ABSTRACT
The intestinal microbiota is an important environmental factor implicated in CRC development. Intriguingly, modulation of DNA methylation by gut microbiota has been reported in preclinical models, although the relationship between tumor-infiltrating bacteria and CIMP status is currently unexplored. In this study, we investigated tumor-associated bacteria in 203 CRC tumor cases and validated the findings using The Cancer Genome Atlas datasets. We assessed the abundance of Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae through qPCR analysis and observed enrichment of all four bacterial species in CRC samples. Notably, except for E. coli, all exhibited significant enrichment in cases of CIMP. This enrichment was primarily driven by a subset of cases distinguished by high levels of these bacteria, which we labeled as "Superhigh". The bacterial Superhigh status showed a significant association with CIMP (odds ratio 3.1, p-value = 0.013) and with MLH1 methylation (odds ratio 4.2, p-value = 0.0025). In TCGA CRC cases (393 tumor and 45 adj. normal), bacterial taxa information was extracted from non-human whole exome sequencing reads, and the bacterial Superhigh status was similarly associated with CIMP (odds ratio 2.9, p < 0.001) and MLH1 methylation (odds ratio 3.5, p < 0.001). Finally, 16S ribosomal RNA gene sequencing revealed high enrichment of Bergeyella spp. C. concisus, and F. canifelinum in CIMP-Positive tumor cases. Our findings highlight that specific bacterial taxa may influence DNA methylation, particularly in CpG islands, and contribute to the development and progression of CIMP in colorectal cancer.
Subject(s)
Bacteria , Colorectal Neoplasms , CpG Islands , DNA Methylation , Gastrointestinal Microbiome , Humans , Colorectal Neoplasms/microbiology , Colorectal Neoplasms/genetics , Bacteria/genetics , Bacteria/classification , Bacteria/isolation & purification , Female , Male , Middle Aged , Bacteroides fragilis/genetics , Bacteroides fragilis/isolation & purification , Aged , PhenotypeABSTRACT
Introduction: Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed. Methods: Here, we leveraged our Collaborative Cross mouse model of developmental vitamin D deficiency (DVD) to investigate the role of DVD in dysregulating the molecular mechanisms underlying liver disease. We defined the effects on the adult liver transcriptome and metabolome and examined the role of epigenetic dysregulation. Given that the parental origin of the genome (POG) influences response to DVD, we used our established POG model [POG1-(CC011xCC001)F1 and POG2-(CC001xCC011)F1] to identify interindividual differences. Results: We found that DVD altered the adult liver transcriptome, primarily downregulating genes controlling liver development, response to injury/infection (detoxification & inflammation), cholesterol biosynthesis, and energy production. In concordance with these transcriptional changes, we found that DVD decreased liver cell membrane-associated lipids (including cholesterol) and pentose phosphate pathway metabolites. Each POG also exhibited distinct responses. POG1 exhibited almost 2X more differentially expressed genes (DEGs) with effects indicative of increased energy utilization. This included upregulation of lipid and amino acid metabolism genes and increased intermediate lipid and amino acid metabolites, increased energy cofactors, and decreased energy substrates. POG2 exhibited broader downregulation of cholesterol biosynthesis genes with a metabolomics profile indicative of decreased energy utilization. Although DVD primarily caused loss of liver DNA methylation for both POGs, only one epimutation was shared, and POG2 had 6.5X more differentially methylated genes. Differential methylation was detected at DEGs regulating developmental processes such as amino acid transport (POG1) and cell growth & differentiation (e.g., Wnt & cadherin signaling, POG2). Conclusions: These findings implicate a novel role for maternal vitamin D in programming essential offspring liver functions that are dysregulated in liver disease. Importantly, impairment of these processes was not rescued by vitamin D treatment at weaning, suggesting these effects require preventative measures. Substantial differences in POG response to DVD demonstrate that the parental genomic context of exposure determines offspring susceptibility.
Subject(s)
Cholesterol , Energy Metabolism , Liver , Vitamin D Deficiency , Animals , Mice , Liver/metabolism , Vitamin D Deficiency/metabolism , Vitamin D Deficiency/genetics , Cholesterol/metabolism , Cholesterol/biosynthesis , Female , Inflammation/metabolism , Male , Mice, Inbred C57BL , Transcriptome , Epigenesis, GeneticABSTRACT
Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as "pks" encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.
