Distinct microbial assemblages associated with genetic selection for high- and low- muscle yield in rainbow trout.

BACKGROUND: Fish gut microbial assemblages play a crucial role in the growth rate, metabolism, and immunity of the host. We hypothesized that the gut microbiota of rainbow trout was correlated with breeding program based genetic selection for muscle yield. To test this hypothesis, fecal samples from 19 fish representing an F2 high-muscle genetic line (ARS-FY-H) and 20 fish representing an F1 low-muscle yield genetic line (ARS-FY-L) were chosen for microbiota profiling using the 16S rRNA gene. Significant differences in microbial assemblages between these two genetic lines might represent the effect of host genetic selection in structuring the gut microbiota of the host. RESULTS: Tukey's transformed inverse Simpson indices indicated that high muscle yield genetic line (ARS-FY-H) samples have higher microbial diversity compared to those of the low muscle yield genetic line (ARS-FY-L) (LMM, χ2(1) =14.11, p < 0.05). The fecal samples showed statistically distinct structure in microbial assemblages between the genetic lines (F1,36 = 4.7, p < 0.05, R2 = 11.9%). Functional profiling of bacterial operational taxonomic units predicted characteristic functional capabilities of the microbial communities in the high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic line samples. CONCLUSION: The significant differences of the microbial assemblages between high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic lines indicate a possible effect of genetic selection on the microbial diversity of the host. The functional composition of taxa demonstrates a correlation between bacteria and improving the muscle accretion in the host, probably, by producing various metabolites and enzymes that might aid in digestion. Further research is required to elucidate the mechanisms involved in shaping the microbial community through host genetic selection.

Analysis of the fecal microbiota of fast- and slow-growing rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Diverse microbial communities colonizing the intestine of fish contribute to their growth, digestion, nutrition, and immune function. We hypothesized that fecal samples representing the gut microbiota of rainbow trout could be associated with differential growth rates observed in fish breeding programs. If true, harnessing the functionality of this microbiota can improve the profitability of aquaculture. The first objective of this study was to test this hypothesis if gut microbiota is associated with fish growth rate (body weight). Four full-sibling families were stocked in the same tank and fed an identical diet. Two fast-growing and two slow-growing fish were selected from each family for 16S rRNA microbiota profiling. Microbiota diversity varies with different DNA extraction methods. The second objective of this study was to compare the effects of five commonly used DNA extraction methods on the microbiota profiling and to determine the most appropriate extraction method for this study. These methods were Promega-Maxwell, Phenol-chloroform, MO-BIO, Qiagen-Blood/Tissue, and Qiagen-Stool. Methods were compared according to DNA integrity, cost, feasibility and inter-sample variation based on non-metric multidimensional scaling ordination (nMDS) clusters. RESULTS: Differences in DNA extraction methods resulted in significant variation in the identification of bacteria that compose the gut microbiota. Promega-Maxwell had the lowest inter-sample variation and was therefore used for the subsequent analyses. Beta diversity of the bacterial communities showed significant variation between breeding families but not between the fast- and slow-growing fish. However, an indicator analysis determined that cellulose, amylose degrading and amino acid fermenting bacteria (Clostridium, Leptotrichia, and Peptostreptococcus) are indicator taxa of the fast-growing fish. In contrary, pathogenic bacteria (Corynebacterium and Paeniclostridium) were identified as indicator taxa for the slow-growing fish. CONCLUSION: DNA extraction methodology should be carefully considered for accurate profiling of the gut microbiota. Although the microbiota was not significantly different between the fast- and slow-growing fish groups, some bacterial taxa with functional implications were indicative of fish growth rate. Further studies are warranted to explore how bacteria are transmitted and potential usage of the indicator bacteria of fast-growing fish for development of probiotics that may improve fish health and growth.

Characterisation of sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum causing Bacterial Cold Water Disease in rainbow trout.

