Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development.

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

The impact of organic vs. inorganic selenium on dairy goat productivity and expression of selected genes in milk somatic cells.

The aim of this study was to determine the effect of diet supplemented with selenized yeast (Se-yeast) on milk yield and milk composition of goats and expression of casein and mammary-gland-immune system genes in milk somatic cells (MSC). Twenty-four dairy goats in their second to fourth lactations were divided into control and experimental groups, balanced according to lactation number and breed (Polish White or Fawn Improved). Morning milk and blood samples were collected four times during lactation (on the 21st, 70th, 120th, 180th day after kidding). The control and experimental groups were fed diets with 0.7 mg inorganic Se/goat/day (sodium selenite) or 0.6 mg organic Se/goat/day (selenized yeast), respectively. Milk, fat and protein yields during lactation as well as average somatic cell count, fat, protein and lactose contents in milk were evaluated. Microelements in milk and blood serum and biochemical parameters in blood serum were determined at the beginning and the end of the experiment. The expression levels of the genes encoding αS1-casein (CSN1S1), αS2-casein (CSN1S2), κ-casein (CSN3), interleukin 8 (IL-8), serum amyloid A3 (SAA3), interleukin 1ß (IL-1ß), bactenecin 7.5 (BAC7.5), bactenecin 5 (BAC5), ß2-defensin (GBD2), hepcidin (HAMP), chemokine 4 (CCL4), tumour necrosis factor α (TNFα), toll-like receptor 2 (TLR2), cathelicidin-7 (MAP34) and cathelicidin-6 (MAP28) were determined in MSC. Milk, fat, and protein yields were higher and somatic cell count (SCC expressed as natural logarithm) was lower in the milk of goats fed organic Se. The Se concentration in milk was twice as high in the organic vs. inorganic treatment groups at the end of the experiment, while there were no differences in studied biochemical parameters between groups. The transcript levels of CSN1S2 and BAC7.5 were higher and IL-8 was lower in MSC of Se-yeast treated groups. Such results may indicate better health status of mammary glands of goats treated with organic Se as well as positive impact of selenized yeast on the goat's milk composition. Differences in the IL-1ß and IL-8 transcript levels were also noted between the stages of lactation, with the highest expression at the peak of lactation (day 70), highlighting the metabolic burden at this time. We concluded that the Se-yeast supplementation improved the productivity and health status of goats and could have significant economic impact on farmer's income.

Genetically Encoded Cyclic Peptide Libraries: From Hit to Lead and Beyond.

With the increasing utilization of high-throughput screening for lead identification in drug discovery, the need for easily constructed and diverse libraries which cover significant chemical space is greater than ever. Cyclic peptides address this need; they combine the advantageous properties of peptides (ease of production, high diversity, high potential specificity) with increased resistance to proteolysis and often increased biological activity (due to conformational locking). There are a number of techniques for the generation and screening of cyclic peptide libraries. As drug discovery moves toward tackling challenging targets, such as protein-protein interactions, cyclic peptide libraries are expected to continue producing hits where small molecule libraries may be stymied. However, it is important to design robust systems for the generation and screening of these large libraries, and to be able to make sense of structure-activity relationships in these highly variable scaffolds. There are a plethora of possible modifications that can be made to cyclic peptides, which is both a weakness and a strength of these scaffolds; high variability will allow more precise tuning of leads to targets, but exploring the whole range of modifications may become an overwhelming challenge.

Design and Application of a DNA-Encoded Macrocyclic Peptide Library.

A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.

Therapeutic cyclic lipopeptides mining from microbes: latest strides and hurdles.

Infectious diseases impose serious public health burdens and often have devastating consequences. The cyclic lipopeptides elaborated by bacteria Bacillus, Paenibacillus, Pseudomonas, Streptomyces, Serratia, Propionibacterium and fungus Fusarium are very crucial in restraining the pathogens. Composed of a peptide and a fatty acyl moiety these amphiphilic metabolites exhibit broad spectrum antimicrobial effects. Among the plethora of cyclic lipopeptides, only selective few have emerged as robust antibiotics. For their functional vigor, polymyxin, daptomycin, surfactin, iturin, fengysin, paenibacterin and pseudofactin have been integrated in mainstream healthcare. Daptomycin has been a significant part of antimicrobial arsenal since the past decade. As the magnitude of drug resistance rises in unprecedented manner, the urgency of prospecting novel cyclic lipopeptides is being perceived. Intense research has revealed the implication of these bioactive compounds stretching beyond antibacterial and antifungal. Anticancer, immunomodulatory, prosthetic parts disinfection and vaccine adjuvancy are some of the validated prospects. This review discusses the emerging applications, mechanisms governing the biological actions, role of genomics in refining structure and function, semi-synthetic analog discovery, novel strain isolation, setbacks etc. Though its beyond the scope of the current topic, for holistic purpose, the role of lipopeptides in bioremediation and crop biotechnology has been briefly outlined. This updated critique is expected to galvanize innovations and diversify therapeutic recruitment of microbial lipopeptides.

Cyclotide discovery in Gentianales revisited--identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.

Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

Identification and quantification of cyclolinopeptides in five flaxseed cultivars.

Cyclolinopeptides are a group of naturally occurring hydrophobic cyclic peptides found in flaxseed and flax oil that have immunosuppressive activity. This study describes the measurement of flaxseed cyclolinopeptide concentrations using an internal standard HPLC method. In addition, the concentration of cyclolinopeptides in the seed of Canadian flax cultivars grown at two locations over two years is reported. The data are consistent with the formation of flaxseed cyclolinopeptides from two ribosome-derived precursors. Each precursor protein includes the sequences corresponding to three cyclolinopeptides from which those cyclolinopeptides are presumably derived by precursor processing. The concentrations of cyclolinopeptides C and E, which are encoded by the same gene sequence, are highly correlated, and the concentrations of cyclolinopeptides D, F, and G, which are encoded by a second gene sequence, are also highly correlated. The strong correlation between the cyclolinopeptides arising from the same gene may prove to be important in understanding how peptide concentration is controlled. Additional research may lead to approaches to improve flax either as a platform for peptide production or as a source of oil with improved drying properties and flavor.

Phage display-directed discovery of LEDGF/p75 binding cyclic peptide inhibitors of HIV replication.

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.

Molecular toxicity of earthworms induced by cadmium contaminated soil and biomarkers screening.

Earthworms (Eisenia fetida) were used to study the impact of low-dose cadmium in treated artificial soil (0, 0.6, 3, 6, 15, 30 mg/kg) and contaminated natural soil (1.46 mg/kg). The changes of earthworms' physiological related gene expressions of metallothionein (MT), annetocin, calreticulin and antimicrobial peptides were detected using real-time PCR after a 70-day incubation period. The results showed that low doses of cadmium could up regulate earthworms' MT and down regulate annetocin gene expression and show a significant positive and negative correlation respectively. The expression of two other genes, calreticulin and anti-microbial peptides, was induced at low doses of cadmium (highest gene expression at 0.6 mg/kg for calreticulin and 6 mg/kg for anti-microbial peptides) and inhibited at high doses. No significant correlation was found for these two genes. This study shows that MT and annetocin genes expression found in earthworms in contaminated soil have the potential to be developed as biomarkers of soil cadmium pollution.

Short linear cationic antimicrobial peptides: screening, optimizing, and prediction.

The problem of pathogenic antibiotic-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa is worsening, demonstrating the urgent need for new therapeutics that are effective against multidrug-resistant bacteria. One potential class of substances is cationic antimicrobial peptides. More than 1000 natural occurring peptides have been described so far. These peptides are short (less than 50 amino acids long), cationic, amphiphilic, demonstrate different three-dimensional structures, and appear to have different modes of action. A new screening assay was developed to characterize and optimize short antimicrobial peptides. This assay is based on peptides synthesized on cellulose, combined with a bacterium, where a luminescence gene cassette was introduced. With help of this method tens of thousands of peptides can be screened per year. Information gained by this high-throughput screening can be used in quantitative structure-activity relationships (QSAR) analysis. QSAR analysis attempts to correlate chemical structure to measurement of biological activity using statistical methods. QSAR modeling of antimicrobial peptides to date has been based on predicting differences between peptides that are highly similar. The studies have largely addressed differences in lactoferricin and protegrin derivatives or similar de novo peptides. The mathematical models used to relate the QSAR descriptors to biological activity have been linear models such as principle component analysis or multivariate linear regression. However, with the development of high-throughput peptide synthesis and an antibacterial activity assay, the numbers of peptides and sequence diversity able to be studied have increased dramatically. Also, "inductive" QSAR descriptors have been recently developed to accurately distinguish active from inactive drug-like activity in small compounds. "Inductive" QSAR in combination with more complex mathematical modeling algorithms such as artificial neural networks (ANNs) may yield powerful new methods for in silico identification of novel antimicrobial peptides.

The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth.

Many species of mycobacteria form structured biofilm communities at liquid-air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 microM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes - especially those involved in siderophore synthesis and iron uptake - are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 microM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron-exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

Formation of cyclotides and variations in cyclotide expression in Oldenlandia affinis suspension cultures.

Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37 mg g(-1) dry weight, was accumulated in irradiated cells at 35 mumol m(-2) s(-1). Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.

Strong combined gene-environment effects in anti-cyclic citrullinated peptide-positive rheumatoid arthritis: a nationwide case-control study in Denmark.

OBJECTIVE: To study the role of shared epitope (SE) susceptibility genes, alone and in combination with tobacco smoking and other environmental risk factors, for risk of subtypes of rheumatoid arthritis (RA) defined by the presence or absence of serum antibodies against cyclic citrullinated peptides (CCPs). METHODS: To address these issues, a nationwide case-control study was conducted in Denmark during 2002-2004, comprising incident cases of RA or patients with recently diagnosed RA (309 seropositive and 136 seronegative for IgG antibodies against CCP) and 533 sex- and age-matched population controls. Associations were evaluated by logistic regression analyses, in which odds ratios (ORs) served as measures of relative risk. RESULTS: Compared with individuals without SE susceptibility genes, SE homozygotes had an elevated risk of anti-CCP-positive RA (OR 17.8, 95% confidence interval [95% CI] 10.8-29.4) but not anti-CCP-negative RA (OR 1.07, 95% CI 0.53-2.18). Strong combined gene-environment effects were observed, with markedly increased risks of anti-CCP-positive RA in SE homozygotes who were heavy smokers (OR 52.6, 95% CI 18.0-154), heavy coffee drinkers (OR 53.3, 95% CI 15.5-183), or oral contraceptive users (OR 44.6, 95% CI 15.2-131) compared with SE noncarriers who were not exposed to these environmental risk factors. CONCLUSION: Persons who are homozygous for SE susceptibility genes, notably those who are also exposed to environmental risk factors, have a markedly and selectively increased risk of anti-CCP-positive RA. A distinction between anti-CCP-positive RA and anti-CCP-negative RA seems warranted, because these RA subtypes most likely represent etiologically distinct disease entities.

Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints.

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek).

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC20 generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC20F2 population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC20F2 population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene.

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.