Ascorbate deficiency increases progression of shigellosis in guinea pigs and mice infection models.

Shigella spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon Shigella oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (Shigella sonnei, Shigella flexneri 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased Shigella penetration into the colon epithelium layer in a Gulo-/- mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both Shigella infection strategies and immune responses to Shigella infection.

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.

Intracellular offspring released from SFB filaments are flagellated.

The gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle.

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.

Disentangling Host-Microbiota Regulation of Lipid Secretion by Enterocytes: Insights from Commensals Lactobacillus paracasei and Escherichia coli.

The gut microbiota contributes to nutrients absorption and metabolism by enterocytes, but the molecular mechanisms involved remain poorly understood, and most conclusions are inferred from studies comparing germfree and conventional animals colonized with diverse bacterial species. We selected two model commensal microorganisms, Escherichia coli and Lactobacillus paracasei, to assess the role of the small-intestinal microbiota in modulating lipid absorption and metabolism by the epithelium. Using an integrated approach encompassing cellular and murine models and combining metabolic parameters measurement, lipid droplet imaging, and gene expression analysis, we demonstrated that under homeostatic conditions, L. paracasei promotes fat storage in enterocytes, whereas E. coli enhances lipid catabolism and reduces chylomicron circulating levels. The Akt/mammalian target of sirolimus (mTOR) pathway is inhibited by both bacterial species in vitro, indicating that several regulatory pathways are involved in the distinct intracellular lipid outcomes associated with each bacterial species. Moreover, soluble bacterial factors partially reproduce the effects observed with live microorganisms. However, reduction of chylomicron circulating levels in E. coli-colonized animals is lost under high-fat-diet conditions, whereas it is potentiated by L. paracasei colonization accompanied by resistance to hypercholesterolemia and excess body weight gain.IMPORTANCE The specific contribution of each bacterial species within a complex microbiota to the regulation of host lipid metabolism remains largely unknown. Using two model commensal microorganisms, L. paracasei and E. coli, we demonstrated that both bacterial species impacted host lipid metabolism in a diet-dependent manner and, notably, that L. paracasei-colonized mice but not E. coli-colonized mice resisted high-fat-diet-induced body weight gain. In addition, we set up cellular models of fatty acid absorption and secretion by enterocytes cocultured with bacteria and showed that, in vitro, both L. paracasei and E. coli inhibited lipid secretion, through increased intracellular fat storage and enhanced lipid catabolism, respectively.

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.

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.

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.

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.

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.

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.

Penicillin resistance compromises Nod1-dependent proinflammatory activity and virulence fitness of neisseria meningitidis.

Neisseria meningitidis is a life-threatening human bacterial pathogen responsible for pneumonia, sepsis, and meningitis. Meningococcal strains with reduced susceptibility to penicillin G (Pen(I)) carry a mutated penicillin-binding protein (PBP2) resulting in a modified peptidoglycan structure. Despite their antibiotic resistance, Pen(I) strains have failed to expand clonally. We analyzed the biological consequences of PBP2 alteration among clinical meningococcal strains and found that peptidoglycan modifications of the Pen(I) strain resulted in diminished in vitro Nod1-dependent proinflammatory activity. In an influenza virus-meningococcal sequential mouse model mimicking human disease, wild-type meningococci induced a Nod1-dependent inflammatory response, colonizing the lungs and surviving in the blood. In contrast, isogenic Pen(I) strains were attenuated for such response and were out-competed by meningococci sensitive to penicillin G. Our results suggest that antibiotic resistance imposes a cost to the success of the pathogen and may potentially explain the lack of clonal expansion of Pen(I) strains.

Epithelial cell proliferation arrest induced by lactate and acetate from Lactobacillus casei and Bifidobacterium breve.

In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA) were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.

A crypt-specific core microbiota resides in the mouse colon.