Subject(s)
Colonic Neoplasms , Colorectal Neoplasms , Humans , Mice , Animals , Mutagens/toxicity , Mutagens/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , DNA Mismatch Repair/genetics , Mutation , Colorectal Neoplasms/genetics , Colonic Neoplasms/pathologyABSTRACT
Despite advances in our understanding of the human microbiome, there exist significant knowledge gaps in our understanding of the skin microbiome of the preterm neonate. Herein, we describe skin microbiome sampling of six preterm neonates at multiple timepoints, and compare the skin microbiome samples to environmental (crib/isolette swabs) and negative controls. Samples of the same type (skin, crib, control) were more similar than when compared by week or by patient.
Subject(s)
Infant, Premature , Microbiota , Infant, Newborn , Humans , SkinABSTRACT
Pancreatic acinar cells display a remarkable degree of plasticity and can dedifferentiate into ductal-like progenitor cells by a process known as acinar ductal metaplasia (ADM). ADM is believed to be one of the earliest precursor lesions toward the development of pancreatic ductal adenocarcinoma and maintaining the pancreatic acinar cell phenotype suppresses tumor formation. The effects of a novel pStat3 inhibitor (LLL12B) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were investigated using 3-D cultures from p48Cre/+ and p48Cre/+LSL-KrasG12D/+ (KC) mice. LLL12B and TSA inhibited ADM in both KC and p48Cre/+ mouse pancreatic organoids. Furthermore, treatment with LLL12B or TSA on dedifferentiated acini from p48Cre/+ and KC mice that had undergone ADM produced morphologic and gene expression changes that suggest a reversal of ADM. Validation experiments using qRT-PCR (p48Cre/+ and KC) and RNA sequencing (KC) of the LLL12B and TSA treated cultures showed that the ADM reversal was more robust for the TSA treatments. Pathway analysis showed that TSA inhibited Spink1 and PI3K/AKT signaling during ADM reversal. The ability of TSA to reverse ADM was also observed in primary human acinar cultures. We report that pStat3 and HDAC inhibition can attenuate ADM in vitro and reverse ADM in the context of wild-type Kras. Our findings suggest that pharmacological inhibition or reversal of pancreatic ADM represents a potential therapeutic strategy for blocking aberrant ductal reprogramming of acinar cells.
ABSTRACT
Preclinical data demonstrate that the gut microbiota can promote pancreatic ductal adenocarcinoma (PDAC), but mechanisms remain unclear. We hypothesized that intestinal microbiota alters anti-tumor innate immunity response to facilitate PDAC progression. Human PDAC L3.6pl cells were heterotopically implanted into Rag1-/- mice after microbiota depletion with antibiotics, while syngeneic murine PDAC Pan02 cells were implanted intrapancreatic into germ-free (GF) C57BL/6 J mice. Natural killer (NK) cells and their IFNγ expression were quantitated by flow cytometry. NK cells were depleted in vivo using anti-Asialo GM1 antibody to confirm the role of NK cells. Bacteria-free supernatant from SPF and GF mice feces was used to test its effect on NK-92MI cell anti-tumor response in vitro. SPF and ex-GF mice (reconstituted with SPF microbiota) developed larger PDAC tumors with decreased NK cell tumor infiltration and IFNγ expression versus GF-Rag1-/-. Microbiota-induced PDAC tumorigenesis was attenuated by antibiotic exposure, a process reversed following NK cell depletion in both Rag1-/- and C57BL/6 J mice. Compared to GF, SPF-Rag1-/- abiotic stool culture supernatant inhibited NK-92MI cytotoxicity, migration, and anti-cancer related gene expression. Gut microbiota promotes PDAC tumor progression through modulation of the intratumoral infiltration and activity of NK cells.