Flavobacterium psychrophilum (Fp) causes Bacterial Cold Water Disease in salmonids. During host-pathogen interactions, gram-negative bacteria, such as Fp, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs' small RNAs (sRNAs) in the Fp-rainbow trout host-pathogen interactions. sRNAs carried within OMVs were isolated from Fp. RNA-Seq datasets from whole-cell Fp and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV-packaged sRNAs were located in the pathogenicity islands of Fp. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA-Seq of host and pathogen transcriptomes on day 5 post-infection of Fp -resistant and -susceptible rainbow trout genetic lines revealed correlated expression of OMV-packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV-treated cells, similar to the Fp -resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV-treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.

Bilirubin is a new ligand for nuclear receptor Liver Receptor Homolog-1.

The nuclear receptor Liver Receptor Homolog-1 (LRH-1, NR5A2 ) binds to phospholipids that regulate important LRH-1 functions in the liver. A recent compound screen unexpectedly identified bilirubin, the product of liver heme metabolism, as a possible ligand for LRH-1. Here, we show unconjugated bilirubin directly binds LRH-1 with apparent K d =9.3uM, altering LRH-1 interaction with all transcriptional coregulator peptides tested. Bilirubin decreased LRH-1 protease sensitivity, consistent with MD simulations predicting bilirubin stably binds LRH-1 within the canonical ligand binding site. Bilirubin activated a luciferase reporter specific for LRH-1, dependent on co-expression with the bilirubin membrane transporter SLCO1B1 , but bilirubin failed to activate ligand-binding genetic mutants of LRH-1. Gene profiling in HepG2 cells shows bilirubin selectively regulated transcripts from endogenous LRH-1 ChIP-seq target genes, which was significantly attenuated by either genetic knockdown of LRH-1, or by a specific chemical competitor of LRH-1. Gene set enrichment suggests bilirubin and LRH-1 share roles in cholesterol metabolism and lipid efflux, thus we propose a new role for LRH-1 in directly sensing intracellular levels of bilirubin.

IPMK regulates HDAC3 activity and histone H4 acetylation in human cells.

Histone deacetylases (HDACs) repress transcription by catalyzing the removal of acetyl groups from histones. Class 1 HDACs are activated by inositol phosphate signaling molecules in vitro , but it is unclear if this regulation occurs in human cells. Inositol Polyphosphate Multikinase (IPMK) is required for production of inositol hexakisphosphate (IP6), pentakisphosphate (IP5) and certain tetrakisphosphate (IP4) species, all known activators of Class 1 HDACs in vitro . Here, we generated IPMK knockout (IKO) human U251 glioblastoma cells, which decreased cellular inositol phosphate levels and increased histone H4-acetylation by mass spectrometry. ChIP-seq showed IKO increased H4-acetylation at IKO-upregulated genes, but H4-acetylation was unchanged at IKO-downregulated genes, suggesting gene-specific responses to IPMK knockout. HDAC deacetylase enzyme activity was decreased in HDAC3 immunoprecipitates from IKO vs . wild-type cells, while deacetylase activity of other Class 1 HDACs had no detectable changes in activity. Wild-type IPMK expression in IKO cells fully rescued HDAC3 deacetylase activity, while kinase-dead IPMK expression had no effect. Further, the deficiency in HDAC3 activity in immunoprecipitates from IKO cells could be fully rescued by addition of synthesized IP4 (Ins(1,4,5,6)P4) to the enzyme assay, while control inositol had no effect. These data suggest that cellular IPMK-dependent inositol phosphates are required for full HDAC3 enzyme activity and proper histone H4-acetylation. Implications for targeting IPMK in HDAC3-dependent diseases are discussed.

Design, synthesis and cellular characterization of a new class of IPMK kinase inhibitors.

Many genetic studies have established the kinase activity of inositol phosphate multikinase (IPMK) is required for the synthesis of higher-order inositol phosphate signaling molecules, the regulation of gene expression and control of the cell cycle. These genetic studies await orthogonal validation by specific IPMK inhibitors, but no such inhibitors have been synthesized. Here, we report complete chemical synthesis, cellular characterization, structure-activity relationships and rodent pharmacokinetics of a novel series of highly potent IPMK inhibitors. The first-generation compound 1 (UNC7437) decreased cellular proliferation and tritiated inositol phosphate levels in metabolically labeled human U251-MG glioblastoma cells. Compound 1 also regulated the transcriptome of these cells, selectively regulating genes that are enriched in cancer, inflammatory and viral infection pathways. Further optimization of compound 1 eventually led to compound 15 (UNC9750), which showed improved potency and pharmacokinetics in rodents. Compound 15 specifically inhibited cellular accumulation of InsP 5 , a direct product of IPMK kinase activity, while having no effect on InsP 6 levels, revealing a novel metabolic signature detected for the first time by rapid chemical attenuation of cellular IPMK activity. These studies designed, optimized and synthesized a new series of IPMK inhibitors, which reduces glioblastoma cell growth, induces a novel InsP 5 metabolic signature, and reveals novel aspects inositol phosphate cellular metabolism and signaling.

Dual RNA-Seq of Flavobacterium psychrophilum and Its Outer Membrane Vesicles Distinguishes Genes Associated with Susceptibility to Bacterial Cold-Water Disease in Rainbow Trout (Oncorhynchus mykiss).

Flavobacterium psychrophilum (Fp), the causative agent of Bacterial Cold-Water disease in salmonids, causes substantial losses in aquaculture. Bacterial outer membrane vesicles (OMVs) contain several virulence factors, enzymes, toxins, and nucleic acids and are expected to play an essential role in host-pathogen interactions. In this study, we used transcriptome sequencing, RNA-seq, to investigate the expression abundance of the protein-coding genes in the Fp OMVs versus the Fp whole cell. RNA-seq identified 2190 transcripts expressed in the whole cell and 2046 transcripts in OMVs. Of them, 168 transcripts were uniquely identified in OMVs, 312 transcripts were expressed only in the whole cell, and 1878 transcripts were shared in the two sets. Functional annotation analysis of the OMV-abundant transcripts showed an association with the bacterial translation machinery and histone-like DNA-binding proteins. RNA-Seq of the pathogen transcriptome on day 5 post-infection of Fp-resistant versus Fp-susceptible rainbow trout genetic lines revealed differential gene expression of OMV-enriched genes, suggesting a role for the OMVs in shaping the host-microbe interaction. Interestingly, a cell wall-associated hydrolase (CWH) gene was the most highly expressed gene in OMVs and among the top upregulated transcripts in susceptible fish. The CWH sequence was conserved in 51 different strains of Fp. The study provides insights into the potential role of OMVs in host-pathogen interactions and explores microbial genes essential for virulence and pathogenesis.

New High-Throughput Screen Discovers Novel Ligands of Full-Length Nuclear Receptor LRH-1.

Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is a lipid-regulated transcription factor and an important drug target for several liver diseases. Advances toward LRH-1 therapeutics have been driven recently by structural biology, with fewer contributions from compound screening. Standard LRH-1 screens detect compound-induced interaction between LRH-1 and a transcriptional coregulator peptide, an approach that excludes compounds that regulate LRH-1 through alternative mechanisms. Here, we developed a FRET-based LRH-1 screen that simply detects compound binding to LRH-1, applying it to discover 58 new compounds that bind the canonical ligand-binding site in LRH-1 (2.5% hit rate), also supported by computational docking. Four independent functional screens identified 15 of these 58 compounds to also regulate LRH-1 function in vitro or in living cells. Although one of these 15 compounds, abamectin, directly binds LRH-1 and regulates full-length LRH-1 in cells, abamectin failed to regulate the isolated ligand-binding domain in standard coregulator peptide recruitment assays using PGC1α, DAX-1, or SHP. Abamectin treatment of human liver HepG2 cells selectively regulated endogenous LRH-1 ChIP-seq target genes and pathways associated with known LRH-1 functions in bile acid and cholesterol metabolism. Thus, the screen reported here can discover compounds not likely to have been identified in standard LRH-1 compound screens but which bind and regulate full-length LRH-1 in cells.