Gut mucosa alterations and loss of segmented filamentous bacteria in type 1 diabetes are associated with inflammation rather than hyperglycaemia.

OBJECTIVE: Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic ß-cells producing insulin. Both T1D patients and animal models exhibit gut microbiota and mucosa alterations, although the exact cause for these remains poorly understood. We investigated the production of key cytokines controlling gut integrity, the abundance of segmented filamentous bacteria (SFB) involved in the production of these cytokines, and the respective role of autoimmune inflammation and hyperglycaemia. DESIGN: We used several mouse models of autoimmune T1D as well as mice rendered hyperglycaemic without inflammation to study gut mucosa and microbiota dysbiosis. We analysed cytokine expression in immune cells, epithelial cell function, SFB abundance and microbiota composition by 16S sequencing. We assessed the role of anti-tumour necrosis factor α on gut mucosa inflammation and T1D onset. RESULTS: We show in models of autoimmune T1D a conserved loss of interleukin (IL)-17A, IL-22 and IL-23A in gut mucosa. Intestinal epithelial cell function was altered and gut integrity was impaired. These defects were associated with dysbiosis including progressive loss of SFB. Transfer of diabetogenic T-cells recapitulated these gut alterations, whereas induction of hyperglycaemia with no inflammation failed to do so. Moreover, anti-inflammatory treatment restored gut mucosa and immune cell function and dampened diabetes incidence. CONCLUSION: Our results demonstrate that gut mucosa alterations and dysbiosis in T1D are primarily linked to inflammation rather than hyperglycaemia. Anti-inflammatory treatment preserves gut homeostasis and protective commensal flora reducing T1D incidence.

Colorectal cancer specific conditions promote Streptococcus gallolyticus gut colonization.

Colonization by Streptococcus gallolyticus subsp. gallolyticus (SGG) is strongly associated with the occurrence of colorectal cancer (CRC). However, the factors leading to its successful colonization are unknown, and whether SGG influences the oncogenic process or benefits from the tumor-prone environment to prevail remains an open question. Here, we elucidate crucial steps that explain how CRC favors SGG colonization. By using mice genetically prone to CRC, we show that SGG colonization is 1,000-fold higher in tumor-bearing mice than in normal mice. This selective advantage occurs at the expense of resident intestinal enterococci. An SGG-specific locus encoding a bacteriocin ("gallocin") is shown to kill enterococci in vitro. Importantly, bile acids strongly enhance this bacteriocin activity in vivo, leading to greater SGG colonization. Constitutive activation of the Wnt pathway, one of the earliest signaling alterations in CRC, and the decreased expression of the bile acid apical transporter gene Slc10A2, as an effect of the Apc founding mutation, may thereby sustain intestinal colonization by SGG. We conclude that CRC-specific conditions promote SGG colonization of the gut by replacing commensal enterococci in their niche.

High-fat diet modifies the PPAR-γ pathway leading to disruption of microbial and physiological ecosystem in murine small intestine.

Diet is among the most important factors contributing to intestinal homeostasis, and basic functions performed by the small intestine need to be tightly preserved to maintain health. Little is known about the direct impact of high-fat (HF) diet on small-intestinal mucosal defenses and spatial distribution of the microbiota during the early phase of its administration. We observed that only 30 d after HF diet initiation, the intervillous zone of the ileum-which is usually described as free of bacteria-became occupied by a dense microbiota. In addition to affecting its spatial distribution, HF diet also drastically affected microbiota composition with a profile characterized by the expansion of Firmicutes (appearance of Erysipelotrichi), Proteobacteria (Desulfovibrionales) and Verrucomicrobia, and decrease of Bacteroidetes (family S24-7) and Candidatus arthromitus A decrease in antimicrobial peptide expression was predominantly observed in the ileum where bacterial density appeared highest. In addition, HF diet increased intestinal permeability and decreased cystic fibrosis transmembrane conductance regulator (Cftr) and the Na-K-2Cl cotransporter 1 (Nkcc1) gene and protein expressions, leading to a decrease in ileal secretion of chloride, likely responsible for massive alteration in mucus phenotype. This complex phenotype triggered by HF diet at the interface between the microbiota and the mucosal surface was reversed when the diet was switched back to standard composition or when mice were treated for 1 wk with rosiglitazone, a specific agonist of peroxisome proliferator-activated receptor-γ (PPAR-γ). Moreover, weaker expression of antimicrobial peptide-encoding genes and intervillous bacterial colonization were observed in Ppar-γ-deficient mice, highlighting the major role of lipids in modulation of mucosal immune defenses.