Subject(s)
Carcinoma, Pancreatic Ductal , Gastrointestinal Microbiome , Pancreatic Neoplasms , Animals , Carcinogenesis , Carcinoma, Pancreatic Ductal/pathology , Homeodomain Proteins/genetics , Humans , Killer Cells, Natural , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/pathology , Pancreatic NeoplasmsABSTRACT
BACKGROUND: The human microbiome can contribute to pathogeneses of many complex diseases by mediating disease-leading causal pathways. However, standard mediation analysis methods are not adequate to analyze the microbiome as a mediator due to the excessive number of zero-valued sequencing reads in the data and that the relative abundances have to sum to one. The two main challenges raised by the zero-inflated data structure are: (a) disentangling the mediation effect induced by the point mass at zero; and (b) identifying the observed zero-valued data points that are not zero (i.e., false zeros). METHODS: We develop a novel marginal mediation analysis method under the potential-outcomes framework to address the issues. We also show that the marginal model can account for the compositional structure of microbiome data. RESULTS: The mediation effect can be decomposed into two components that are inherent to the two-part nature of zero-inflated distributions. With probabilistic models to account for observing zeros, we also address the challenge with false zeros. A comprehensive simulation study and the application in a real microbiome study showcase our approach in comparison with existing approaches. CONCLUSIONS: When analyzing the zero-inflated microbiome composition as the mediators, MarZIC approach has better performance than standard causal mediation analysis approaches and existing competing approach.
Subject(s)
Microbiota , Models, Statistical , Computer Simulation , Humans , Microbiota/genetics , Research DesignABSTRACT
BACKGROUND: Recent studies show that human gut microbial composition can determine whether a patient is a responder or non-responder to immunotherapy but have not identified a common microbial signal shared by responding patients. The functional relationship between immunity, intestinal microbiota, and NSCLC response to immune checkpoint inhibitor/inhibition (ICI) in an American cohort remains unexplored. METHODS: RNAlater-preserved fecal samples were collected from 65 pre-treatment (baseline) and post-treatment stage III/IV NSCLC patients undergoing ICI therapy, categorized as responders or non-responders according to RECIST criteria. Pooled and individual responder and non-responder microbiota were transplanted into a gnotobiotic mouse model of lung cancer and treated with ICIs. 16S rDNA and RNA sequencing was performed on patient fecal samples, 16S rDNA sequencing on mouse fecal samples, and flow cytometric analysis on mouse tumor tissue. RESULTS: Responder patients have both a different microbial community structure than non-responders (P = 0.004) and a different bacterial transcriptome (PC2 = 0.03) at baseline. Taxa significantly enriched in responders include amplicon sequence variants (ASVs) belonging to the genera Ruminococcus, Akkermansia, and Faecalibacterium. Pooled and individual responder microbiota transplantation into gnotobiotic mice decreased tumor growth compared to non-responder colonized mice following ICI (P = 0.023, P = 0.019, P = 0.008, respectively). Responder tumors showed an increased anti-tumor cellular phenotype following ICI treatment. Responder mice are enriched with ASVs belonging to the genera Bacteroides, Blautia, Akkermansia, and Faecalibacterium. Overlapping taxa mapping between human and mouse cohorts correlated with tumor size and weight revealed a network highlighting responder-associated ASVs belonging to the genera Colidextribacter, Frisingicoccus, Marvinbryantia, and Blautia which have not yet been reported. CONCLUSIONS: The role of isolate-specific function and bacterial gene expression in gut microbial-driven responsiveness to ICI has been underappreciated. This work supports further investigation using isolate-driven models to characterize the mechanisms underlying this phenomenon.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Gastrointestinal Microbiome , Lung Neoplasms , Animals , Humans , Immune Checkpoint Inhibitors , Lung Neoplasms/drug therapy , Mice , Programmed Cell Death 1 Receptor , United StatesABSTRACT
The microbiota comprises the microorganisms that live in close contact with the host, with mutual benefit for both counterparts. The contribution of the gut microbiota to the emergence of castration-resistant prostate cancer (CRPC) has not yet been addressed. We found that androgen deprivation in mice and humans promotes the expansion of defined commensal microbiota that contributes to the onset of castration resistance in mice. Specifically, the intestinal microbial community in mice and patients with CRPC was enriched for species capable of converting androgen precursors into active androgens. Ablation of the gut microbiota by antibiotic therapy delayed the emergence of castration resistance even in immunodeficient mice. Fecal microbiota transplantation (FMT) from CRPC mice and patients rendered mice harboring prostate cancer resistant to castration. In contrast, tumor growth was controlled by FMT from hormone-sensitive prostate cancer patients and Prevotella stercorea administration. These results reveal that the commensal gut microbiota contributes to endocrine resistance in CRPC by providing an alternative source of androgens.
