E40 glutenase detoxification capabilities of residual gluten immunogenic peptides in in vitro gastrointestinal digesta of food matrices made of soft and durum wheat.

Gluten degrading enzymes, which are commonly referred to as "glutenases," represent attractive candidates for the development of a pharmacological treatment of gluten related disorders, such as coeliac disease (CeD). Endoprotease-40 (E40), a novel glutenase secreted by the actinomycete Actinoallomurus A8 and recombinantly produced in S. lividans TK24, was shown to be active at pH 3 to 6 (optimum pH 5), resistant to pepsin and trypsin degradation, able to destroy immunotoxicity of both gliadin 33-mer peptide and whole proteins and to strongly reduce the response of specific T cells when added to gliadin in in vitro gastrointestinal digestion. This study aims to functionally assess the capabilities of Endoprotease-40 (E40) to detoxify residual gluten immunogenic peptides in gastrointestinal digesta of food matrices made of soft and durum wheat. The INFOGEST harmonized protocols were applied to the multicompartmental model of simulated human gastrointestinal digestion, for the quantitative assessment of residual gluten in liquid (beer) and solid (bread and pasta) foods, made of either soft or durum wheat. Proteomic and immunological techniques, and functional assays on intestinal T cell lines from celiac disease patients were used to identify gluten-derived immunogenic peptide sequences surviving in gastric and gastrointestinal digesta after the addition of E40 at increasing enzyme: wheat proteins ratios. During the gastric phase (2 h incubation time), the addition of E40 demonstrated an extensive (≥ 95%) dose-dependent detoxification of whole gluten in real food matrices. Overall, the residual gluten content was found at, or even below, the 20 ppm gluten-free threshold for soft and durum wheat-based food. Furthermore, unlike in untreated gastrointestinal digesta, none of the immunodominant α-gliadin peptides survived in E40-treated digesta. Traces of ω- and γ-gliadin derived immunogenic peptides were still detected in E40-treated digesta, but unable to stimulate celiac-intestinal T cells. In conclusion, E40 is a promising candidate for the oral enzymatic therapy of CeD, as a stand-alone enzyme being efficient along the complete gastrointestinal digestion of gluten.

Reticulibacter mediterranei gen. nov., sp. nov., within the new family Reticulibacteraceae fam. nov., and Ktedonospora formicarum gen. nov., sp. nov., Ktedonobacter robiniae sp. nov., Dictyobacter formicarum sp. nov. and Dictyobacter arantiisoli sp. nov., belonging to the class Ktedonobacteria.

The aerobic, Gram-positive, mesophilic Ktedonobacteria strains, Uno17T, SOSP1-1T, 1-9T, 1-30T and 150040T, formed mycelia of irregularly branched filaments, produced spores or sporangia, and numerous secondary metabolite biosynthetic gene clusters. The five strains grew at 15-40 °C (optimally at 30 °C) and pH 4.0-8.0 (optimally at pH 6.0-7.0), and had 7.21-12.67 Mb genomes with 49.7-53.7 mol% G+C content. They shared MK9(H2) as the major menaquinone and C16 : 1-2OH and iso-C17 : 0 as the major cellular fatty acids. Phylogenetic and phylogenomic analyses showed that Uno17T and SOSP1-9T were most closely related to members of the genus Dictyobacter, with 94.43-96.21 % 16S rRNA gene similarities and 72.16-81.56% genomic average nucleotide identity. The strain most closely related to SOSP1-1T and SOSP1-30T was Ktedonobacter racemifer SOSP1-21T, with 91.33 and 98.84 % 16S rRNA similarities, and 75.13 and 92.35% average nucleotide identities, respectively. Strain 150040T formed a distinct clade within the order Ktedonobacterales, showing <90.47 % 16S rRNA gene similarity to known species in this order. Based on these results, we propose: strain 150040T as Reticulibacter mediterranei gen. nov., sp. nov. (type strain 150 040T=CGMCC 1.17052T=BCRC 81202T) within the family Reticulibacteraceae fam. nov. in the order Ktedonobacterales; strain SOSP1-1T as Ktedonospora formicarum gen. nov., sp. nov. (type strain SOSP1-1T=CGMCC 1.17205T=BCRC 81203T) and strain SOSP1-30T as Ktedonobacter robiniae sp. nov. (type strain SOSP1-30T=CGMCC 1.17733T=BCRC 81205T) within the family Ktedonobacteraceae; strain Uno17T as Dictyobacter arantiisoli sp. nov. (type strain Uno17T=NBRC 113155T=BCRC 81116T); and strain SOSP1-9T as Dictyobacter formicarum sp. nov. (type strain SOSP1-9T=CGMCC 1.17206T=BCRC 81204T) within the family Dictyobacteraceae.