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

BACKGROUND: A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities. RESULTS: Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems. CONCLUSIONS: Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

Gut microbiota profile and the influence of nutritional status on bacterial distribution in diabetic and healthy Tunisian subjects.

Gut microbiota plays a key role in the regulation of metabolism and immunity. We investigated the profile of gut microbiota and the impact of dietary intake on gut bacterial distribution in diabetic and healthy Tunisian subjects, aiming to identify a dysbiotic condition, hence opening the way to restore eubiosis and facilitate return to health. In the present research, we enrolled 10 type 1 diabetic (T1D), 10 type 2 diabetic (T2D) patients and 13 healthy (H) subjects. Illumina Miseq technology was used to sequence V3-V4 hypervariable regions of bacterial 16SrRNA gene. Data were analyzed referring to QIIME 2 pipeline. RStudio software was used to explore the role of nutrition in gut bacterial distribution. At the phylum level, we identified an imbalanced gut microbiota composition in diabetic patients marked by a decrease in the proportion of Firmicutes and an increase in the abundance of Bacteroidetes compared with H subjects. We observed higher amounts of Fusobacteria and a decline in the levels of TM7 phyla in T1D patients compared with H subjects. However, we revealed a decrease in the proportions of Verrucomicrobia in T2D patients compared with H subjects. At the genus level, T2D subjects were more affected by gut microbiota alteration, showing a reduction in the relative abundance of Faecalibacterium, Akkermansia, Clostridium, Blautia and Oscillibacter, whereas T1D group shows a decrease in the proportion of Blautia. The gut bacteria distribution was mainly affected by fats and carbohydrates consumption. Gut microbiota composition was altered in Tunisian diabetic patients and affected by dietary habits.

The temporal balance between self-renewal and differentiation of human neural stem cells requires the amyloid precursor protein.

Neurogenesis in the developing human cerebral cortex occurs at a particularly slow rate owing in part to cortical neural progenitors preserving their progenitor state for a relatively long time, while generating neurons. How this balance between the progenitor and neurogenic state is regulated, and whether it contributes to species-specific brain temporal patterning, is poorly understood. Here, we show that the characteristic potential of human neural progenitor cells (NPCs) to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the amyloid precursor protein (APP). In contrast, APP is dispensable in mouse NPCs, which undergo neurogenesis at a much faster rate. Mechanistically, APP cell-autonomously contributes to protracted neurogenesis through suppression of the proneurogenic activator protein-1 transcription factor and facilitation of canonical WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.

RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding.

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.

Crypt- and Mucosa-Associated Core Microbiotas in Humans and Their Alteration in Colon Cancer Patients.

We have previously identified a crypt-specific core microbiota (CSCM) in the colons of healthy laboratory mice and related wild rodents. Here, we confirm that a CSCM also exists in the human colon and appears to be altered during colon cancer. The colonic microbiota is suggested to be involved in the development of colorectal cancer (CRC). Because the microbiota identified in fecal samples from CRC patients does not directly reflect the microbiota associated with tumor tissues themselves, we sought to characterize the bacterial communities from the crypts and associated adjacent mucosal surfaces of 58 patients (tumor and normal homologous tissue) and 9 controls with normal colonoscopy results. Here, we confirm that bacteria colonize human colonic crypts in both control and CRC tissues, and using laser-microdissected tissues and 16S rRNA gene sequencing, we further show that right and left crypt- and mucosa-associated bacterial communities are significantly different. In addition to Bacteroidetes and Firmicutes, and as with murine proximal colon crypts, environmental nonfermentative Proteobacteria are found in human colonic crypts. Fusobacterium and Bacteroides fragilis are more abundant in right-side tumors, whereas Parvimonas micra is more prevalent in left-side tumors. More precisely, Fusobacterium periodonticum is more abundant in crypts from cancerous samples in the right colon than in associated nontumoral samples from adjacent areas but not in left-side colonic samples. Future analysis of the interaction between these bacteria and the crypt epithelium, particularly intestinal stem cells, will allow deciphering of their possible oncogenic potential.IMPORTANCE Due to the huge number of bacteria constituting the human colon microbiota, alteration in the balance of its constitutive taxa (i.e., dysbiosis) is highly suspected of being involved in colorectal oncogenesis. Indeed, bacterial signatures in association with CRC have been described. These signatures may vary if bacteria are identified in feces or in association with tumor tissues. Here, we show that bacteria colonize human colonic crypts in tissues obtained from patients with CRC and with normal colonoscopy results. Aerobic nonfermentative Proteobacteria previously identified as constitutive of the crypt-specific core microbiota in murine colonic samples are similarly prevalent in human colonic crypts in combination with other anaerobic taxa. We also show that bacterial signatures characterizing the crypts of colonic tumors vary depending whether right-side or left-side tumors are analyzed.