Subject(s)
Androgens/biosynthesis , Bacteria/metabolism , Gastrointestinal Microbiome/physiology , Host Microbial Interactions , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/microbiology , Aged , Aged, 80 and over , Androgen Antagonists/therapeutic use , Animals , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/genetics , Cell Line, Tumor , Fecal Microbiota Transplantation , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasms, Experimental , Prevotella/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Symbiosis , Xenograft Model Antitumor AssaysABSTRACT
To investigate the relationship between intestinal microbiota and SARS-CoV-2-mediated pathogenicity in a United States, majority African American cohort. We prospectively collected fecal samples from 50 SARS-CoV-2 infected patients, 9 SARS-CoV-2 recovered patients, and 34 uninfected subjects seen by the hospital with unrelated respiratory medical conditions (controls). 16S rRNA sequencing and qPCR analysis was performed on fecal DNA/RNA. The fecal microbial composition was found to be significantly different between SARS-CoV-2 patients and controls (PERMANOVA FDR-P = .004), independent of antibiotic exposure. Peptoniphilus, Corynebacterium and Campylobacter were identified as the three most significantly enriched genera in COVID-19 patients compared to controls. Actively infected patients were also found to have a different gut microbiota than recovered patients (PERMANOVA FDR-P = .003), and the most enriched genus in infected patients was Campylobacter, with Agathobacter and Faecalibacterium being enriched in the recovered patients. No difference in microbial community structure between recovered patients and uninfected controls was observed, nor a difference in alpha diversity between the three groups. 24 of the 50 COVID-19 patients (48%) tested positive via RT-qPCR for fecal SARS-CoV-2 RNA. A significant difference in gut microbial composition between SARS-CoV-2 positive and negative samples was observed, with Klebsiella and Agathobacter being enriched in the positive cohort. No significant associations between microbiome composition and disease severity was found. The intestinal microbiota is sensitive to the presence of SARS-CoV-2, with increased relative abundance of genera (Campylobacter, Klebsiella) associated with gastrointestinal (GI) disease. Further studies are needed to investigate the functional impact of SARS-CoV-2 on GI health.
Subject(s)
COVID-19/microbiology , Gastrointestinal Microbiome , Aged , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , COVID-19/diagnosis , COVID-19/virology , Cohort Studies , Feces/microbiology , Feces/virology , Female , Humans , Male , Middle Aged , RNA, Ribosomal, 16S/genetics , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Severity of Illness Index , United States/epidemiologyABSTRACT
BACKGROUND: Hereditary alpha-tryptasemia (HαT) is characterized by elevated basal serum tryptase due to increased copies of the TPSAB1 gene. Individuals with HαT frequently present with multisystem complaints, including anaphylaxis and seemingly functional gastrointestinal (GI) symptoms. OBJECTIVE: We sought to determine the prevalence of HαT in an irritable bowel syndrome cohort and associated immunologic characteristics that may distinguish patients with HαT from patients without HαT. METHODS: Tryptase genotyping by droplet digital PCR, flow cytometry, cytometry by time-of-flight, immunohistochemistry, and other molecular biology techniques was used. RESULTS: HαT prevalence in a large irritable bowel syndrome cohort was 5% (N = 8/158). Immunophenotyping of HαT PBMCs (N ≥ 27) revealed increased total and class-switched memory B cells. In the small bowel, expansion of tissue mast cells with expression of CD203c, HLA-DR, and FcεRI, higher intestinal epithelial cell pyroptosis, and increased class-switched memory B cells were observed. IgG profiles in sera from individuals with HαT (N = 21) significantly differed from those in individuals with quiescent Crohn disease (N = 20) and non-HαT controls (N = 19), with increased antibodies directed against GI-associated proteins identified in individuals with HαT. CONCLUSIONS: Increased mast cell number and intestinal epithelial cell pyroptosis in the small intestine, and class-switched memory B cells in both the gut and peripheral blood associated with IgG reactive to GI-related proteins, distinguish HαT from functional GI disease. These innate and adaptive immunologic findings identified in association with HαT are suggestive of subclinical intestinal inflammation in symptomatic individuals.
