Comparative Genomics and Physiology of Akkermansia muciniphila Isolates from Human Intestine Reveal Specialized Mucosal Adaptation

Akkermansia muciniphila is a champion of mucin degradation in the human gastrointestinal tract. Here, we report the isolation of six novel strains from healthy human donors and their genomic, proteomic and physiological characterization in comparison to the type-strains A. muciniphila Muc(T) and A. glycaniphila Pyt(T). Complete genome sequencing revealed that, despite their large genomic similarity (>97.6%), the novel isolates clustered into two distinct subspecies of A. muciniphila: Amuc1, which includes the type-strain Muc(T), and AmucU, a cluster of unassigned strains that have not yet been well characterized. CRISPR analysis showed all strains to be unique and confirmed that single healthy subjects can carry more than one A. muciniphila strain. Mucin degradation pathways were strongly conserved amongst all isolates, illustrating the exemplary niche adaptation of A. muciniphila to the mucin interface. This was confirmed by analysis of the predicted glycoside hydrolase profiles and supported by comparing the proteomes of A. muciniphila strain H2, belonging to the AmucU cluster, to Muc(T) and A. glycaniphila Pyt(T) (including 610 and 727 proteins, respectively). While some intrinsic resistance was observed among the A. muciniphila straind, none of these seem to pose strain-specific risks in terms of their antibiotic resistance patterns nor a significant risk for the horizontal transfer of antibiotic resistance determinants, opening the way to apply the type-strain Muc(T) or these new A. muciniphila strains as next generation beneficial microbes.Peer reviewe

The Use of Defined Microbial Communities To Model Host-Microbe Interactions in the Human Gut

The human intestinal ecosystem is characterized by a complex interplay between different microorganisms and the host. The high variation within the human population further complicates the quest toward an adequate understanding of this complex system that is so relevant to human health and well-being. To study host-microbe interactions, defined synthetic bacterial communities have been introduced in gnotobiotic animals or in sophisticated in vitro cell models. This review reinforces that our limited understanding has often hampered the appropriate design of defined communities that represent the human gut microbiota. On top of this, some communities have been applied to in vivo models that differ appreciably from the human host. In this review, the advantages and disadvantages of using defined microbial communities are outlined, and suggestions for future improvement of host-microbe interaction models are provided. With respect to the host, technological advances, such as the development of a gut-on-a-chip system and intestinal organoids, may contribute to more-accurate in vitro models of the human host. With respect to the microbiota, due to the increasing availability of representative cultured isolates and their genomic sequences, our understanding and controllability of the human gut "core microbiota" are likely to increase. Taken together, these advancements could further unravel the molecular mechanisms underlying the human gut microbiota superorganism. Such a gain of insight would provide a solid basis for the improvement of pre-, pro-, and synbiotics as well as the development of new therapeutic microbes.</p

Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro

Akkermansia muciniphila is a well-studied anaerobic bacterium specialized in mucus degradation and associated with human health. Because of the structural resemblance of mucus glycans and free human milk oligosaccharides (HMOs), we studied the ability of A. muciniphila to utilize human milk oligosaccharides. We found that A. muciniphila was able to grow on human milk and degrade HMOs. Analyses of the proteome of A. muciniphila indicated that key-glycan degrading enzymes were expressed when the bacterium was grown on human milk. Our results display the functionality of the key-glycan degrading enzymes (alpha -l-fucosidases, beta -galactosidases, exo-alpha -sialidases and beta -acetylhexosaminidases) to degrade the HMO-structures 2 ' -FL, LNT, lactose, and LNT2. The hydrolysation of the host-derived glycan structures allows A. muciniphila to promote syntrophy with other beneficial bacteria, contributing in that way to a microbial ecological network in the gut. Thus, the capacity of A. muciniphila to utilize human milk will enable its survival in the early life intestine and colonization of the mucosal layer in early life, warranting later life mucosal and metabolic health.Peer reviewe

miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells

SummaryTo investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance

Intercellular communication between artificial cells by allosteric amplification of a molecular signal

Multicellular organisms rely on intercellular communication to coordinate the behaviour of individual cells, which enables their differentiation and hierarchical organization. Various cell mimics have been developed to establish fundamental engineering principles for the construction of artificial cells displaying cell-like organization, behaviour and complexity. However, collective phenomena, although of great importance for a better understanding of life-like behaviour, are underexplored. Here, we construct collectives of giant vesicles that can communicate with each other through diffusing chemical signals that are recognized and processed by synthetic enzymatic cascades. Similar to biological cells, the Receiver vesicles can transduce a weak signal originating from Sender vesicles into a strong response by virtue of a signal amplification step, which facilitates the propagation of signals over long distances within the artificial cell consortia. This design advances the development of interconnected artificial cells that can exchange metabolic and positional information to coordinate their higher-order organization

Characterization of the molecular mechanisms underlying increased mucus production of HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation

The multifunctional intestinal mucus layer plays a crucial role in human health. Our understanding of the human colonic mucus layer in terms of structure, function and has been largely dependent on expensive and advanced ex vivo or in vitro models, which often require high expertise. The mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used in more simple in vitro models, but produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured in Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2 and had a thicker mucus layer compared to conventional culturing methods. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased mucus production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell inserts for 15 days were subject to transcriptome analysis to investigate underlying molecular pathways at gene expression level. We also further characterized the model by measuring transepithelial resistance and pH and lactate production of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms in which mucus production is enhanced under SWMS and propose potential applications of this model in future studies. Overall design: HT29-MTX-E12 and Caco-2 cells were seeded on Transwell membranes. One day after seeding, cells were further cultured for 14 days under either SWMS conditions (i.e. low apical volume and continuous shaking at 65 rpm) or static conditions (similar conditions as during seeding). After harvesting, RNA isolated from cells was subjected to microarray analysis

Characterization of the molecular mechanisms underlying increased mucus production of HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation

The multifunctional intestinal mucus layer plays a crucial role in human health. Our understanding of the human colonic mucus layer in terms of structure, function and has been largely dependent on expensive and advanced ex vivo or in vitro models, which often require high expertise. The mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used in more simple in vitro models, but produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured in Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2 and had a thicker mucus layer compared to conventional culturing methods. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased mucus production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell inserts for 15 days were subject to transcriptome analysis to investigate underlying molecular pathways at gene expression level. We also further characterized the model by measuring transepithelial resistance and pH and lactate production of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms in which mucus production is enhanced under SWMS and propose potential applications of this model in future studies. Overall design: HT29-MTX-E12 and Caco-2 cells were seeded on Transwell membranes. One day after seeding, cells were further cultured for 14 days under either SWMS conditions (i.e. low apical volume and continuous shaking at 65 rpm) or static conditions (similar conditions as during seeding). After harvesting, RNA isolated from cells was subjected to microarray analysis

The Use of Defined Microbial Communities To Model Host-Microbe Interactions in the Human Gut

The human intestinal ecosystem is characterized by a complex interplay between different microorganisms and the host. The high variation within the human population further complicates the quest toward an adequate understanding of this complex system that is so relevant to human health and well-being. To study host-microbe interactions, defined synthetic bacterial communities have been introduced in gnotobiotic animals or in sophisticated in vitro cell models. This review reinforces that our limited understanding has often hampered the appropriate design of defined communities that represent the human gut microbiota. On top of this, some communities have been applied to in vivo models that differ appreciably from the human host. In this review, the advantages and disadvantages of using defined microbial communities are outlined, and suggestions for future improvement of host-microbe interaction models are provided. With respect to the host, technological advances, such as the development of a gut-on-a-chip system and intestinal organoids, may contribute to more-accurate in vitro models of the human host. With respect to the microbiota, due to the increasing availability of representative cultured isolates and their genomic sequences, our understanding and controllability of the human gut "core microbiota" are likely to increase. Taken together, these advancements could further unravel the molecular mechanisms underlying the human gut microbiota superorganism. Such a gain of insight would provide a solid basis for the improvement of pre-, pro-, and synbiotics as well as the development of new therapeutic microbes.</p

Systematic comparison of transcriptomes of Caco-2 cells cultured under different cellular and physiological conditions

There is a need for standardized in vitro models emulating the functionalities of the human intestinal tract to study human intestinal health without the use of laboratory animals. The Caco-2 cell line is a well-accepted and highly characterized intestinal barrier model, which has been intensively used to study intestinal (drug) transport, host–microbe interactions and chemical or drug toxicity. This cell line has been cultured in different in vitro models, ranging from simple static to complex dynamic microfluidic models. We aimed to investigate the effect of these different in vitro experimental variables on gene expression. To this end, we systematically collected and extracted data from studies in which transcriptome analyses were performed on Caco-2 cells grown on permeable membranes. A collection of 13 studies comprising 100 samples revealed a weak association of experimental variables with overall as well as individual gene expression. This can be explained by the large heterogeneity in cell culture practice, or the lack of adequate reporting thereof, as suggested by our systematic analysis of experimental parameters not included in the main analysis. Given the rapidly increasing use of in vitro cell culture models, including more advanced (micro) fluidic models, our analysis reinforces the need for improved, standardized reporting protocols. Additionally, our systematic analysis serves as a template for future comparative studies on in vitro transcriptome and other experimental data