UNLABELLED: In an attempt to explore the microbial content of functionally critical niches of the mouse gastrointestinal tract, we targeted molecular microbial diagnostics of the crypts that contain the intestinal stem cells, which account for epithelial regeneration. As current evidence indicates, the gut microbiota affects epithelial regeneration; bacteria that are likely to primarily participate in this essential step of the gut, microbiota cross talk, have been identified. We show in this article that only the cecal and colonic crypts harbor resident microbiota in the mouse and that regardless of the line and breeding origin of these mice, this bacterial population is unexpectedly dominated by aerobic genera. Interestingly, this microbiota resembles the restricted microbiota found in the midgut of invertebrates; thus, the presence of our so-called "crypt-specific core microbiota" (CSCM) in the mouse colon potentially reflects a coevolutionary process under selective conditions that can now be addressed. We suggest that CSCM could play both a protective and a homeostatic role within the colon. This article is setting the bases for such studies, particularly by providing a bona fide--and essentially cultivable--crypt microbiota of reference. IMPORTANCE: Metagenomic typing of the whole-gut luminal microbiome was recently provided, revealing great opportunities for physiological and physiopathological analysis of the host-microbiota interface. On this basis, it appears increasingly important to analyze which niches of the gut exposed to a particular microbiota are of major functional importance, specifically focusing on the crypt, which accounts for permanent epithelial renewal, and to analyze how this microbiota compares to its luminal counterpart in composition and quantity. Crypt-specific core microbiotas may show themselves as important elements regarding crypt protection and homeostasis of its functions.

Morphogenetic competence of HNF4 alpha-deficient mouse hepatic cells.

BACKGROUND/AIMS: To specify roles of HNF 4 alpha in mouse liver development, we have analyzed the ex vivo morphogenetic potential of HNF4 alpha-null embryonic hepatic cells. METHODS: Using mice with floxed or deficiency alleles of HNF4 alpha, hepatic cells lacking this transcription factor were explanted into primary culture and derived into cell lines. RESULTS: Contrary to behavior in vivo where HNF4 alpha-null liver cells fail to show normal polarity and epithelialization, e18.5 hepatic cells in primary culture from mutant embryos show restoration of apical expression of tight junction protein-1 and of transcripts for E-cadherin. Clones of control and HNF4 alpha-null cell lines were indistinguishable, even when differentiation of bile canalicular formation was induced. HNF4 alpha-null and control cell lines showed similar potential to colonize livers of the murine ALB-uPA/SCID model of liver regeneration, but null cells formed only bile ducts and not clusters of hepatocytes. Finally, analysis of mutant embryonic livers revealed a transcriptional signature consistent with a stress response, which could underlie the morphogenetic defects observed in vivo. CONCLUSIONS: We conclude that the lack of epithelialization characteristic of the HNF4 alpha-null embryonic liver is due, at least in part, to non-cell autonomous defects, and that null cells do not suffer intrinsic defects in polarization.

Maturation of paneth cells induces the refractory state of newborn mice to Shigella infection.

The intestinal tract of adult mice is naturally resistant to infection by Shigella, the causative agent of bacillary dysentery in humans. Conversely, newborn mice are highly susceptible to intragastric Shigella infection and develop inflammatory lesions of the jejunal mucosa, very similar to those observed in the colon of dysenteric patients. However, the susceptibility period is short and one week after birth, animals have acquired a status of resistance characteristic of adult animals. To identify the developmental changes controlling the switch from disease susceptibility to resistance, we performed global gene expression analysis on noninfected and infected intestinal tissues taken from 4-day- and 7-day-old animals. Transcriptomic analysis of 4-day-old mice infected with the invasive Shigella strain showed a profile reflecting a strong inflammatory response with no evidence for retro-control, suggesting that the invasive process had occurred, whereas inflammation had been controlled after infection with the noninvasive strain. Differences in gene expression profiles between noninfected 4-day- and 7-day-old mice corresponded mainly to genes encoding anti-microbial peptides and proteases, suggesting that these molecules could be candidates for host antimicrobial resistance in the course of shigellosis. Indeed, expression of genes specific of Paneth cells was higher in 7-day- than in 4-day-old mice, and histological analysis indicated that Paneth cells were present only at day 7. Finally, using Sox9(flox/flox)-vil-cre mice, we showed that depletion of Paneth cells restored the susceptibility to Shigella of 7-day-old mice, clearly indicating that Paneth cells development is crucial for the clearance of intestinal infection.