Subject(s)
Gastrointestinal Diseases , Genetic Diseases, Inborn , Immunoglobulin G/immunology , Intestine, Small/immunology , Mastocytosis , Tryptases , Adult , Epithelial Cells/immunology , Female , Gastrointestinal Diseases/blood , Gastrointestinal Diseases/genetics , Gastrointestinal Diseases/immunology , Gastrointestinal Diseases/pathology , Genetic Diseases, Inborn/blood , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/immunology , Genetic Diseases, Inborn/pathology , Genotype , Humans , Immunoglobulin G/blood , Intestine, Small/cytology , Intestine, Small/pathology , Male , Mast Cells/immunology , Mastocytosis/blood , Mastocytosis/genetics , Mastocytosis/immunology , Mastocytosis/pathology , Middle Aged , Pyroptosis , Tryptases/blood , Tryptases/genetics , Young AdultABSTRACT
BACKGROUND: Oat has been widely accepted as a key food for human health. It is becoming increasingly evident that individual differences in metabolism determine how different individuals benefit from diet. Both host genetics and the gut microbiota play important roles on the metabolism and function of dietary compounds. OBJECTIVES: To investigate the mechanism of individual variations in response to whole-grain (WG) oat intake. METHODS: We used the combination of in vitro incubation assays with human gut microbiota, mouse and human S9 fractions, chemical analyses, germ-free (GF) mice, 16S rRNA sequencing, gnotobiotic techniques, and a human feeding study. RESULTS: Avenanthramides (AVAs), the signature bioactive polyphenols of WG oat, were not metabolized into their dihydro forms, dihydro-AVAs (DH-AVAs), by both human and mouse S9 fractions. DH-AVAs were detected in the colon and the distal regions but not in the proximal and middle regions of the perfused mouse intestine, and were in specific pathogen-free (SPF) mice but not in GF mice. A kinetic study of humans fed oat bran showed that DH-AVAs reached their maximal concentrations at much later time points than their corresponding AVAs (10.0-15.0 hours vs. 4.0-4.5 hours, respectively). We observed interindividual variations in the metabolism of AVAs to DH-AVAs in humans. Faecalibacterium prausnitzii was identified as the individual bacterium to metabolize AVAs to DH-AVAs by 16S rRNA sequencing analysis. Moreover, as opposed to GF mice, F. prausnitzii-monocolonized mice were able to metabolize AVAs to DH-AVAs. CONCLUSIONS: These findings demonstrate that the presence of intestinal F. prausnitzii is indispensable for proper metabolism of AVAs in both humans and mice. We propose that the abundance of F. prausnitzii can be used to subcategorize individuals into AVA metabolizers and nonmetabolizers after WG oat intake. This study was registered at clinicaltrials.gov as NCT04335435.
Subject(s)
Avena , Faecalibacterium prausnitzii , Gastrointestinal Microbiome , ortho-Aminobenzoates/metabolism , Animals , Avena/chemistry , Diet , Humans , Mice , RNA, Ribosomal, 16S/geneticsABSTRACT
Fatigue is one of the most prevalent and distressing complications among hematopoietic stem cell transplantation (HCT) survivors, negatively affecting physical, social, and emotional domains of quality of life. Chronic systemic inflammation has been linked to alterations in nervous system activity and initiation of distressing symptoms, such as fatigue. Damage to gut mucosa due to alteration in gut microbiota (GM) composition and microbial translocation has been shown to increase systemic proinflammatory cytokines. The aim of this study was to evaluate the relationship between fatigue and GM by measuring the differences in GM composition in HCT survivors with and without persistent fatigue. This cross-sectional study included 30 adults who underwent HCT for a hematologic disease and were at least 1 year post-HCT. Patients with chronic graft-versus-host disease were excluded. Fatigue severity was assessed by the Brief Fatigue Inventory (BFI). Based on the BFI score, patients were grouped into 2 categories: 0 to 3 (without fatigue) and ≥4 (with fatigue). The V1 to V3 region of the 16S rRNA gene from fecal specimens was sequenced using the Illumina MiSeq. Sequencing reads were processed, denoised, and replicated, chimeras were filtered, amplicon sequence variants (ASVs) were generated, and taxonomy was assigned using DADA2. Beta diversity analysis through principal coordinate analysis was generated using the Bray-Curtis dissimilarity matrix, and the difference was tested using linear model with generalized least squares in R. An alpha diversity analysis was performed using Chao1. Linear discriminant analysis effect size (LEfSe) was used to find markers that differ between the 2 groups. Based on the BFI results, patients were categorized into 2 cohorts: with fatigue (n = 14) and without fatigue (n = 16). The 2 cohorts were similar in terms of demographics, disease, and transplant characteristics. Based on the GM analysis, there was a significant difference in GM composition (beta diversity) between the 2 cohorts (P = .001). Alpha diversity (richness) was also significantly lower in survivors with fatigue (P =.002). LEfSe analysis identified 46 discriminative features (P < .05; linear discriminant analysis score >2) whose relative abundance varied significantly among individuals with fatigue and those without fatigue. Ten ASVs were associated with the patients with fatigue, and 36 ASVs were associated with those without fatigue. Several ASVs enriched in survivors with fatigue included organisms such as Klebsiella and Enterococcus, which have been implicated in inflammatory bowel diseases. The ASVs enriched in the cohort without fatigue were members of the Ruminococcaceae family (Oscillospira spp) and the Lachnospiraceae family (Fusicatenibacter and Coprococcus spp), which are known to have the ability to ferment complex plant carbohydrates. These findings show an association between GM composition and fatigue and suggest a microbial contribution to clinically significant fatigue post-HCT, which may guide the development of new approaches to treating fatigue based on manipulation of the GM.
