Project description:The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion-sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B12). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems. Keywords: Other 57 samples analyzed, 1 of these is the reference (input) sample
Project description:The influence of the microbiota on viral transmission and replication is well appreciated. However, its impact on retroviral pathogenesis outside of transmission/replication control remained unknown. Using Murine Leukemia Virus (MuLV), we found that some commensal bacteria promoted the development of leukemia induced by this retrovirus. The promotion of leukemia development by commensals was due to suppression of the adaptive immune response through upregulation of several negative regulators of immunity. These negative regulators included Serpinb9b and Rnf128, which are associated with a poor prognosis of some spontaneous human cancers. Upregulation of Serpinb9b was mediated by sensing of bacteria by NOD1/NOD2/RIPK2 pathway. This work describes a novel mechanism by which the microbiota enhances tumorigenesis within gut-distant organs and points at potential new targets for cancer therapy.
Project description:The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion-sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B12). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems. Keywords: Other
Project description:M cells are the main site of bacterial translocation in the intestine. We used the in vitro M cell model to study the effect of the commensal bacteria; Lactobacillus salivarius, Eschericha coli and Bacteroides fragilis, on M cell gene expression. Bacterial translocation across the gut mucosa has traditionally been based on the detection of commensals in the mesenteric lymph node. Differential rates of commensal translocation have been reported in vivo, however fewer studies have examined translocation of commensals at the level of the gut epithelial M cell. In this study we employed an in vitro M cell model to quantify translocation of various bacteria. C2BBe1 cells were differentiated into M cells and the gene expression profile and transport kinetics of different bacterial strains, namely Lactobacillus salivarius, Escherichia coli, and Bacteroides fragilis, was assessed. For comparison with M cell uptake, the THP-1 monocytic cell line was used to analyze bacterial internalization and resulting cytokine production. The commensal bacterial strains were translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency while L. salivarius translocated with less efficiency. In contrast, L. salivarius was internalized by THP-1 cells to a higher degree than B. fragilis or E. coli and was associated with a different cytokine profile. Microarray analysis showed both common and differential gene expression amongst the bacteria and control polystyrene beads. In the presence of bacteria, but not beads, upregulated genes were mainly involved in transcription regulation and dephosphorylation, e.g. EGR1, JUN; whereas proinflammatory and stress response genes were primarily upregulated by E. coli and B. fragilis, but not L. salivarius nor beads, e.g. IL8, TNFAIP3. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses. C2bbe1 cells were converted to M cells (C2M) following 21 days of culture on Transwells in the presence of Raji B cells. C2M cells were co-cultured alone, Lactobacillus salivarius, Eschericha coli, Bacteroides fragilis and control beads. Total RNA was extracted and processed for Affymetrix array hybridisation
Project description:Single-cell RNA sequencing is increasingly applied to bacterial model species, but a protocol for obligate anaerobic gut commensals belonging to the Bacteroidota phylum has not previously been established. Here, we present a technique to profile global gene expression of individual Bacteroides thetaiotaomicron cells. We describe steps for transcriptome stabilization and FACS-based collection and lysis of single Bacteroides cells. We then detail procedures for reverse transcription, sensitive depletion of ribosomal sequences, and cDNA library generation. This experimental pipeline has potential to uncover cell-to-cell variability in predominant bacterial members of the healthy human gut microbiota.
Project description:M cells are the main site of bacterial translocation in the intestine. We used the in vitro M cell model to study the effect of the commensal bacteria; Lactobacillus salivarius, Eschericha coli and Bacteroides fragilis, on M cell gene expression. Bacterial translocation across the gut mucosa has traditionally been based on the detection of commensals in the mesenteric lymph node. Differential rates of commensal translocation have been reported in vivo, however fewer studies have examined translocation of commensals at the level of the gut epithelial M cell. In this study we employed an in vitro M cell model to quantify translocation of various bacteria. C2BBe1 cells were differentiated into M cells and the gene expression profile and transport kinetics of different bacterial strains, namely Lactobacillus salivarius, Escherichia coli, and Bacteroides fragilis, was assessed. For comparison with M cell uptake, the THP-1 monocytic cell line was used to analyze bacterial internalization and resulting cytokine production. The commensal bacterial strains were translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency while L. salivarius translocated with less efficiency. In contrast, L. salivarius was internalized by THP-1 cells to a higher degree than B. fragilis or E. coli and was associated with a different cytokine profile. Microarray analysis showed both common and differential gene expression amongst the bacteria and control polystyrene beads. In the presence of bacteria, but not beads, upregulated genes were mainly involved in transcription regulation and dephosphorylation, e.g. EGR1, JUN; whereas proinflammatory and stress response genes were primarily upregulated by E. coli and B. fragilis, but not L. salivarius nor beads, e.g. IL8, TNFAIP3. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.