Shigella sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy.

Shigella is a leading cause of dysentery worldwide, with the majority of infections caused by two subgroups, S. flexneri and S. sonnei. Although S. flexneri has been highly prevalent in low-income countries, global development has brought an increase in S. sonnei at the expense of S. flexneri. However, the mechanisms behind this shift are not understood. Here we report that S. sonnei, but not S. flexneri, encodes a type VI secretion system (T6SS) that provides a competitive advantage in the gut. S. sonnei competes against E. coli and S. flexneri in mixed cultures, but this advantage is reduced in T6SS mutant strains. In addition, S. sonnei can persist as well as outcompete E. coli and S. flexneri in mice in a T6SS-dependent manner. These findings suggest that S. sonnei has a competitive advantage over S. flexneri and potentially explain the increasing global prevalence of S. sonnei.

Lipopolysaccharide from Crypt-Specific Core Microbiota Modulates the Colonic Epithelial Proliferation-to-Differentiation Balance.

We identified a crypt-specific core microbiota (CSCM) dominated by strictly aerobic, nonfermentative bacteria in murine cecal and proximal colonic (PC) crypts and hypothesized that, among its possible functions, it may affect epithelial regeneration. In the present work, we isolated representative CSCM strains using selective media based upon our initial 16S rRNA-based molecular identification (i.e., Acinetobacter, Delftia, and Stenotrophomonas). Their tropism for the crypt was confirmed, and their influence on epithelial regeneration was demonstrated in vivo by monocolonization of germfree mice. We also showed that lipopolysaccharide (LPS), through its endotoxin activity, was the dominant bacterial agonist controlling proliferation. The relevant molecular mechanisms were analyzed using colonic crypt-derived organoids exposed to bacterial sonicates or highly purified LPS as agonists. We identified a Toll-like receptor 4 (TLR4)-dependent program affecting crypts at different stages of epithelial differentiation. LPS played a dual role: it repressed cell proliferation through RIPK3-mediated necroptosis of stem cells and cells of the transit-amplifying compartment and concurrently enhanced cell differentiation, particularly the goblet cell lineage.IMPORTANCE The LPS from crypt-specific core microbiota controls intestinal epithelium proliferation through necroptosis of stem cells and enhances cell differentiation, mainly the goblet cell lineage.

Complete Genome Sequence of Delftia tsuruhatensis CM13 Isolated from Murine Proximal Colonic Tissue.

We report here the complete genome sequence of Delftia tsuruhatensis CM13, isolated from murine proximal colonic tissue. The genome assembly using PacBio single-molecule real-time sequencing resulted in a single scaffold of 7.19 Mb.

Modeling alternate RNA structures in genomic sequences.

We introduce the concept of RNA multistructures, which is a formal grammar-based framework specifically designed to model a set of alternate RNA secondary structures. Such alternate structures can either be a set of suboptimal foldings, or distinct stable folding states, or variants within an RNA family. We provide several such examples and propose an efficient algorithm to search for RNA multistructures within a genomic sequence.

Draft Genome Sequences of Acinetobacter parvus CM11, Acinetobacter radioresistens CM38, and Stenotrophomonas maltophilia BR12, Isolated from Murine Proximal Colonic Tissue.

Here, we report three genome sequences of bacteria isolated from murine proximal colonic tissue and identified as Acinetobacter parvus CM11, Acinetobacter radioresistens CM38, and Stenotrophomonas maltophilia BR12.

RNA locally optimal secondary structures.

RNA locally optimal secondary structures provide a concise and exhaustive description of all possible secondary structures of a given RNA sequence, and hence a very good representation of the RNA folding space. In this paper, we present an efficient algorithm that computes all locally optimal secondary structures for any folding model that takes into account the stability of helical regions. This algorithm is implemented in a software called regliss that runs on a publicly accessible web server.