Subject(s)
Dysbiosis , Fatigue , Gastrointestinal Microbiome , Hematopoietic Stem Cell Transplantation , Adult , Cross-Sectional Studies , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Quality of Life , RNA, Ribosomal, 16S , SurvivorsSubject(s)
Faecalibacterium prausnitzii , Gastrointestinal Microbiome , Graft vs Host Disease/microbiology , Intestinal Diseases/microbiology , Phylogeny , Adolescent , Adult , Chronic Disease , Cross-Sectional Studies , Faecalibacterium prausnitzii/genetics , Faecalibacterium prausnitzii/metabolism , Female , Graft vs Host Disease/genetics , Humans , Intestinal Diseases/genetics , MaleABSTRACT
BACKGROUND: Older adults have worse outcomes after sepsis than young adults. Additionally, alterations of the gut microbiota have been demonstrated to contribute to sepsis-related mortality. We sought to determine if there were alterations in the gut microbiota with a novel sepsis model in old adult mice, which enter a state of persistent inflammation, immunosuppression, and catabolism (PICS), as compared with young adult mice, which recover with the sepsis model. METHODS: Mixed sex old (â¼20 mo) and young (â¼4 mo) C57Bl/6J mice underwent cecal ligation and puncture with daily chronic stress (CLP+DCS) and were compared with naive age-matched controls. Mice were sacrificed at CLP+DCS day 7 and feces collected for bacterial DNA isolation. The V3-V4 hypervariable region was amplified, 16S rRNA gene sequencing performed, and cohorts compared. α-Diversity was assessed using Chao1 and Shannon indices using rarefied counts, and ß-diversity was assessed using Bray-Curtis dissimilarity. RESULTS: Naive old adult mice had significantly different α and ß-diversity compared with naive adult young adult mice. After CLP+DCS, there was a significant shift in the α and ß-diversity (FDRâ=â0.03 for both) of old adult mice (naive vs. CLP+DCS). However, no significant shift was displayed in the microbiota of young mice that underwent CLP+DCS in regards to α-diversity (FDRâ=â0.052) and ß-diversity (FDRâ=â0.12), demonstrating a greater overall stability of their microbiota at 7 days despite the septic insult. The taxonomic changes in old mice undergoing CLP+DCS were dominated by decreased abundance of the order Clostridiales and genera Oscillospira. CONCLUSION: Young adult mice maintain an overall microbiome stability 7 days after CLP+DCS after compared with old adult mice. The lack of microbiome stability could contribute to PICS and worse long-term outcomes in older adult sepsis survivors. Further studies are warranted to elucidate mechanistic pathways and potential therapeutics.
Subject(s)
Gastrointestinal Microbiome/physiology , Sepsis/microbiology , Age Factors , Animals , Female , Male , Mice , Mice, Inbred C57BLABSTRACT
The target of inference in microbiome analyses is usually relative abundance (RA) because RA in a sample (e.g., stool) can be considered as an approximation of RA in an entire ecosystem (e.g., gut). However, inference on RA suffers from the fact that RA are calculated by dividing absolute abundances (AAs) over the common denominator (CD), the summation of all AA (i.e., library size). Because of that, perturbation in one taxon will result in a change in the CD and thus cause false changes in RA of all other taxa, and those false changes could lead to false positive/negative findings. We propose a novel analysis approach (IFAA) to make robust inference on AA of an ecosystem that can circumvent the issues induced by the CD problem and compositional structure of RA. IFAA can also address the issues of overdispersion and handle zero-inflated data structures. IFAA identifies microbial taxa associated with the covariates in Phase 1 and estimates the association parameters by employing an independent reference taxon in Phase 2. Two real data applications are presented and extensive simulations show that IFAA outperforms other established existing approaches by a big margin in the presence of unbalanced library size. Supplementary materials for this article are available online.
