Ashley R. Wolf

Background: Stool-based molecular tests provide noninvasive options for pediatric tuberculosis (TB) diagnosis but have lower sensitivity compared to sputum-based tests. Untargeted metagenomic next-generation sequencing (mNGS) on stool could improve sensitivity and identify new gene targets for molecular testing. Methods: We performed shotgun mNGS on DNA isolated from stool samples of children undergoing assessment for pulmonary TB in Uganda. We defined the performance of mNGS to identify Mycobacterium tuberculosis (Mtb) against a microbiological reference standard (MRS, TB if sputum Xpert Ultra or culture positive) and a composite reference standard (TB if confirmed or unconfirmed TB). We also compared the accuracy of mNGS against stool-based Xpert Ultra. Finally, we identified enriched genomic loci among Mtb-classified reads. Results: We analyzed 176 stool samples of children with a median age of 3.6 years (interquartile range [IQR], 1–6 years). Against the MRS, the sensitivities of mNGS with positive TB defined as ≥1, 2, or 5 sequence fragments were 35.5% (95% confidence interval [CI] 19%–55%), 25.7% (12%–45%), and 19.4% (7%–37%), respectively, and specificities 92.6% (87%–96%), 97% (93%–99%), and 99.3% (96%–100%). Stool Xpert Ultra had similar sensitivity (22.6%) to stool mNGS considering all samples tested. In a head-to-head comparison, stool mNGS had lower sensitivity than stool Xpert Ultra (38.5% vs 53.8%, difference −15.3%, 95% CI 14–68 vs 25–81). mNGS utilized genes encoding rRNA, virulence proteins, and membrane proteins not targeted in current PCR-based platforms. Conclusions: Metagenomic sequencing of stool DNA did not increase sensitivity of TB detection, but identified novel targets for molecular testing that may support development of more sensitive tests.

Protein and glycan antigens synthesized by gut microbes stimulate circulating and secreted antibody production. Despite continuous exposure of hosts to the plant glycans that constitute dietary fiber, it remains unclear whether these foreign structures induce mucosal immune responses. We report that humans and mice generate antibodies specific for common glycans in plant foods. Oral exposure to individual fiber types induced T cell-independent, glycan-specific IgM and IgA. The induction of anti-fiber antibodies required colonization by particular microbes, since germ-free mice and mice harboring representatives of several bacterial phyla failed to respond. The OMM12 model community was sufficient to rescue antibody induction, and dietary fiber glycans were detected on the surfaces of OMM12 microbes, suggesting a route by which bacteria trigger anti-fiber immune responses. Our results reveal a direct impact of dietary fiber on the adaptive immune system with implications for host control of fiber breakdown by bacteria in the gut lumen.

Prenatal undernutrition and low birth weight are associated with risk of type 2 diabetes and obesity. Prenatal caloric restriction results in low birth weight, glucose intolerance, obesity, and reduced plasma bile acids (BAs) in offspring mice. Because BAs can regulate systemic metabolism and glucose homeostasis, we hypothesized that BA supplementation could prevent diet-induced obesity and glucose intolerance in this model of developmental programming. Pregnant dams were food restricted by 50% from gestational days 12.5 to 18.5. Offspring of both undernourished (UN) and control (C) dams given unrestricted diets were weaned to high-fat diets with or without supplementation with 0.25% w/w ursodeoxycholic acid (UDCA), yielding four experimental groups: C, UN, C + UDCA, and UN + UDCA. Glucose homeostasis, BA composition, liver and intestinal gene expression, and microbiota composition were analyzed in the four groups. Although UDCA supplementation ameliorated diet-induced obesity in C mice, there was no effect in UN mice. UDCA similarly lowered fasting insulin, and improved glucose tolerance, pyruvate tolerance, and liver steatosis in C, but not UN, animals. BA composition differed significantly, and liver and ileal expression of genes involved in BA metabolism (Cyp7b1, Shp) were differentially induced by UDCA in C vs UN animals. Bacterial taxa in fecal microbiota correlated with treatment groups and metabolic parameters. In conclusion, prenatal undernutrition alters responsiveness to the metabolic benefits of BA supplementation, with resistance to the weight-lowering and insulin-sensitizing effects of UDCA supplementation. Our findings suggest that BA metabolism may be a previously unrecognized contributor to developmentally programmed diabetes risk.

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.

Background: Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results: We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions: We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation.