GMMAD: a comprehensive database of human gut microbial metabolite associations with diseases.

BACKGROUND: The natural products, metabolites, of gut microbes are crucial effect factors on diseases. Comprehensive identification and annotation of relationships among disease, metabolites, and microbes can provide efficient and targeted solutions towards understanding the mechanism of complex disease and development of new markers and drugs. RESULTS: We developed Gut Microbial Metabolite Association with Disease (GMMAD), a manually curated database of associations among human diseases, gut microbes, and metabolites of gut microbes. Here, this initial release (i) contains 3,836 disease-microbe associations and 879,263 microbe-metabolite associations, which were extracted from literatures and available resources and then experienced our manual curation; (ii) defines an association strength score and a confidence score. With these two scores, GMMAD predicted 220,690 disease-metabolite associations, where the metabolites all belong to the gut microbes. We think that the positive effective (with both scores higher than suggested thresholds) associations will help identify disease marker and understand the pathogenic mechanism from the sense of gut microbes. The negative effective associations would be taken as biomarkers and have the potential as drug candidates. Literature proofs supported our proposal with experimental consistence; (iii) provides a user-friendly web interface that allows users to browse, search, and download information on associations among diseases, metabolites, and microbes. The resource is freely available at http://guolab.whu.edu.cn/GMMAD . CONCLUSIONS: As the online-available unique resource for gut microbial metabolite-disease associations, GMMAD is helpful for researchers to explore mechanisms of disease- metabolite-microbe and screen the drug and marker candidates for different diseases.

Discovery of Drug Candidates for Specific Human Disease Based on Natural Products of Gut Microbes.

The beneficial metabolites of the microbiome could be used as a tool for screening drugs that have the potential for the therapy of various human diseases. Narrowing down the range of beneficial metabolite candidates in specific diseases was primarily a key step for further validation in model organisms. Herein, we proposed a reasonable hypothesis that the metabolites existing commonly in multiple beneficial (or negatively associated) bacteria might have a high probability of being effective drug candidates for specific diseases. According to this hypothesis, we screened metabolites associated with seven human diseases. For type I diabetes, 45 out of 88 screened metabolites had been reported as potential drugs in the literature. Meanwhile, 18 of these metabolites were specific to type I diabetes. Additionally, metabolite correlation could reflect disease relationships in some sense. Our results have demonstrated the potential of bioinformatics mining gut microbes' metabolites as drug candidates based on reported numerous microbe-disease associations and the Virtual Metabolic Human database. More subtle methods would be developed to ensure more accurate predictions.

Synthesis and structure-activity relationship study of a potent MHO7 analogue as potential anti-triple negative breast cancer agent.

Triple-negative breast cancer (TNBC) is the most aggressive, high recurrence and metastatic breast cancer subtype. There are few safe and effective therapeutic drugs for treatment of TNBC. The marine natural product MHO7 has been determined to be a potential antitumor agent. However, its moderate activity and complex structure hampered its clinical application. In this study, a series of novel derivatives with modification on C24 of MHO7 were first synthesized. Some of the analogues were significantly more potent than MHO7 against all selected breast cancer cell lines. Among them, compound 4m had the best activity, and its IC50 value against TNBC was up to 0.51 µM. A whole-genome transcriptomic analysis shown that the mechanism of compound 4m against TNBC cells was similar with that of parent compound MHO7. Subsequent cellular mechanism studies showed that compound 4m could induce apoptosis of MDA-MB-231 cells through mitochondria pathway and cause G1 phase arrest. Moreover, 4m could disrupt the expressions of MAPK/Akt pathway-associated proteins (p-p38 and p-Akt) and remarkably increase the ratio of Bax to Bcl-2 and activate cleaved caspase 3/9/PARP. Importantly, 4m could influence the expression of Smad 7, and p-Smad 3 to inhibit TNBC cells metastasis. Stability assays in rat plasma and liver microsomes indicated that 4m still have room for further optimization. And the results of the online molinspiration software predicted that 4m has desirable physicochemical properties but some properties still have violation from the Lipinski rule of five. Overall, the modification on C24 of MHO7 was a promising way for developing novel anti-TNBC agents with considerable potential for optimization.

Conjugated linoleic acid attenuates 2,4-dinitrofluorobenzene-induced atopic dermatitis in mice through dual inhibition of COX-2/5-LOX and TLR4/NF-κB signaling.

Conjugated linoleic acid (CLA), commonly found in beef, lamb and dairy products, has been reported to exhibit anti-inflammatory and antipruritus effects and to inhibit the release of chemical mediators such as histamine and eicosanoid in laboratory rodents. The chief objective of the study is to assess the efficacy of CLA on atopic dermatitis (AD) in mice and to explore possible mechanisms with CLA treatments. To develop a new therapy for AD, the anti-AD potential of CLA was investigated by inducing AD-like skin lesions in mice using 2,4-dinitrofluorobenzene. We evaluated dermatitis severity; histopathological changes; serum levels of T helper (Th) cytokines (interferon-γ, interleukin-4); changes in protein expression by western blotting and immunohistochemistry staining for cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), toll like receptor 4 (TLR-4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB) and tumor necrosis factor α (TNF-α); and production of the proinflammatory lipid mediators, such as prostaglandin E2 and leukotriene B4, in the skin lesions. Treatment with CLA ameliorated the development of AD-like clinical symptoms and effectively inhibited epidermal hyperplasia and infiltration of mast cells and CD4+ T cells in the AD mouse skin. Total serum immunoglobulin E levels and the expression levels of Th1/Th2 cytokines and lipid mediators in dorsal skin were dramatically suppressed by CLA. Furthermore, CLA down-regulated the expressions of COX-2, 5-LOX, TLR4, MyD88, NF-κB and TNF-α. Taken together, our findings demonstrate the potential usefulness of CLA as an anti-inflammatory dietary supplement or drug for the prevention and management of AD skin diseases by modulating the COX-2/5-LOX and TLR4/MyD88/NF-κB signaling pathways.

[The third-generation sequencing combined with targeted capture technology for high-resolution HLA typing and MHC region haplotype identification].

The high-resolution and accurate typing of human leukocyte antigen (HLA) is of great significance for the study of tissue matching in organ transplantation and the correlation between HLA and disease. In this study, the peripheral blood of 12 patients with primary hepatocellular carcinoma was used to compare the advantages and disadvantages of the next- and third-generation sequencing technology for high-resolution HLA typing. In addition, probe capture technology was used to capture the MHC region of YH and HeLa standard cell lines, and a primary hepatocellular carcinoma patient. The captured products were sequenced using PacBio platform to assess the potential of ultra-long reads sequencing technology for analysis of the entire MHC region. Our results showed that: (1) the next- and third-generation sequencing technology can both achieve 6-8 digit high resolution in HLA typing. However, the coverage of the third-generation is significantly better than the next-generation sequencing technology. (2) The ultra-long reads of the third generation sequencing can directly span the entire amplicon region, which has obvious advantages for haplotype phasing, with 92.79% of the HLA genes having accurate phasing results, which is much higher than the 75.65% from the next-generation data. (3) The long-reads from the third generating sequencing can not only be used to assemble the MHC region but also the ability to phase the entire MHC region of 3.6 Mb, thereby helping to clarify the localization information of the mutation sites, alleles and non-coding regions on each MHC haplotype, and providing a theoretical basis for the study of immune and other related diseases.

Micromonospora mangrovi sp. nov., isolated from mangrove soil.