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-γ when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.

Design of gliadin peptide analogues with low affinity for the celiac disease associated HLA-DQ2 protein.

The HLA-DQ2.5 receptors bind gluten-derived peptides and present them to the T cells in the intestinal mucosa thus inducing the development of immune responses typical of the celiac disease. On the basis of the X-ray structure of the domain of HLA-DQ2.5 bound to the DQ2.5-glia-α1a epitope, fifteen peptides were designed with the aim of lowering the epitope binding affinity, thus reducing the autoimmune response. Hydroxylation of Pro residues was proposed as a suitable functionalization given that both enzymatic and chemical synthetic methods are available. Then, a computational study on the effects of Pro hydroxylation on HLA-DQ2.5 binding was performed by molecular docking. A docking protocol able to reproduce the binding geometry of the known crystallographic complex was set up and applied to the designed DQ2.5-glia-α1a analogues. Among them, the one including four di-hydroxylated Pro residues was predicted to lower the binding affinity to the greatest extent. Therefore, the same functionalization was also computationally tested for other celiac disease relevant epitopes, DQ2.5-glia-α1b and DQ2.5-glia-α2, and their ability for inhibiting the binding to HLA-DQ2.5 was confirmed. On this basis, these hydroxylated peptides are expected to significantly affect the gluten activity involved in celiac disease and, after experimental validation, a synthetic method will be developed for introducing this gluten modification directly in flour. The proposed approach is a promising tool to study the binding of other gliadin and glutenin derived T-cell epitopes as well as their variants.

The genus Actinoallomurus and some of its metabolites.

In the search for novel antibiotics, natural products continue to represent a valid source of bioactive molecules. During a program aimed at identifying previously unreported taxa of actinomycetes as potential source of novel compounds, we isolated hundreds of different representatives of a new group, initially designated as 'Alpha' and independently described as Actinoallomurus. We report on a PCR-specific method for the detection of this taxon, on appropriate growth conditions and on a pilot-screening program on 78 strains. The strains produce antibacterial or antifungal compounds at a relatively high frequency. Four strains were characterized in further detail: one produced the aromatic polyketide benanomicin B and its dexylosyl derivative; a second strain produced N-butylbenzenesulfonamide; a third strain was an efficient converter of soymeal isoflavonoids from soymeal constituents; and a fourth strain produced several coumermycin-related aminocoumarins, with coumermycin A2 as the major peak, and with some new congeners as minor components of the complex. These data suggest that Actinoallomurus strains possess several pathways for secondary metabolism and represent an attractive source in the search for novel antibiotics.

Novel members of the family Micromonosporaceae, Rugosimonospora acidiphila gen. nov., sp. nov. and Rugosimonospora africana sp. nov.

Two novel Gram-positive-staining, acidophilic strains were isolated from soil samples. Both show typical features of filamentous actinomycetes. On the basis of 16S rRNA gene sequence analysis, the strains are members of the family Micromonosporaceae. The two strains contain hydroxydiaminopimelic acid, glycine, alanine and glutamic acid in the peptidoglycan. Fatty acid profiles clearly differentiate the two strains: cyclohexyl C(17 : 0), i-C(16 : 0) and ai-C(17 : 0) are predominant in Delta1(T), while the major components for Delta3(T) are ai-C(17 : 0) and i-C(16 : 0). The two strains also differ in their major menaquinones, MK-9(H(8), H(4), H(6)) for Delta1(T) and MK-9(H(8), H(6)) for Delta3(T), and in phospholipid patterns; Delta1(T) displays phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, methyl phosphatidylethanolamine and an unknown aminophospholipid, while Delta3(T) also contains minor amounts of several unknown phospholipids in addition to these phospholipids. The whole-cell sugars of both strains are galactose, arabinose and xylose. The G+C content of the DNA is 72.7 mol% for Delta1(T) and 71.9 mol% for Delta3(T). On the basis of chemotaxonomic, physiological and phylogenetic data, we propose Rugosimonospora gen. nov. to accommodate the two strains, with the description of Rugosimonospora acidiphila gen. nov., sp. nov. (the type species; type strain Delta1(T) =DSM 45227(T) =NBRC 104874(T)) and Rugosimonospora africana sp. nov. (type strain Delta3(T) =DSM 45228(T) =NBRC 104875(T)).

A novel lantibiotic acting on bacterial cell wall synthesis produced by the uncommon actinomycete Planomonospora sp.

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.

Catenulispora acidiphila gen. nov., sp. nov., a novel, mycelium-forming actinomycete, and proposal of Catenulisporaceae fam. nov.

