Cinzia Dedi

Genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences

Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage-oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogenetically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut-specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative ‘viral-enterotypes’ among this fraction of the human gut virome.

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome.

An unusual ring―A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17beta-acetoxy-5 alpha-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17beta side-chain. This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17beta-acetoxy-5 alpha-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17beta-alcohol of testosterone. All steroidal metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.

Predominant allylic hydroxylation at carbons 6 and 7 of 4 and 5-ene functionalized steroids by the thermophilic fungus Rhizomucor tauricus IMI23312

This paper demonstrates for the first time transformation of a series of steroids (progesterone, androst-4-en-3,17-dione, testosterone, pregnenolone and dehydroepiandrosterone) by the thermophilic fungus Rhizomucor tauricus. All transformations were found to be oxidative (monohydroxylation and dihydroxylation) with allylic hydroxylation the predominant route of attack functionalizing the steroidal skeleta. Timed experiments demonstrated that dihydroxylation of progesterone, androst-4-en-3,17-dione and pregnenolone all initiated with hydroxylation on ring-B followed by attack on ring-C. Similar patterns of steroidal transformation to those observed with R. tauricus have been observed with some species of thermophilic Bacilli and mesophilic fungi. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The application of thermophilic fungi to steroid transformation may represent a potentially rich source for the generation of new steroidal compounds as well as for uncovering inter and intraspecies similarities and differences in steroid metabolism.

Evaluation of environmental scanning electron microscopy for analysis of Proteus mirabilis crystalline biofilms in situ on urinary catheters

Proteus mirabilis is a common cause of catheter-associated urinary tract infections and frequently leads to blockage of catheters due to crystalline biofilm formation. Scanning electron microscopy (SEM) has proven to be a valuable tool in the study of these unusual biofilms, but entails laborious sample preparation that can introduce artefacts, undermining the investigation of biofilm development. In contrast, environmental scanning electron microscopy (ESEM) permits imaging of unprocessed, fully hydrated samples, which may provide much insight into the development of P. mirabilis biofilms. Here, we evaluate the utility of ESEM for the study of P. mirabilis crystalline biofilms in situ, on urinary catheters. In doing so, we compare this to commonly used conventional SEM approaches for sample preparation and imaging. Overall, ESEM provided excellent resolution of biofilms formed on urinary catheters and revealed structures not observed in standard SEM imaging or previously described in other studies of these biofilms. In addition, we show that energy-dispersive X-ray spectroscopy (EDS) may be employed in conjunction with ESEM to provide information regarding the elemental composition of crystalline structures and demonstrate the potential for ESEM in combination with EDS to constitute a useful tool in exploring the mechanisms underpinning crystalline biofilm formation.

Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance

Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

Transformation of some 3α-substituted steroids by Aspergillus tamarii KITA reveals stereochemical restriction of steroid binding orientation in the minor hydroxylation pathway

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17beta-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11beta-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3beta-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3alpha-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11beta-position (normal binding), indicating that the 3alpha-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1beta-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11beta-hydroxyl completely inhibited Baeyer-Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11beta-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3alpha-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3alpha-acetate inhibited the Baeyer-Villiger monooxygenase with its activity being observed 24h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17beta-alcohol for the first time in this organism.

Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by quantitative PCR controlled Cell-SELEX

In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.

Transformation of a series of saturated isomeric steroidal diols by Aspergillus tamarii KITA reveals a precise stereochemical requirement for entrance into the lactonization pathway

Four isomers of 5α-androstan-3,17-diol have been transformed by the filamentous fungus Aspergillus tamarii, an organism which has the ability to convert progesterone to testololactone in high yield through an endogenous four step enzymatic pathway. The only diol handled within the lactonization pathway was 5α-androstan-3α,17β-diol which, uniquely underwent oxidation of the 17β-alcohol to the 17-ketone prior to its Baeyer-Villiger oxidation and the subsequent production of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one. This demonstrated highly specific stereochemical requirements of the 17β-hydroxysteroid dehydrogenase for oxidation of this specific steroidal diol to occur. In contrast, the other three diols were transformed within the hydroxylation pathway resulting in functionalization at C-11β. Only 5α-androstan-3β,17α-diol could bind to the hydroxylase in multiple binding modes undergoing monohydroxylation in 6β and 7β positions. Evidence from this study has indicated that hydroxylation of saturated steroidal lactones may occur following binding of ring-D in its open form in which an α-alcohol is generated with close spatial parity to the C-17α hydroxyl position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR and DEPT analysis and further characterized using infra-red, elemental analysis and accurate mass measurement.

Transformation of structurally diverse steroidal analogues by the fungus Corynespora cassiicola CBS 161.60 results in generation of 8β-monohydroxylated metabolites with evidence in favour of 8β-hydroxylation through inverted binding in the 9α-hydroxylase

Corynespora cassiicola has a unique but unexplored ability amongst fungi, in that it can hydroxylate 17α-hydroxyprogesterone at the highly hindered C-8 position of the steroid nucleus. In order to gain greater understanding of the mechanistic basis and capability of the 8β-hydroxylase we have transformed a range of structurally diverse androgens and progestogens with this organism. This has revealed that both steroid types can be hydroxylated at the 8β-position. The collective data has demonstrated the first time that 8β-hydroxylation occurs through inverted binding within a 9α-hydroxylase of the fungus. In the case of the progestogens, for this to occur, the presence of 17α-oxygen functionality (alcohol or epoxide) was essential. Remarkably monohydroxylation of 17α-hydroxyprogesterone at carbons 8β and 15β has strongly indicated that the responsible hydroxylase has 2 different binding sites for the ring-A ketone. Unusually, with one exception, all hydroxylation occurred at axial protons and in the case of the progestogens, all above the plane of the ring system. In general all maximally oxidised metabolites contained four oxygen atoms. The importance of these findings in relation to 8β-hydroxylation of these steroids is discussed.