A novel actinomycete strain 2803GPT1-18(T) was isolated from a composite mangrove soil sample collected from Beihai, Guangxi province, China. Phylogenetic analysis of the 16S rRNA gene sequence of strain 2803GPT1-18(T) indicated high similarity with 'Micromonospora harpali' NEAU-JC6(T) (99.2 %), Micromonospora haikouensis 232617(T) (99.1 %), Micromonospora wenchangensis 2602GPT1-05(T) (99.1 %), Micromonospora schwarzwaldensis HKI0641(T) (99.1 %). The gyrB gene sequence analysis also indicated that strain 2803GPT1-18(T) should be assigned to the genus Micromonospora but different from any established Micromonospora species. The strain harbored meso-DAP and glycine as major cell wall amino acids, MK-10(H6) (53.5 %), MK-9(H6) (25.1 %) and MK-9(H4) (13.4 %) as predominant menaquinones. The characteristic whole cell sugars are arabinose, xylose, glucose, galactose and mannose. The polar lipid profile comprises phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and unidentified polar lipids. The major cellular fatty acids present are iso-C16:0 (44.2 %) and iso-C15:0 (12.4 %). The DNA G+C content is 71.2 mol%. Furthermore, a combination of DNA-DNA relatedness and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from the closely related species. On the basis of these phenotypic and genotypic data, strain 2803GPT1-18(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora mangrovi sp. nov. is proposed. The type strain is 2803GPT1-18(T) (=CCTCC AA2012012(T) = DSM45761(T)).

Genotype-driven isolation of enterocin with novel bioactivities from mangrove-derived Streptomyces qinglanensis 172205.

The type II polyketide synthase (PKS) natural product enterocin (1) was isolated from a mangrove-derived novel species Streptomyces qinglanensis 172205 guided by genome sequence, and its putative biosynthetic gene cluster was revealed. Its natural analogues 5-deoxyenterocin (2) and wailupemycin A-C (3-5) were also identified by tandem mass spectrometry. By feeding experiments with aryl acids, strain 172205 was proved to incorporate partial exogenous starter units into enterocin- and wailupemycin-based analogues, thus being a new and suitable microorganism for engineering unnatural enc-derived polyketide metabolites. In addition, biological assays indicated that enterocin showed obvious inhibitory activity against ß-amyloid protein (Aß1-42) fibrillation and moderate cytotoxicity against HeLa and HepG2 for the first time.

Draft Genome Sequence of Streptomyces sp. Strain CT34, Isolated from a Ghanaian Soil Sample.

Presented here is a draft genome sequence of Streptomyces sp. strain CT34, which produces a novel ribosomally synthesized and posttranslationally modified peptide (RiPP). Analysis of the deduced open reading frame set identified the putative RiPP biosynthesis gene cluster, as well as other secondary metabolite gene clusters.

Streptomyces ferrugineus sp. nov., isolated from mangrove soil in Thailand.

Bacterial strain HV38(T) was isolated from mangrove soil, which was collected from Thailand. Chemotaxonomic and morphological characteristics were found to be typical of members of the genus Streptomyces. The strain was found to form a distinct phyletic line in the Streptomyces 16S rRNA gene tree and to be closely associated with the type strains of Streptomyces coeruleofuscus CGMCC 4.1667(T) (98.84 % sequence similarity), Streptomyces chromofuscus CGMCC 4.1451(T) (98.63 %) and Streptomyces albidoflavus CGMCC 4.1291(T) (98.56 %). The major menaquinones were identified as MK-9(H8) and MK-9(H10). Its major cellular fatty acids were found to be iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:1ω8c, C16:0, anteiso-C16:1ω8c, iso-C16:0 and anteiso-C16:0. The DNA-DNA hybridization values between strain HV38(T) with S. coeruleofuscus CGMCC 4.1667(T), S. chromofuscus CGMCC 4.1451(T) and S. albidoflavus CGMCC 4.1291(T) were 32.7 ± 0.9, 21.8 ± 0.3 and 19.9 ± 0.9 %, respectively, which clearly supported the conclusion that they belong to separate genomic species. Cumulatively, the data indicated that strain HV38(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces ferrugineus sp. nov. is proposed. The type strain is HV38(T) (=CCTCC AA2014009(T )= DSM 42152(T)).

Natural products from mangrove actinomycetes.

Mangroves are woody plants located in tropical and subtropical intertidal coastal regions. The mangrove ecosystem is becoming a hot spot for natural product discovery and bioactivity survey. Diverse mangrove actinomycetes as promising and productive sources are worth being explored and uncovered. At the time of writing, we report 73 novel compounds and 49 known compounds isolated from mangrove actinomycetes including alkaloids, benzene derivatives, cyclopentenone derivatives, dilactones, macrolides, 2-pyranones and sesquiterpenes. Attractive structures such as salinosporamides, xiamycins and novel indolocarbazoles are highlighted. Many exciting compounds have been proven as potential new antibiotics, antitumor and antiviral agents, anti-fibrotic agents and antioxidants. Furthermore, some of their biosynthetic pathways have also been revealed. This review is an attempt to consolidate and summarize the past and the latest studies on mangrove actinomycetes natural product discovery and to draw attention to their immense potential as novel and bioactive compounds for marine drugs discovery.

Genome-scale screening of drug-target associations relevant to Ki using a chemogenomics approach.

The identification of interactions between drugs and target proteins plays a key role in genomic drug discovery. In the present study, the quantitative binding affinities of drug-target pairs are differentiated as a measurement to define whether a drug interacts with a protein or not, and then a chemogenomics framework using an unbiased set of general integrated features and random forest (RF) is employed to construct a predictive model which can accurately classify drug-target pairs. The predictability of the model is further investigated and validated by several independent validation sets. The built model is used to predict drug-target associations, some of which were confirmed by comparing experimental data from public biological resources. A drug-target interaction network with high confidence drug-target pairs was also reconstructed. This network provides further insight for the action of drugs and targets. Finally, a web-based server called PreDPI-Ki was developed to predict drug-target interactions for drug discovery. In addition to providing a high-confidence list of drug-target associations for subsequent experimental investigation guidance, these results also contribute to the understanding of drug-target interactions. We can also see that quantitative information of drug-target associations could greatly promote the development of more accurate models. The PreDPI-Ki server is freely available via: http://sdd.whu.edu.cn/dpiki.

Micromonospora wenchangensis sp. nov., isolated from mangrove soil.

An actinomycete, strain 2602GPT1-05(T), was isolated from a composite mangrove soil sample collected from Wenchang, Hainan province, China. Strain 2602GPT1-05(T) showed closest 16S rRNA gene sequence similarity to Micromonospora haikouensis 232617(T) (99.05 %), and phylogenetically clustered with Micromonospora haikouensis 232617(T), Micromonospora matsumotoense IMSNU 22003(T) (98.7 %) and Micromonospora rifamycinica AM105(T) (98.6 %) based on the 16S rRNA and gyrB gene sequence phylogenetic analysis. The strain harboured meso-DAP and glycine as major cell-wall amino acids, and MK-10(H6) and MK-9(H6) as predominant menaquinones. The characteristic whole-cell sugars were xylose, arabinose, glucose and galactose. The polar lipid profile comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositol mannosides, unknown phospholipid and an unknown phosphoglycolipid. The major cellular fatty acids were C18 : 1ω9c, iso-C15 : 0, 10-methyl C18 : 0 (tuberculostearic acid), C16 : 0, C18 : 0 and iso-C16 : 0. The DNA G+C content was 71.7 mol%. Furthermore, some physiological and biochemical properties and low DNA-DNA relatedness values enabled the strain to be differentiated from members of closely related species. On the basis of these phenotypic, genotypic and chemotaxonomic data, strain 2602GPT1-05(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora wenchangensis sp. nov. is proposed. The type strain is 2602GPT1-05(T) ( = CCTCC AA 2012002(T) = DSM 45709(T)).

[Study on electroporation of rare Actinomycete Micromonospora sp. 40027 with Streptomyces plasmid pSET152].

Electroporation of Micromonospora sp. 40027 isolated from soil was studied with Streptomyces plasmid pSET152, an integrative vector commonly used in Streptomyces genetic manipulation. Transformant was not obtained by electroporation with germinated spores of Micromonospora sp. 40027 as recipient, but plasmid pSET152 can be electroporated into the fresh mycelium of Micromonospora sp. 40027, and the highest electroporation efficiency was yielded under the electric field strength of 13kV/cm. Plasmid stability experiment and southern blot showed that pSET152 could stably exist in the Micromonospora sp. 40027, and was integrated into its chromosome via the attP site, originated from Streptomyces phage phiC31. These data suggested that plasmid pSET152 was successfully electroporated into Micromonospora, and that the integrase gene and attP site of Streptomyces phage phiC31 could play the same role in Micromonospora.

[Studies on the isolation and biological characterization of actinophages infecting Micromonospora sp. 40027].

Three actinophages, phiHAU7, phiHAU9 and phiHAU11, were isolated from soil using Micromonospora sp. 40027 as a indicator strain. Three phages showed very narrow host-ranges. Three phages could infect both Micromonospora sp. 40027 and Micromonospora A-M-01, phiHAU9 and phiHAU11 could also form plaques on Micromonospora purprea. All of the three phage particles have the hexagonal heads and tails; the plaque formation of three phages on Micromonospora sp. 40027 were best on DNA medium with addition of 32 mM Ca2+ and 30 mM Mg2+; phiHAU7 was stable at pH 6-12, and other phages were stable at pH 6-10; the suitable incubation temperature for the propagation of three phages was between 28 degrees C - 37 degrees C; 53% of phiHAU7 remained viable and none of other phages was alive when they were incubated at 60 degrees C for 30 min. Restriction digestion analysis of genomes of three phages indicated that they were all double-stranded DNA with cohesive ends, their genome size are ca. 60 kb, 58 kb and 55 kb, respectively.

[Cloning of bldAa and the effect on morphological differentiation and avermectins production in Streptomyces avermitilis NRRL8165].

bldA encodes the only tRNA that efficiently translates the rare UUA leucine codon in Streptomyces coelicolor. bldA inactivation leaded to defection in morphological development and production of two of four known antibiotics in S. coelicolor. A bldA homologue, termed bldA. , has been identified in the sequenced genome of Streptomyces avermitilis MA4680. To investigate the function of bldA., genomic DNA of S. avermitilis NRRL8165 was digested with BamH I and the 5 - 6kb was fractioned and ligated with the BamH I digested E. coli plasmid vector pIJ4642 to yield a sub-library. A clone containing bldAa and its flanking sequence was obtained by screening from this genome sub-library. pHL358, a bldA, replacement plasmid, was constructed using the lambdaRED mediated PCR-targeting technique, and conjugated into S. avermitilis NRRL8165.Three bldA-disruption mutant strains (named TW10) were obtained, which showed a bald phenotype, indicating that bldA, controlled the morphological differentiation of S. avermitilis . HPLC analysis of the TW10 fermentation culture showed that TW10 did not synthesize avermectins anymore, suggesting that the synthesis of avermectins were dominated by bldAa . There are TTA codons within aveA3 and aveR of the avermectin biosynthesis gene cluster, suggesting that the translation of the two genes may depend on bldAa, which were consistent with the experimental results.

[Activation of silent antibiotic synthesis in Streptomyces lividans by disruption of a negative regulator nsdA, a gene conserved in Streptomyces].

The global regulatory gene, nsdA, negatively regulates antibiotics production in Streptomyces coelicolor. Southern blot experiment, using an nsdA fragment of S. coelicolor as probe, indicated that nsdA gene existed in many Streptomyces. Primers were designed based on the published sequences of S. coelicolor and S. avermitilis. PCR amplification and sequencing showed that nsdA in Streptomyces was conservative and that of S. lividans ZX64 has a 100% identity in the nucleotide sequence comparing with that of S. coelicolor A3 (2). The nsdA disrupted mutant of S. lividans was constructed named as WQ2. WQ2 was able to produce actinorhodin but the wild-type strain ZX64 did not, which has a silent gene cluster contributing to the biosynthesis of actinorhodin. However, the ability was lost when another copy of the wild nsdA gene was introduced into WQ2. All the results above indicate that nsdA homologous gene is wildly existent and conserved in Streptomyces. And it plays a role in negatively regulating the actinorhodin synthesis in S. lividans and disruption of it can activate the silent gene cluster.

cvhA gene of Streptomyces hygroscopicus 10-22 encodes a negative regulator for mycelia development.

A five-gene cluster cvhABCDE was identified from Streptomyces hygroscopicus 10-22. As the first gene of this cluster, cvhA encoded a putative sensor histidine kinase with a predicted sensor domain consisting of two trans-membrane segments at the N-terminus and a conserved HATPase_c domain at the C-terminus. The C-terminus polypeptide of CvhA expressed in Escherichia coli was purified and shown to be autophosphorylated with [gamma-32P]ATP in vitro. The phosphoryl group was acid-labile and basic-stable, which supported histidine as the phosphorylation residue. No obvious difference of mycelia development was observed between the null mutant of cvhA generated by targeted gene replacement and the wild-type parental strain 10-22 grown on solid soya flour medium with 2%-8% glucose or sucrose, but the cvhA mutant could form much more abundant aerial mycelia and spores than the wild-type strain on solid soya flour medium supplemented with 6%-8% mannitol, 6%-8% sorbitol, 4%-6% mannose, or 4%-6% fructose. This phenotype was complemented by the cloned wild-type cvhA gene, and no difference was observed for growth curves of the cvhA mutant and the wild strain in liquid minimal medium with the tested sugars at a concentration of 4%, 6% and 8%. We thus propose that CvhA is likely a sensor histidine kinase and negatively regulates the morphological differentiation in a sugar-dependent manner in S. hygroscopicus 10-22.

Identification and characterization of hmr19 gene encoding a multidrug resistance efflux protein from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22.

The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane alpha-helical spanners, belonging to the drug:H(+) antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [(3)H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

[Construction of a temperature inducible shuttle expression vector and its application in Streptomyces].

pHZ1080, an E. coli-Streptomyces shuttle expression vector was constructed in order to explore the utilization of lambda phage regulated expression elements in Streptomyces. A 2.7 kb polyketide synthase (PKS) gene from Streptomyces sp. FR-008 was inserted into downstream of lambda phage promoter (PR) to give the shuttle plasmid, pHZ1067. The PKS protein was expressed in Streptomyces lividans carrying pHZ1067 in a heat-dependent manner, as it did in E. coli. The PKS protein expressed in both hosts with same molecular weight was detected by SDS-PAGE and Western-blot. The successful heat-induced expression of PKS suggested that pHZ1080 was useful and convenient for heat-induced expression of heterologous genes in both E. coli and Streptomyces.

[Determination of nanchangmycin and meilingmycin by high performance liquid chromatography].

A high performance liquid chromatographic method had been established for the separation and determination of two antibiotics produced by Streptomyces nanchangensis, polyether nanchangmycin and 16-membered macrolide meilingmycin. The latter is composed of several components. The operating conditions were Waters XTerra RP18 column (3.9 mm i.d. x 150 mm, 5 microns) at 25 degrees C, mobile phase linear gradient elution of acetonitrile-water with 57:43 (volume ratio) during 0 min-30 min, 70:30 during 30 min-32 min, 80:20 during 32 min-34 min, 90:10 during 34 min-50 min at a flow rate of 0.8 mL/min, and photodiode array detector at 234 nm. This method is accurate, rapid and simple, and can be used for the integrated analysis of the two different natural compounds, polyethers and macrolides.