A novel, Gram-positive bacterial strain was isolated from forest soil. Among species with validly published names, the 16S rRNA gene sequence is related most closely (approx. 93 % similarity) to that of Sporichthya polymorpha DSM 43042(T). However, differently from this species, it forms both vegetative and aerial mycelia. The aerial hyphae are straight to slightly flexuous, starting to septate to form chains of more than 20 cylindrical spores with a rugose surface. The strain is acidophilic, with a pH range for robust growth between 4.3 and 6.8 and an optimum around 6.0. The peptidoglycan type is A3gamma ll-Dpm-Gly. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and two unknown phospholipids. Predominant menaquinones are MK-9(H(6)) and -9(H(4)), and iso- and anteiso-branched C(16 : 0) and C(17 : 0) are the main cellular fatty acids. The DNA G+C content is 71.9 mol%. The distinct phylogenetic position and the unusual combination of chemotaxonomic characteristics justify the proposal of Catenulispora gen. nov., with the type species Catenulispora acidiphila sp. nov. (type strain, ID139908(T) =DSM 44928(T)=NRRL B-24433(T)). Catenulisporaceae fam. nov. is also proposed.

Actinospica robiniae gen. nov., sp. nov. and Actinospica acidiphila sp. nov.: proposal for Actinospicaceae fam. nov. and Catenulisporinae subord. nov. in the order Actinomycetales.

Two novel Gram-positive, acidophilic bacterial strains were isolated from forest soil. According to their 16S rRNA gene sequences, these strains are related closely to each other and form a distinct cluster within the order Actinomycetales. They show the typical features of filamentous actinomycetes, with branched vegetative hyphae and production of aerial hyphae. The distinct phylogenetic positions and the combination of chemotaxonomic characteristics of these strains justify the proposal of Actinospica gen. nov. Both strains display 3-hydroxydiaminopimelic acid plus traces of meso-diaminopimelic acid, the phospholipids diphosphatidylglycerol, phosphatidylethanolamine, methylphosphatidylethanolamine and phosphatidylinositol, the predominant cellular fatty acids i-C(15 : 0), i-C(16 : 0) and ai-C(15 : 0) and the whole-cell sugars mannose and rhamnose. They differ in the fatty acid profiles, in the quantitative ratios of the major menaquinones MK-9(H(4)), MK-9(H(6)) and MK-9(H(8)) and in the occurrence of additional whole-cell sugars (arabinose and xylose in strain GE134766(T) and galactose in strain GE134769(T)). Differences in the phenotypic characteristics and in the 16S rRNA gene sequences suggest the description of two species, Actinospica robiniae gen. nov., sp. nov. (the type species) and Actinospica acidiphila sp. nov., with the type strains GE134769(T) (=DSM 44927(T)=NRRL B-24432(T)) and GE134766(T) (=DSM 44926(T)=NRRL B-24431(T)), respectively. The DNA G+C contents of strains GE134769(T) and GE134766(T) are 70.8 and 69.2 mol%, respectively. Due to the large phylogenetic distance from known actinomycete genera, it is proposed to accommodate Actinospica gen. nov. in Actinospicaceae fam. nov. In addition, Catenulisporineae subord. nov. is proposed to harbour Actinospicaceae fam. nov. and the newly proposed family Catenulisporaceae, described in the accompanying paper.

New lineage of filamentous, spore-forming, gram-positive bacteria from soil.

A novel bacterial strain that was isolated from an Italian soil and was designated SOSP1-21T forms branched mycelia in solid and liquid media and has a filamentous morphology similar to that of some genera belonging to the Actinobacteria. Electron microscopy showed that this organism has a grape-like appearance, resulting from interlacing of spores originating from sporophoric hyphae. Ten strains that are morphologically related to SOSP1-21T were recovered from soil. Phylogenetic analyses of 16S rRNA gene segments confirmed the relatedness of these strains to SOSP1-21T and indicated that the newly isolated strains form separate clades in a deeply branching lineage. The closest matches for the 16S rRNA sequences of all the strains (around 79% identity) were matches with representatives of the Chloroflexi, although the affiliation with this division was not supported by high bootstrap values. The strains are mesophilic aerobic heterotrophs and are also capable of growing under microaerophilic conditions. They all stain gram positive. Strain SOSP1-21T contains ornithine, alanine, glutamic acid, serine, and glycine as the peptidoglycan amino acids. In addition, an unusual level of C16:1 2OH (30%) was found in the cellular fatty acids. The G+C content of SOSP1-21T genomic DNA is 53.9%, and MK-9(H2) was the only menaquinone detected. All these data suggest that SOSP1-21T and the related strains may constitute a new division of filamentous, spore-forming, gram-positive bacteria. We propose the name Ktedobacter racemifer gen. nov., sp. nov. for strain SOSP1-21T.

Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes.

The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia, have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil (Jospeh et al., Appl Environ Microbiol 69, 7210-7215, 2003). Thus, these previously uncultured actinomycetes share several features with Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics.

Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp.

In the course of a microbial product screening aimed at the discovery of novel antibiotics acting on bacterial protein synthesis, a complex of three structurally related tetrapeptides, namely, GE81112 factors A, B, and B1, was isolated from a Streptomyces sp. The screening was based on a cell-free assay of bacterial protein synthesis driven by a model mRNA containing natural initiation signals. In this study we report the production, isolation, and structure determination of these novel, potent and selective inhibitors of cell-free bacterial protein synthesis, which stably bind the 30S ribosomal subunit and inhibit the formation of fMet-puromycin. They did not inhibit translation by yeast ribosomes in vitro. Spectroscopic analyses revealed that they are tetrapeptides constituted by uncommon amino acids. While GE81112 factors A, B, and B1 were effective in inhibiting bacterial protein synthesis in vitro, they were less active against Gram-positive and Gram-negative bacterial cells. Cells grown in minimal medium were more susceptible to the compounds than those grown in rich medium, and this is most likely due to competition or regulation by medium components during peptide uptake. The novelty of the chemical structure and of the specific mode of action on the initiation phase of bacterial protein synthesis makes GE81112 a unique scaffold for designing new drugs.

Conexibacter woesei gen. nov., sp. nov., a novel representative of a deep evolutionary line of descent within the class Actinobacteria.

A novel Gram-positive bacterial strain was isolated from forest soil. According to its 16S rRNA sequence, this strain is a deep-rooting member of the class Actinobacteria. The 16S rRNA sequence is most closely related (approximately 94% identity) to clones of uncultured bacteria detected in different terrestrial environments, while showing only a remote relationship (approximately 90% identity or less) to sequences of cultured species. Cells of the first cultured representative of this phylogenetic cluster are small, short rods that are motile by peritrichous flagella, catalase- and oxidase-positive and grow under aerobic conditions. In liquid culture, flagella from different cells can aggregate to form networks, clearly visible under the light microscope. The peptidoglycan contains meso-diaminopimelic acid and is directly cross-linked (type A1gamma). Mycolic acids are not present. The polar lipids are phosphatidylinositol and an unidentified phospholipid. Menaquinone MK-7(H4) was detected as the predominant isoprenoid quinone. Oleic, 14-methylpentadecanoic, hexadecanoic and omega6c-heptadecenoic acids are the predominant components of the cellular fatty acid profile. The DNA G + C content is 71 mol%. The distinct phylogenetic position and the unusual combination of chemotaxonomic characteristics justify the proposal of a new genus and species, Conexibacter woesei gen. nov., sp. nov., with the type strain ID131577T (=DSM 14684T =JCM 11494T).

Microbial technologies for the discovery of novel bioactive metabolites.

Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development and application of molecular methods for the detection of uncommon genera of actinomycetes in soil DNA and for the rapid dereplication of actinomycete isolates. The results indicate a substantial presence in many soils of the uncommon genera and a large diversity of isolated actinomycetes. However, while uncommon actinomycete strains may provide an increased chance of yielding novel structures, their genetics and physiology are poorly understood. To speed up their manipulation, we have developed vectors capable of stably maintaining large segments of actinomycete DNA in Escherichia coli and of integrating site specifically in the Streptomyces genome. These vectors are suitable for the reconstruction of gene clusters from smaller segment of cloned DNA, the preparation of large-insert libraries from unusual actinomycete strains and the construction of environmental libraries.

New PCR primers for the selective amplification of 16S rDNA from different groups of actinomycetes.

Actinomycetes play a relevant role in soil ecology and are also of important biotechnological interest as they produce several bioactive metabolites. Within the filamentous actinomycetes, it would be desirable to recognize and characterize environmental samples containing unusual genera. To this end, we have developed selective primer sets for polymerase chain reaction (PCR) amplification of 16S rDNA from the Actinomycetales families Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae and Thermomonosporaceae, and from the genus Dactylosporangium. Each primer set, evaluated on genomic DNA from reference strains, showed high specificity and good sensitivity. After amplification of DNA extracted from soil samples, the sequence of the cloned PCR products confirmed the specific amplification of the desired group of sequences in at least 95% of the clones for each primer set. The application of these primers to environmental samples showed the frequent occurrence of these groups in soil samples and also revealed sequences that can be attributed to new groups of actinomycetes.