Functional characterization of O‐methyltransferases used to catalyse site‐specific methylation in the post‐tailoring steps of pradimicin biosynthesis

Aims

To identify the roles of the two O‐methyltransferase homologous genes pdmF and pdmT in the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157‐2.

Methods and Results

Pradimicins are pentangular polyphenol antibiotics synthesized by bacterial type II polyketide synthases (PKSs) and tailoring enzymes. Pradimicins are naturally derivatized by combinatorial O‐methylation at two positions (i.e., 7‐OH and 11‐OH) of the benzo[α]naphthacenequinone structure. PdmF and PdmT null mutants (PFKO and PTKO) were generated. PFKO produced the 11‐O‐demethyl shunt metabolites 11‐O‐demethylpradimicinone II ( 1 ), 11‐O‐demethyl‐7‐methoxypradimicinone II ( 2 ), 11‐O‐demethylpradimicinone I ( 3 ) and 11‐O‐demethylpradimicin A ( 4 ), while PTKO generated the 7‐O‐demethyl derivatives pradimicinone II ( 5 ) and 7‐hydroxypradimicin A ( 6 ). Pradimicinones 1 , 2 , 3 , and 5 were fed to a heterologous host Escherichia coli harbouring expression plasmid pET‐22b::pdmF or pET‐28a::pdmT. PdmF catalysed 11‐O‐methylation of pradimicinones 1 , 2 , and 3 regardless of O‐methylation at the C‐7 position, while PdmT was unable to catalyse 7‐O‐methylation when the C‐11 hydroxyl group was methylated ( 5 ).

Conclusions

PdmF and PdmT were involved in 11‐O‐ and 7‐O‐methylations of the benzo[α]naphthacenequinone moiety of pradimicin, respectively. Methylation of the C‐7 hydroxyl group precedes methylation of the C‐11 hydroxyl group in pradimicin biosynthesis.

Significance and Impact of the Study

This is the first reported demonstration of the functions of PdmF and PdmT for regiospecific O‐methylation, which contributes to better understanding of the post‐PKS modifications in pradimicin biosynthesis as well as to rational engineering of the pradimicin biosynthetic machinery.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

Synthesis of 6‐[4‐(4‐Propoxyphenyl)piperazin‐1‐yl]‐9H‐purine Derivatives as Antimycobacterial and Antifungal Agents: In Vitro Evaluation and In Silico Study

A series of novel alkyl substituted purines were synthesized. 6‐[4‐(4‐Propoxyphenyl)piperazin‐1‐yl]‐9H‐purine was used as the key starting material, which was synthesized via a multistep protocol and finally subjected for N‐alkylation with various alkyl halides with an aim to get prospective antimicrobial agents. The structures of the novel compounds were established by substantiating them through spectral techniques like ¹H‐NMR, ¹³C‐NMR, FT‐IR and EI‐MS. They were explored for antitubercular activity against Mycobacterium tuberculosis H37RV. Furthermore, they were checked for their antimicrobial activity concerning bacterial and fungal strains. The title compounds exhibited considerable antimicrobial activity without any significant toxicity. In silico studies depicted their good binding profile against Mycobacterium tuberculosis enoyl reductase (InhA; PDB ID: 4TZK) and Candida albicans dihydrofolate reductase (PDB ID: 1AI9). The title compounds obeyed Lipinski's parameters and have exhibited good drug‐like properties.     

Antimicrobial activity of the indolicidin‐derived novel synthetic peptide In‐58

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin‐derived novel synthetic peptide In‐58. In‐58 was generated by replacing all tryptophan residues on phenylalanine in D‐configuration; the α‐amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In‐58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In‐58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD’::lux), we investigated the action of indolicidin and In‐58 at the subcellular level. At subinhibitory concentrations, indolicidin and In‐58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Systemic administration of bone marrow‐derived cells leads to better uterine engraftment than use of uterine‐derived cells or local injection

Stem cells are recruited to the uterus where they differentiate into endometrial cells and have been suggested as potential therapy for uterine injury such as Asherman's syndrome. However, it is unknown whether local intrauterine injection may result in better stem cell engraftment of the uterus compared with systemic administration, and whether uterine‐derived cells (UDCs) may confer an advantage over BM‐derived cells (BMDCs). Mice underwent local injury to a single uterine horn. Green fluorescent protein (GFP)‐expressing BMDCs, UDCs or saline (control) were injected either intravenously or locally (uterine lumen) into wild‐type recipients. Two or 3 weeks post‐transplant, uterine tissues were collected for fluorescence‐activated cell sorting (FACS) and immunohistochemistry/immunofluorescence studies. Mice injected intravenously with BMDCs or UDCs had increased GFP⁺ cells recruitment to the non‐injured or injured uterus compared to those injected locally. No significant differences were noted in GFP⁺ cell recruitment to the injured versus non‐injured horn. In addition, systemic injection of BMDCs led to greater recruitment of GFP⁺ cells at 2 weeks and 3 weeks compared with UDCs. Immunohistochemical staining demonstrated that GFP⁺ cells were found in stroma but not in epithelium or blood vessels. Immunofluorescence analysis revealed that GFP⁺ cells were mostly CD45‐negative, and negative for CD31 and cytokeratin, confirming their stromal identity. In conclusion, the systemic route of administration results in better recruitment of BMDCs or UDCs to the injured uterus than local injection. In addition, BMDCs recruitment to the uterus is greater than UDCs. These findings inform the development of stem cell‐based therapies targeting the uterus.     

