK)1 pathway. Thus, IL-15 promotes terminally differentiated inflammatory Th17 cells in the intestine raising the possibility that IL-15 may be a target for UC treatments.

immunology↗

bioRxiv2022

Robust Harmonization of Microbiome Studies by Phylogenetic Scaffolding with MaLiAmPi

Microbiome science is difficult to translate back to patients due to an inability to harmonize 16S rRNA gene-based microbiome data, as differences in the technique will result in different amplicon sequence variants (ASV) from the same microbe. Here we demonstrate that placement of ASV onto a common phylogenetic tree of full-length 16S rRNA alleles can harmonize microbiome studies. Using in silico data approximating 100 healthy human stool microbiomes we demonstrated that phylogenetic placement of ASV can recapitulate the true relationships between communities as compared closed-OTU based approaches (Spearman R 0.8 vs 0.2). Using real data from thousands of human gut and vaginal microbiota, we demonstrate phylogenetic placement, but not closed OTUs, were able to group communities by origin (stool vs vaginal) without being confounded by technique and integrate new data into existing ordination/clustering models for precision medicine. This enables meta-analysis of microbiome studies and the microbiome as a biomarker.

bioinformatics↗

bioRxiv2022

Mucolytic bacteria license pathobionts to acquire host-derived nutrients during dietary nutrient restriction

Pathobionts employ unique metabolic adaptation mechanisms to maximize their growth in disease conditions. Adherent-invasive Escherichia coli (AIEC), a pathobiont enriched in the gut mucosa of patients with inflammatory bowel disease (IBD), utilizes diet-derived L-serine to adapt to the inflamed gut. Therefore, the restriction of dietary L-serine starves AIEC and limits its fitness advantage. Here, we find that AIEC can overcome this nutrient limitation by switching the nutrient source from the diet to the host cells in the presence of mucolytic bacteria. During diet-derived L-serine restriction, the mucolytic symbiont Akkermansia muciniphila promotes the encroachment of AIEC to the epithelial niche by degrading the mucus layer. In the epithelial niche, AIEC acquires L-serine from the colonic epithelium and thus proliferates. Our work suggests that the indirect metabolic network between pathobionts and commensal symbionts enables pathobionts to overcome nutritional restriction and thrive in the gut.

microbiology↗

bioRxiv2021

Comparative analysis of public RNA-sequencing data from human intestinal enteroid (HIEs) infected with enteric RNA viruses identifies universal and virus-specific epithelial responses

Acute gastroenteritis (AGE) has a significant disease burden on society. Noroviruses, rotaviruses and astroviruses are important viral causes of AGE but are relatively understudied enteric pathogens. Recent developments in novel biomimetic human models of enteric disease are opening new possibilities for studying human-specific hostmicrobe interactions. Human intestinal enteroids (HIE), which are epithelium-only intestinal organoids derived from stem cells isolated from human intestinal biopsy tissues, have been successfully used to culture representative norovirus, rotavirus and astrovirus strains. Previous studies investigated host-virus interactions at the intestinal epithelial interface by individually profiling the epithelial transcriptional response to a member of each virus family by RNA sequencing (RNA-seq). We used these publicly available datasets to uniformly analyze these data and identify shared and unique transcriptional changes in the human intestinal epithelium upon human enteric virus infections.

microbiology↗

bioRxiv2020

Gene-level metagenomics identifies genome islands associated with immunotherapy response

Researchers must be able to generate experimentally testable hypotheses from sequencing-based observational microbiome experiments to discover the mechanisms underlying the influence of gut microbes on human health. We describe a novel bioinformatics tool for identifying testable hypotheses based on gene-level metagenomic analysis of WGS microbiome data (geneshot). By applying geneshot to two independent previously published cohorts, we identified microbial genomic islands consistently associated with response to immune checkpoint inhibitor (ICI)-based cancer treatment in culturable type strains. The identified genomic islands are within operons involved in type II secretion, TonB-dependent transport, and bacteriophage growth. These results, as well as the underlying methodology, inform further mechanistic studies and facilitate the development of microbiome-enhanced therapeutics.

bioinformatics↗

bioRxiv2020

Organoid-derived adult human colonic epithelium responds to co-culture with a probiotic strain of Bifidobacterium longum

In germ-free animals the lack of microbes within the gut results in a propensity to mucosal inflammation among other immune deficits. This suggests microbes are essential for the healthy function of the human gut, but we have lacked a reproducible mechanistic model of interactions between human colonic epithelium and the anaerobic microbes within the gut. To establish the physiological effect of a common anaerobe in the human gut, we co-cultured a probiotic strain of Bifidobacterium longum (35624) with organoid-derived adult human colonic epithelium in asymmetric gas conditions (anoxic apically, 5% oxygen basolaterally) and compared to axenic ( germ-free) epithelium. Bacteria proliferated and retained their normal cellular morphology in the presence of the human colonic epithelium. The human colonic mucosa retained trans-epithelial electrical resistance (TEER) consistent with an intact epithelium but lower than in axenic conditions. Changes in TEER corresponded to changes in Claudin-family gene expression. Inflammation was repressed in co-culture as compared to axenic, with reduced expression of executor and pyroptosis caspases; reduced expression of activators and increased expression of inhibitors of NFKB; reduced expression of toll-like-receptors (TLRs) and increased expression of TOLLIP (a negative regulator of TLRs). Consistent with the presence of actively fermenting bacteria that produce lactate and acetate but do not produce butyrate, PPARA expression was increased while PPARG expression was reduced. As in germ-free animal experiments, axenic human colonic mucosa is poised for inflammation. Co-culture with Bifidobacterium longum resolved the pro-inflammatory state while modulating barrier function (via Claudin genes) and cellular energetics (via PPARA and PPARG genes). ImportanceExperiments in animals demonstrate the importance of microbes with the gut for the health of the gut. Many of the microbes within the healthy gut are strictly anaerobic and cannot grow in the presence of oxygen; human tissues require some oxygen to live. Here we observe the effect of growing an anaerobic bacterium - Bifidobacterium longum - typically found in the human gut with human colonic tissue. We observed that the human tissue responded favorably to being cultivated with the microbe as compared to being alone. This experiment confirms human colonic tissues reduce their inflammation in the presence of a bacteria typically found in the gut.

systems biology↗

bioRxiv2018

Functional Analysis of Metagenomes by Likelihood Inference (FAMLI) Successfully Compensates for Multi-Mapping Short Reads from Metagenomic Samples

High-throughput sequencing can establish the functional capacity of a microbial community by cataloging the protein-coding sequences (CDS) present in the metagenome of the community. The relative performance of different computational methods for identifying CDS from whole-genome shotgun sequencing (WGS) is not fully established.\n\nHere we present an automated benchmarking workflow, using synthetic shotgun sequencing reads for which we know the true CDS content of the underlying communities, to determine the relative performance (sensitivity, positive predictive value or PPV, and computational efficiency) of different metagenome analysis tools for extracting the CDS content of a microbial community.\n\nAssembly-based methods are limited by coverage depth, with poor sensitivity for CDS at < 5X depth of sequencing, but have excellent PPV. Mapping-based techniques are more sensitive at low coverage depths, but can struggle with PPV. We additionally describe an expectation maximization based iterative algorithmic approach which we show to successfully improve the PPV of a mapping based technique while retaining improved sensitivity and computational efficiency.

bioinformatics↗