Riboswitches are newly discovered regulatory elements which control a wide set of basic metabolic pathways. They consist solely of RNA, sense their ligand in a preformed binding pocket and perform a conformational switch in response to ligand binding resulting in altered gene expression. We have utilized the enormous potential of RNA for molecular sensing and conformational changes to develop novel molecular switches with predetermined structural transitions in response to the binding of a small molecule. To validate these in vivo, we exploit the distance-dependent inhibitory potential of secondary structure elements placed close to the bacterial ribosome binding site. We created a translational control element by combining the theophylline aptamer with a helical communication module for which a ligand-dependent one-nucleotide slipping mechanism had been proposed. This structural element was inserted at a position just interfering with translation in the non ligand-bound form. Addition of the ligand then shifts the inhibitory element to a distance which permits efficient translation. We present here a novel regulatory mechanism in the first rationally designed, in vivo active RNA switch. Its use of a slippage mechanism to control gene expression makes it different from natural riboswitches which are based on sequestration or antitermination.

SummaryDietary InsP6 can modulate eukaryotic cell proliferation and has complex nutritive consequences, but its metabolism in the mammalian gastrointestinal tract is poorly understood. Therefore, we performed phylogenetic analyses of the gastrointestinal microbiome in order to search for candidate InsP6 phosphatases. We determined that prominent gut bacteria express homologs of the mammalian InsP6 phosphatase (MINPP) and characterized the enzyme from Bacteroides thetaiotaomicron (BtMinpp). We show that BtMinpp has exceptionally high catalytic activity, which we rationalize on the basis of mutagenesis studies and by determining its crystal structure at 1.9 Å resolution. We demonstrate that BtMinpp is packaged inside outer membrane vesicles (OMVs) protecting the enzyme from degradation by gastrointestinal proteases. Moreover, we uncover an example of cross-kingdom cell-to-cell signaling, showing that the BtMinpp-OMVs interact with intestinal epithelial cells to promote intracellular Ca2+ signaling. Our characterization of BtMinpp offers several directions for understanding how the microbiome serves human gastrointestinal physiology.

As part of their life cycle, Gram-negative bacteria produce and release microvesicles (outer membrane vesicles, OMVs) consisting of spherical protrusions of the outer membrane that encapsulate periplasmic contents. OMVs produced by commensal bacteria in the gastrointestinal (GI) tract of animals are dispersed within the gut lumen with their cargo and enzymes being distributed across and throughout the GI tract. Their ultimate destination and fate is unclear although they can interact with and cross the intestinal epithelium using different entry pathways and access underlying immune cells in the lamina propria. OMVs have also been found in the bloodstream from which they can access various tissues and possibly the brain. The nanosize and non-replicative status of OMVs together with their resistance to enzyme degradation and low pH, alongside their ability to interact with the host, make them ideal candidates for delivering biologics to mucosal sites, such as the GI and the respiratory tract. In this mini-review, we discuss the fate of OMVs produced in the GI tract of animals with a focus on vesicles released by Bacteroides species and the use of OMVs as vaccine delivery vehicles and other potential applications.

Gram-negative bacteria ubiquitously produce and release nano-size, non-replicative outer membrane vesicles (OMVs). In the gastrointestinal (GI-) tract, OMVs generated by members of the intestinal microbiota are believed to contribute to maintaining the intestinal microbial ecosystem and mediating bacteria-host interactions, including the delivery of bacterial effector molecules to host cells to modulate their physiology. Bacterial OMVs have also been found in the bloodstream although their origin and fate are unclear. Here we have investigated the interactions between OMVs produced by the major human gut commensal bacterium, Bacteroides thetaiotaomicron (Bt), with cells of the GI-tract. Using a combination of in vitro culture systems including intestinal epithelial organoids and in vivo imaging we show that intestinal epithelial cells principally acquire Bt OMVs via dynamin-dependent endocytosis followed by intracellular trafficking to LAMP-1 expressing endo-lysosomal vesicles and co-localization with the perinuclear membrane. We observed that Bt OMVs can also transmigrate through epithelial cells via a paracellular route with in vivo imaging demonstrating that within hours of oral administration Bt OMVs can be detected in systemic tissues and in particular, the liver. Our findings raise the intriguing possibility that OMVs may act as a long-distance microbiota-host communication system.