Altered allocation to roots and shoots in the endophyte‐infected seedlings of Puccinellia distans (Poaceae)

Endophytes play an important role in ecological and evolutionary processes in plants and have marked economic value. Seed‐transmitted fungal endophytes are conventionally regarded as mutualistic symbionts, but their fitness consequences for the offspring of the host are not clear. Puccinellia distans infected with the fungus Epichloë typhina (E+) produces seeds that are several times smaller than normal (E?). This observation suggests that the E+ seedlings face a developmental disadvantage. Our growth chamber experiments compared the germination rates of the small E+ and large E? seeds of P. distans and examined the biomass allocation of seedlings to roots and shoots. The E+ seedlings germinated more slowly and maintained shorter shoots and a smaller root biomass for 30–50 days after sowing. Despite this disadvantage, the E+ plants more quickly increased their total size, attaining a larger shoot and whole‐plant biomass. The shoot:root biomass ratio increased more rapidly through time in the E+ seedlings, attaining a value nine times higher in the E+ than the E? group 50 days after sowing. Such differences between the E+ and E? seedlings were not explained by the growth allometry between shoots and roots. The seedlings of P. distans infected with the Epichloë endophyte were initially handicapped by their postponed emergence, but this disadvantage was quickly overcome by their superior growth capacity. The decrease in the relative allocation to roots may indicate that endophytes increase the performance of roots as resource‐acquiring organs and/or reduce the role of roots in protection against herbivores.     

Absence of in vitro innate immunomodulation by insect‐derived short proline‐rich antimicrobial peptides points to direct antibacterial action in vivo

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A network pharmacology‐based approach to explore the effects of Chaihu Shugan powder on a non‐alcoholic fatty liver rat model through nuclear receptors

The pathogenesis of non‐alcoholic fatty liver disease (NAFLD) is still not fully understood, and currently, no effective pharmacotherapy is available. Nuclear receptors (NRs) are important biological participants in NAFLD that exhibit great therapeutic potential. Chaihu Shugan powder (CSP) is a traditional Chinese medicine (TCM) formula that has a wide therapeutic spectrum including NAFLD, but the effective components and functional mechanisms of CSP are unclear. We adopted a network pharmacology approach using multiple databases for Gene Ontology (GO) enrichment analysis and the molecular complex detection (MCODE) method for a protein‐protein interaction (PPI) analysis, and we used molecular docking method to screen the NR targets and determine the corresponding CSP components. The screening results were validated through a NAFLD rat model that was used to explain the possible relationship between CSP and NAFLD. Finally, we screened PPARγ, FXR, PPARα, RARα and PPARδ as target genes and quercetin, kaempferol, naringenin, isorhamnetin and nobiletin as target compounds. The five components were detected through high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS), the results of which aligned with the docking experiments of PPARγ, PPARα and PPARδ. After CSP intervention, the NAFLD rat model showed ameliorated effects in terms of bodyweight, hepatic histopathology, and serum and liver lipids, and the mRNA levels of PPARγ, FXR, PPARα and RARα were significantly changed. The results from this study indicate that CSP exhibits healing effects in an NAFLD model and that the network pharmacology approach to screening NR targets and determining the corresponding CSP components is a practical strategy for explaining the mechanism by which CSP ameliorates NAFLD.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Design,expression, and characterization of the hybrid antimicrobial peptide T‐catesbeianin‐1 based on FyuA

The overuse of antibiotics has resulted in the emergence of antibiotic‐resistant bacteria, which presents an urgent need for new antimicrobial agents. At present, antimicrobial peptides have attracted a great deal of attention from researchers. However, antimicrobial peptides often affect a broad range of microorganisms, including the normal flora in a host organism. In the present study, we designed a novel hybrid antimicrobial peptide, expressed the hybrid peptide, and studied its specific target. The hybrid peptide, named T‐catesbeianin‐1, which includes the FyuA‐binding domain of pesticin and the peptide catesbeianin‐1, was designed and expressed in Pichia pastoris X‐33. Then, we determined the antimicrobial activity, cytotoxicity, and specific target of the peptide. T‐catesbeianin‐1 has strong antimicrobial activity and binds to FyuA to inhibit or kill Escherichia coli present in clinical specimens and mixed‐species culture. In summary, these findings suggested that T‐catesbeianin‐1 might be promising and specific antibiotic agent for therapeutic application against fyuA⁺ E. coli.     

CYP105—diverse structures,functions and roles in an intriguing family of enzymes in Streptomyces

The cytochromes P450 (CYP or P450) are a large superfamily of haem‐containing enzymes found in all domains of life. They catalyse a variety of complex reactions, predominantly mixed‐function oxidations, often displaying highly regio‐ and/or stereospecific chemistry. In streptomycetes, they are predominantly associated with secondary metabolite biosynthetic pathways or with xenobiotic catabolism. Homologues of one family, CYP105, have been found in all Streptomyces species thus far sequenced. This review looks at the diverse biological functions of CYP105s and the biosynthetic/catabolic pathways they are associated with. Examples are presented showing a range of biotransformative abilities and different contexts. As biocatalysts capable of some remarkable chemistry, CYP105s have great biotechnological potential and merit detailed study. Recent developments in biotechnological applications which utilize CYP105s are described, alongside a brief overview of the benefits and drawbacks of using P450s in commercial applications. The role of CYP105s in vivo is in many cases undefined and provides a rich source for further investigation into the functions these enzymes fulfil and the metabolic pathways they participate in, in the natural environment.     

Using isolation‐by‐distance‐based approaches to assess the barrier effect of linear landscape elements on badger (Meles meles) dispersal

As the European badger (Meles meles) can be of conservation or management concern, it is important to have a good understanding of the species’ dispersal ability. In particular, knowledge of landscape elements that affect dispersal can contribute to devising effective management strategies. However, the standard approach of using Bayesian clustering methods to correlate genetic discontinuities with landscape elements cannot easily be applied to this problem, as badger populations are often characterized by a strong confounding isolation‐by‐distance (IBD) pattern. We therefore developed a two‐step method that compares the location of pairs of related badgers relative to a putative barrier and utilizes the expected spatial genetic structure characterized by IBD as a null model to test for the presence of a barrier. If a linear feature disrupts dispersal, the IBD pattern characterising pairs of individuals located on different sides of a putative barrier should differ significantly from the pattern obtained with pairs of individuals located on the same side. We used our new approach to assess the impact of rivers and roads of different sizes on badger dispersal in western England. We show that a large, wide river represented a barrier to badger dispersal and found evidence that a motorway may also restrict badger movement. Conversely, we did not find any evidence for small rivers and roads interfering with badger movement. One of the advantages of our approach is that potentially it can detect features that disrupt gene flow locally, without necessarily creating distinct identifiable genetic units.     

Considerations for the process development of insect‐derived antimicrobial peptide production

Antimicrobial peptides (AMPs) could evolve into new therapeutic lead molecules against multi‐resistant bacteria. As insects are a rich source of AMP, the identification and characterization of insect‐derived AMPs is particularly emphasized. One challenge of bringing these molecules into market, e.g., as a drug, is to develop a cost‐efficient large‐scale production process. Due to the fact that a direct AMP isolation from insects is not economical and that chemical synthesis is recommended for peptide sizes below 40 amino acids, a viable option is heterologous AMP production. Therefore, previous knowledge concerning the expression of larger proteins can be adapted, but due to the AMP nature (e.g., small size, bactericide) additional challenges have to be faced during up and downstream processing. Nonetheless the bottleneck for large‐scale AMP production is the same as for proteins; mainly the downstream process. This review introduces opportunities for insect‐derived AMP production, like the choice of the expression system (based on previously derived data), depending on the AMP nature, as well as new purification strategies like elastin‐like peptide/intein based purification strategies. All of these aspects are discussed with regard to large‐scale processes and costs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:1–11, 2015     

Intra‐articular injection of autologous adipose‐derived mesenchymal stem cells in the treatment of knee osteoarthritis

Background

Osteoarthritis (OA) is a chronic degenerative joint disease and is considered to be the fourth leading cause of disability and the second cause of inability to work in men. Recently, adipose‐derived mesenchymal stem cells (AD‐MSCs) came into focus for regenerative medicine as a promising tool for the treatment of OA. The administration of stem cells into impaired joints results in pain relief and improves quality of life, accompanied by restoration of hyaline articular cartilage.

Methods

In the present study, nine patients (including two patients with bilateral symptoms) diagnosed with osteoarthritis (International Knee Documentation grade B in 5 and grade D in six knees) were treated using a single injection of AD‐MSCs at a concentration of 0.5–1.0 × 10⁷ cells and were followed up for 18 months. During follow‐up, all the cases were evaluated clinically by Knee Society score (KSS), Hospital for Special Surgery knee score (HSS‐KS), Tegner–Lysholm (T–L) score and visual analogue scale (VAS) of pain, as well as by plain radiography and by magnetic resonance imaging visualization with 2D Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score assessment.

Results

Significant improvement of all four clinical scores was observed within the first 6 months (KSS for 41.4 points, HSS‐KS for 33.9 points, T–L score for 44.8 points, VAS of pain from 54.5 to 9.3) and improvement persisted throughout the rest of the follow‐up. MOCART score showed significant cartilage restoration (from 43 ± 7.2 to 63 ± 17.1), whereas radiography showed neither improvement, nor further joint degeneration.

Conclusions

The results obtained in the present study provide good basis for prospective randomized controlled clinical trials with respect to the use of AD‐MSCs in the treatment of osteoarthritis.     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.