Nutritional and host environments determine community ecology and keystone species in a synthetic gut bacterial community.

A challenging task to understand health and disease-related microbiome signatures is to move beyond descriptive community-level profiling towards disentangling microbial interaction networks. Using a synthetic gut bacterial community, we aimed to study the role of individual members in community assembly, identify putative keystone species and test their influence across different environments. Single-species dropout experiments reveal that bacterial strain relationships strongly vary not only in different regions of the murine gut, but also across several standard culture media. Mechanisms involved in environment-dependent keystone functions in vitro include exclusive access to polysaccharides as well as bacteriocin production. Further, Bacteroides caecimuris and Blautia coccoides are found to play keystone roles in gnotobiotic mice by impacting community composition, the metabolic landscape and inflammatory responses. In summary, the presented study highlights the strong interdependency between bacterial community ecology and the biotic and abiotic environment. These results question the concept of universally valid keystone species in the gastrointestinal ecosystem and underline the context-dependency of both, keystone functions and bacterial interaction networks.

In vitro interaction network of a synthetic gut bacterial community.

A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies.

Transcription Factor Levels after Forward Programming of Human Pluripotent Stem Cells with GATA1, FLI1, and TAL1 Determine Megakaryocyte versus Erythroid Cell Fate Decision.

The production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or erythroblasts. Using "rainbow" lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.