ABSTRACT
Phase II drug metabolism inactivates xenobiotics and endobiotics through the addition of either a glucuronic acid or sulfate moiety prior to excretion, often via the gastrointestinal tract. While the human gut microbial ß-glucuronidase enzymes that reactivate glucuronide conjugates in the intestines are becoming well characterized and even controlled by targeted inhibitors, the sulfatases encoded by the human gut microbiome have not been comprehensively examined. Gut microbial sulfatases are poised to reactivate xenobiotics and endobiotics, which are then capable of undergoing enterohepatic recirculation or exerting local effects on the gut epithelium. Here, using protein structure-guided methods, we identify 728 distinct microbiome-encoded sulfatase proteins from the 4.8 million unique proteins present in the Human Microbiome Project Stool Sample database and 1766 gut microbial sulfatases from the 9.9 million sequences in the Integrated Gene Catalogue. We purify a representative set of these sulfatases, elucidate crystal structures, and pinpoint unique structural motifs essential to endobiotic sulfate processing. Gut microbial sulfatases differentially process sulfated forms of the neurotransmitters serotonin and dopamine, and the hormones melatonin, estrone, dehydroepiandrosterone, and thyroxine in a manner dependent both on variabilities in active site architecture and on markedly distinct oligomeric states. Taken together, these data provide initial insights into the structural and functional diversity of gut microbial sulfatases, providing a path toward defining the roles these enzymes play in health and disease.
Subject(s)
Bacteria/enzymology , Gastrointestinal Microbiome , Microbiota , Sulfatases/metabolism , Bacteria/chemistry , Bacteria/genetics , Bacteria/metabolism , Catalytic Domain , Feces/microbiology , Genes, Bacterial , Humans , Models, Molecular , Protein Conformation , Sulfatases/chemistry , Sulfatases/geneticsABSTRACT
BACKGROUND: The intestinal tract undergoes a period of cellular maturation during early life, primarily characterized by the organization of epithelial cells into specialized crypt and villus structures. These processes are in part mediated by the acquisition of microbes. Infants delivered at term typically harbor a stable, low diversity microbiota characterized by an overrepresentation of various Bacilli spp., while pre-term infants are colonized by an assortment of bacteria during the first several weeks after delivery. However, the functional effects of these changes on intestinal epithelium homeostasis and maturation remain unclear. To study these effects, human neonate feces were obtained from term and pre-term infants. Fecal 16S rDNA sequencing and global untargeted LC-MS were performed to characterize microbial composition and metabolites from each population. Murine enteral organoids (enteroids) were cultured with 0.22 µm filtered stool supernatant pooled from term or pre-term infants. RESULTS: Term and pre-term microbial communities differed significantly from each other by principle components analysis (PCoA, PERMANOVA p < 0.001), with the pre-term microbiome characterized by increased OTU diversity (Wilcox test p < 0.01). Term communities were less diverse and dominated by Bacilli (81.54%). Pre-term stools had an increased abundance of vitamins, amino acid derivatives and unconjugated bile acids. Pathway analysis revealed a significant increase in multiple metabolic pathways in pre-term samples mapped to E. coli using the KEGG database related to the fermentation of various amino acids and vitamin biosynthesis. Enteroids cultured with supernatant from pre-term stools proliferated at a higher rate than those cultured with supernatant from term stools (cell viability: 207% vs. 147.7%, p < 0.01), grew larger (area: 81,189µm2 vs. 41,777µm2, p < 0.001), and bud at a higher rate (6.5 vs. 4, p < 0.01). Additionally, genes involved in stem cell proliferation were upregulated in pre-term stool treated enteroid cultures (Lgr5, Ephb2, Ascl2 Sox9) but not term stool treated enteroids. CONCLUSIONS: Our findings indicate that microbial metabolites from the more diverse gut microbiome associated with pre-term infants facilitate stem cell proliferation. Therefore, perturbations of the pre-term microbiota may impair intestinal homeostasis.
