Transferrable protection by gut microbes against STING-associated lung disease

STING modulates immunity by responding to bacterial and endogenous cyclic dinucleotides (CDNs). Humans and mice with STING gain-of-function mutations develop a syndrome known as STING-associated vasculopathy with onset in infancy (SAVI), which is characterized by inflammatory or fibrosing lung disease. We hypothesized that hyperresponsiveness of gain-of-function STING to bacterial CDNs might explain autoinflammatory lung disease in SAVI mice. We report that depletion of gut microbes with oral antibiotics (vancomycin, neomycin, and ampicillin [VNA]) nearly eliminates lung disease in SAVI mice, implying that gut microbes might promote STING-associated autoinflammation. However, we show that germ-free SAVI mice still develop severe autoinflammatory disease and that transferring gut microbiota from antibiotics-treated mice to germ-free animals eliminates lung inflammation. Depletion of anaerobes with metronidazole abolishes the protective effect of the VNA antibiotics cocktail, and recolonization with the metronidazole-sensitive anaerobe Bacteroides thetaiotaomicron prevents disease, confirming a protective role of a metronidazole-sensitive microbe in a model of SAVI

Microbiota-produced indole metabolites disrupt mitochondrial function and inhibit Cryptosporidium parvum growth

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. To explore microbial influences on susceptibility, we screened 85 microbiota-associated metabolites for their effects on Cryptosporidium parvum growth in vitro. We identify eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin

A modified bacterial one-hybrid system yields improved quantitative models of transcription factor specificity

We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis—GRaMS (Growth Rate Modeling of Specificity)—that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well-characterized C2H2 zinc-finger TF on both a 28 bp randomized library for the standard B1H method and on 6 bp randomized library for the CV-B1H method for which 45 different experimental conditions were tested: five time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media

Identification of muscle-specific regulatory modules in Caenorhabditis elegans

Transcriptional regulation is the major regulatory mechanism that controls the spatial and temporal expression of genes during development. This is carried out by transcription factors (TFs), which recognize and bind to their cognate binding sites. Recent studies suggest a modular organization of TF-binding sites, in which clusters of transcription-factor binding sites cooperate in the regulation of downstream gene expression. In this study, we report our computational identification and experimental verification of muscle-specific cis-regulatory modules in Caenorhabditis elegans. We first identified a set of motifs that are correlated with muscle-specific gene expression. We then predicted muscle-specific regulatory modules based on clusters of those motifs with characteristics similar to a collection of well-studied modules in other species. The method correctly identifies 88% of the experimentally characterized modules with a positive predictive value of at least 65%. The prediction accuracy of muscle-specific expression on an independent test set is highly significant (P < 0.0001). We performed in vivo experimental tests of 12 predicted modules, and 10 of those drive muscle-specific gene expression. These results suggest that our method is highly accurate in identifying functional sequences important for muscle-specific gene expression and is a valuable tool for guiding experimental designs

Novel transcription regulatory elements in Caenorhabditis elegans muscle genes

We report the identification of three new transcription regulatory elements that are associated with muscle gene expression in the nematode Caenorhabditis elegans. Starting from a subset of well-characterized nematode muscle genes, we identified conserved DNA motifs in the promoter regions using computational DNA pattern-recognition algorithms. These were considered to be putative muscle transcription regulatory motifs. Using the green-fluorescent protein (GFP) as a reporter, experiments were done to determine the biological activity of these motifs in driving muscle gene expression. Prediction accuracy of muscle expression based on the presence of these three motifs was encouraging; nine of 10 previously uncharacterized genes that were predicted to have muscle expression were shown to be expressed either specifically or selectively in the muscle tissues, whereas only one of the nine that scored low for these motifs expressed in muscle. Knockouts of putative regulatory elements in the promoter of the mlc-2 and unc-89 genes show that they significantly contribute to muscle expression and act in a synergistic manner. We find that these DNA motifs are also present in the muscle promoters of C. briggsae, indicating that they are functionally conserved in the nematodes

Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in Caenorhabditis elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. We find that two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naïve blastomeres to muscle when overproduced ectopically in the embryo. In addition, we have used genetic null mutants to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type

Caenorhabditis elegans unc-82 Encodes a Serine/Threonine Kinase Important for Myosin Filament Organization in Muscle During Growth

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome

Single-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry.

As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota