Fxr signaling and microbial metabolism of bile salts in the zebrafish intestine.

Bile salt synthesis, secretion into the intestinal lumen, and resorption in the ileum occur in all vertebrate classes. In mammals, bile salt composition is determined by host and microbial enzymes, affecting signaling through the bile salt-binding transcription factor farnesoid X receptor (Fxr). However, these processes in other vertebrate classes remain poorly understood. We show that key components of hepatic bile salt synthesis and ileal transport pathways are conserved and under control of Fxr in zebrafish. Zebrafish bile salts consist primarily of a C27 bile alcohol and a C24 bile acid that undergo multiple microbial modifications including bile acid deconjugation that augments Fxr activity. Using single-cell RNA sequencing, we provide a cellular atlas of the zebrafish intestinal epithelium and uncover roles for Fxr in transcriptional and differentiation programs in ileal and other cell types. These results establish zebrafish as a nonmammalian vertebrate model for studying bile salt metabolism and Fxr signaling.

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.

Epigenetic control of intestinal barrier function and inflammation in zebrafish.

The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown. Here, we show that loss of function of the epigenetic regulator ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1) in zebrafish leads to a reduction in tnfa promoter methylation and the induction of tnfa expression in intestinal epithelial cells (IECs). The increase in IEC tnfa levels is microbe-dependent and results in IEC shedding and apoptosis, immune cell recruitment, and barrier dysfunction, consistent with chronic inflammation. Importantly, tnfa knockdown in uhrf1 mutants restores IEC morphology, reduces cell shedding, and improves barrier function. We propose that loss of epigenetic repression and TNF induction in the intestinal epithelium can lead to IBD onset.

Microgavage of zebrafish larvae.

The zebrafish has emerged as a powerful model organism for studying intestinal development(1-5), physiology(6-11), disease(12-16), and host-microbe interactions(17-25). Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae(26). Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

Glafenine-induced intestinal injury in zebrafish is ameliorated by µ-opioid signaling via enhancement of Atf6-dependent cellular stress responses.

Beside their analgesic properties, opiates exert beneficial effects on the intestinal wound healing response. In this study, we investigated the role of µ-opioid receptor (MOR) signaling on the unfolded protein response (UPR) using a novel zebrafish model of NSAID-induced intestinal injury. The NSAID glafenine was administered to zebrafish larvae at 5 days post-fertilization (dpf) for up to 24 hours in the presence or absence of the MOR-specific agonist DALDA. By analysis with histology, transmission electron microscopy and vital dye staining, glafenine-treated zebrafish showed evidence of endoplasmic reticulum and mitochondrial stress, with disrupted intestinal architecture and halted cell stress responses, alongside accumulation of apoptotic intestinal epithelial cells in the lumen. Although the early UPR marker BiP was induced with glafenine-induced injury, downstream atf6 and s-xbp1 expression were paradoxically not increased, explaining the halted cell stress responses. The µ-opioid agonist DALDA protected against glafenine-induced injury through induction of atf6-dependent UPR. Our findings show that DALDA prevents glafenine-induced epithelial damage through induction of effective UPR.

New insights into Chlamydia intracellular survival mechanisms.

Chlamydia sp. are responsible for a wide range of diseases of significant clinical and public health importance. In this review, we highlight how recent cellular and functional genomic approaches have significantly increased our knowledge of the pathogenic mechanisms used by these genetically intractable bacteria. As the extensive repertoire of chlamydial proteins that are translocated into the mammalian host is identified and characterized, a molecular understanding of how Chlamydiae co-opt host cellular functions and block innate immune pathways is beginning to emerge.

Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole.

The acquisition of host-derived lipids is essential for the pathogenesis of the obligate intracellular bacteria Chlamydia trachomatis. Current models of chlamydial lipid acquisition center on the fusion of Golgi-derived exocytic vesicles and endosomal multivesicular bodies with the bacteria-containing parasitophorous vacuole ("inclusion"). In this study, we describe a mechanism of lipid acquisition and organelle subversion by C. trachomatis. We show by live cell fluorescence microscopy and electron microscopy that lipid droplets (LDs), neutral lipid storage organelles, are translocated from the host cytoplasm into the inclusion lumen. LDs dock at the surface of the inclusion, penetrate the inclusion membrane and intimately associate with reticulate Bodies, the replicative form of Chlamydia. The inclusion membrane protein IncA, but not other inclusion membrane proteins, cofractionated with LDs and accumulated in the inclusion lumen. Therefore, we postulate that the translocation of LDs may occur at IncA-enriched subdomains of the inclusion membrane. Finally, the chlamydial protein Lda3 may participate in the cooption of these organelles by linking cytoplasmic LDs to inclusion membranes and promoting the removal of the LD protective coat protein, adipocyte differentiation related protein (ADRP). The wholesale transport of LDs into the lumen of a parasitophorous vacuole represents a unique mechanism of organelle sequestration and subversion by a bacterial pathogen.

CD4+ T cells and CXC chemokines modulate the pathogenesis of Staphylococcus aureus wound infections.

T cells are critical for the formation of intraabdominal abscesses by Staphylococcus aureus. We hypothesized that T cells modulate the development of experimental staphylococcal infections by controlling polymorphonuclear leukocyte (PMN) trafficking. In models of staphylococcal s.c. abscess formation, hindpaw infection, and surgical wound infection, S. aureus multiplied in the tissues of WT C57BL/6J mice and elicited a marked inflammatory response. CD4(+) alphabeta T cells homed to the surgical wound infection site of WT animals. In contrast, significantly fewer S. aureus were recovered from the tissues of mice deficient in alphabeta T cells, and the inflammatory response was considerably diminished compared with that of WT animals. Alphabeta T cell receptor (-/-) mice had significantly lower concentrations of PMN-specific CXC chemokines in wound tissue than did WT mice. The severity of the wound infection was enhanced by administration of a CXC chemokine and abrogated by antibodies that blocked the CXC receptor. An acapsular mutant was less virulent than the parental S. aureus strain in both the s.c. abscess and the surgical wound infection models in WT mice. These data reveal an important and underappreciated role for CD4(+) alphabeta T cells in S. aureus infections in controlling local CXC chemokine production, neutrophil recruitment to the site of infection, and subsequent bacterial replication.

Molecular characterization of the capsule locus from non-typeable Staphylococcus aureus.

Most Staphylococcus aureus express a serotype 5 or 8 capsular polysaccharide (CP). However, 20-25% of human isolates and up to 86% of bovine strains of S. aureus are non-typeable (NT), i.e. non-reactive with antibodies to CP types 1, 2, 5 or 8. A vaccine that targets the S. aureus CP would not protect against NT strains. The aim of this study was to characterize NT S. aureus isolates at the molecular level to explain their lack of type 5 or 8 capsule production. The cap5(8) locus was present in all 22 NT clinical isolates from humans, eight of 21 bovine isolates, and in all eight sequenced strains. NT strains positive for the cap5(8) transcript had mutations within essential capsule genes and could be complemented in trans. S. aureus strains with reduced cap5(8) transcript had mutations within the cap5A promoter, decreased RNAIII levels, or a truncated arlR gene product. More than one mutation was identified in several isolates. The cap5(8) locus was replaced by IS257 in 13 of 21 NT bovine isolates of S. aureus. Lack of capsule expression in NT S. aureus can be explained by multiple mechanisms, and the data argue against the existence of capsule serotypes other than 1, 2, 5 and 8.