Subject(s)
Bacteria/classification , Enterocytes/cytology , Metabolomics/methods , Premature Birth/microbiology , RNA, Ribosomal, 16S/genetics , Animals , Animals, Newborn , Bacteria/chemistry , Bacteria/genetics , Bacteria/isolation & purification , Biomarkers/metabolism , Cell Proliferation , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Enterocytes/microbiology , Feces/microbiology , Gastrointestinal Microbiome , Gene Expression Regulation , Humans , Infant, Newborn , Mice , Organ Culture Techniques , Organoids/chemistry , Organoids/cytology , Organoids/microbiology , Phylogeny , Term BirthABSTRACT
Infection is a major cause of morbidity and mortality after hematopoietic cell transplantation (HCT). Gut microbiota (GM) composition and metabolites provide colonization resistance against dominance of potential pathogens, and GM dysbiosis following HCT can be deleterious to immune reconstitution. Little is known about the composition, diversity, and evolution of GM communities in HCT patients and their association with subsequent febrile neutropenia (FN) and infection. Identification of markers before HCT that predict subsequent infection could be useful in developing individualized antimicrobial strategies. Fecal samples were collected prospectively from 33 HCT recipients at serial time points: baseline, post-conditioning regimen, neutropenia onset, FN onset (if present), and hematologic recovery. GM was assessed by 16S rRNA sequencing. FN and major infections (ie, bloodstream infection, typhlitis, invasive fungal infection, pneumonia, and Clostridium difficile enterocolitis) were identified. Significant shifts in GM composition and diversity were observed during HCT, with the largest alterations occurring after initiation of antibiotics. Loss of diversity persisted without a return to baseline at hematologic recovery. GM in patients with FN was enriched in Mogibacterium, Bacteroides fragilis, and Parabacteroides distasonis, whereas increased abundance of Prevotella, Ruminococcus, Dorea, Blautia, and Collinsella was observed in patients without fever. A baseline protective GM profile (BPGMP) was predictive of protection from major infection. The BPGMP was associated with subsequent major infections with 77% accuracy and an area under the curve of 79%, with sensitivity, specificity, and positive and negative predictive values of 0.71, 0.91, 0.77, and 0.87, respectively. Our data show that large shifts in GM composition occur early after HCT, and differences in baseline GM composition are associated with the development of subsequent major infections.
Subject(s)
Gastrointestinal Microbiome , Hematopoietic Stem Cell Transplantation , Bacteroidetes , Feces , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , RNA, Ribosomal, 16S/geneticsABSTRACT
Pain is one of the most common reasons to seek medical attention and chronic pain is a worldwide epidemic. There are currently no relevant biomarkers for the diagnosis of chronic pain, and new therapeutic strategies for chronic pain treatment are desperately needed. The chronic constriction injury (CCI) of the sciatic nerve is a widely used preclinical model of pathological neuropathic pain. Over the past decade, investigators have come to appreciate the many contributions of noncoding RNA including microRNA (miRNA), and other long and short noncoding (nc) RNAs. The development and/or maintenance of chronic pain could be controlled epigenetically through ncRNAs. Here we seek to characterize CNS tissues in a mouse model of neuropathic pain as this may serve to elucidate potential biomarkers relevant to pathological pain in humans. Male C57BL6/J mice (6 CCI and 6 sham procedure) underwent surgery for sciatic nerve ligation with chromic gut sutures. Following 7 days, mechanical allodynia was quantified using the von Frey assay. Mice were then euthanized for collection of spinal cord and sciatic nerve. cDNA was synthesized to 627 unique mature miRNAs from the total RNA. In the CCI mice that displayed mechanical allodynia, 11 and 125 miRNAs were differentially expressed (i.e., greater than 1.5-fold increase or decrease; Pâ¯<â¯0.05) in the spinal cord and sciatic nerve, respectively, as compared to sham controls. Among those differentially expressed miRNAs in the sciatic nerve of CCI mice, the following passed the more stringent Bonfferoni correction: miR-138-3p, miR-138-5p and miR-676-3p, reduced and miR-142-5p, increased. Our data support miRNAs as promising therapeutic targets for the treatment of pathological pain.