RESUMO
Nowadays, not only biologists, but also researchers from other disciplines such as chemistry, pharmacy, material sciences, or physics are working with antimicrobial peptides. This review is written for researchers and students working in or interested in the field of antimicrobial peptides - and especially those who do not have a profound biological background. To lay the ground for a thorough discussion on how AMPs act on cells, the architectures of mammalian and bacterial cell envelopes are described in detail because they are important targets of AMPs and provide the basis for their selectivity. The modes of action of α-helical AMPs (αAMPs) are not limited to different models of membrane permeabilization, but also include the disruption of intracellular processes, as well as the formation of fibrillary structures and their potential implications for antimicrobial activity. As biofilm-related infections are very difficult to treat with conventional antibiotics, they pose a major problem in the clinic. Therefore, this review also discusses the biological background of biofilm infections and the mode of actions of αAMPs against biofilms. The last chapter focusses on the design of αAMPs by providing an overview of historic milestones in αAMP design. It describes how modern αAMP design is aiming to produce peptides suitable to be applied in the clinic. Hence, the article concludes with a section on translational research discussing the prospects of αAMPs and remaining challenges on their way into the clinic.
RESUMO
α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/efeitos dos fármacos , Quimotripsina/química , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/metabolismo , Testes de Sensibilidade Microbiana , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Proteólise , Tripsina/químicaRESUMO
Forage maize is often infected by mycotoxin-producing Fusarium fungi during plant growth, which represent a serious health risk to exposed animals. Deoxynivalenol (DON) and zearalenone (ZEN) are among the most important Fusarium mycotoxins, but little is known about the occurrence of their modified forms in forage maize. To assess the mycotoxin contamination in Northern Germany, 120 natural contaminated forage maize samples of four cultivars from several locations were analysed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) for DON and ZEN and their modified forms deoxynivalenol-3-glucoside (DON3G), the sum of 3- and 15-acetyl-deoxynivalenol (3+15-AcDON), α- and ß-zearalenol (α-ZEL, ß-ZEL). DON and ZEN occurred with high incidences (100 and 96%) and a wide range of concentrations, reaching levels up to 10,972 and 3910 µg/kg, respectively. Almost half of the samples (46%) exceeded the guidance value in complementary and complete feeding stuffs for ZEN (500 µg/kg), and 9% for DON (5000 µg/kg). The DON related mycotoxins DON3G and 3+15-AcDON were also present in almost all samples (100 and 97%) with amounts of up to 3038 and 2237 µg/kg and a wide range of concentrations. For the ZEN metabolites α- and ß-ZEL lower incidences were detected (59 and 32%) with concentrations of up to 423 and 203 µg/kg, respectively. Forage maize samples were contaminated with at least three co-occurring mycotoxins, whereby 95% of all samples contained four or more mycotoxins with DON, DON3G, 3+15-AcDON, and ZEN co-occurring in 93%, together with α-ZEL in 57% of all samples. Positive correlations were established between concentrations of the co-occurring mycotoxins, especially between DON and its modified forms. Averaged over all samples, ratios of DON3G/DON and 3+15-AcDON/DON were similar, 20.2 and 20.5 mol%; cultivar-specific mean ratios ranged from 14.6 to 24.3 mol% and 15.8 to 24.0 mol%, respectively. In total, 40.7 mol% of the measured DON concentration was present in the modified forms DON3G and 3+15-AcDON. The α-ZEL/ZEN ratio was 6.2 mol%, ranging from 5.2 to 8.6 mol% between cultivars. These results demonstrate that modified mycotoxins contribute substantially to the overall mycotoxin contamination in forage maize. To avoid a considerable underestimation, it is necessary to analyse modified mycotoxins in future mycotoxin monitoring programs together with their parent forms.
Assuntos
Fusarium/metabolismo , Tricotecenos/análise , Zea mays/microbiologia , Zearalenona/análise , Ração Animal/microbiologia , Biotransformação , Cromatografia Líquida , Microbiologia de Alimentos , Alemanha , Glucosídeos/análise , Espectrometria de Massas , Medição de Risco , Tricotecenos/toxicidade , Zea mays/crescimento & desenvolvimento , Zearalenona/toxicidade , Zeranol/análogos & derivados , Zeranol/análiseRESUMO
Diazocines are characterized by extraordinary photochemical properties rendering them of particular interest for switching the conformation of biomolecules with visible light. Current developments afford synthetic access to unprecedented diazocine derivatives promising particular opportunities in photocontrol of proteins and biological systems. In this work, the well-established approach of photocontrolling the secondary structure of α-helices was exploited using a diazocine to reversibly fold and unfold the tertiary structure of a small protein. The protein of choice was the globulary folded Trp-cage, a widely used model system for the elucidation of protein folding pathways. A specifically designed, short and rigid dicarboxy-functionalized diazocine-based cross-linker was attached to two solvent-exposed side chains at the α-helix of the miniprotein through the use of a primary amine-selective active ester. This cross-linking strategy is orthogonal to the common cysteine-based chemistry. The cross-linked Trp-cage was successfully photoisomerized and exhibited a strong correlation between protein fold and diazocine isomeric state. As determined by NMR spectroscopy, the cis-isomer stabilized the fold, while the trans-isomer led to complete protein unfolding. The successful switching of the protein fold in principle demonstrates the ability to control protein function, as the activity depends on their structural integrity.
Assuntos
Reagentes de Ligações Cruzadas/química , Luz , Dobramento de Proteína/efeitos da radiação , Isomerismo , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
Fusarium mycotoxins and their derivatives are frequently detected in freshly harvested forage maize. This study assessed the time course effects during ensiling of forage maize on the fate of Fusarium mycotoxins, using laboratory-scale silos and artificially contaminated raw material. A multi-mycotoxin liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was used to determine the levels of deoxynivalenol (DON), zearalenone (ZEN) and their derivatives DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, deepoxy-DON, α-zearalenol and ß-zearalenol. A significant increase of DON was observed during ensiling, whereas the levels of DON-3-glucoside and its acetylated forms proportionally decreased. In contrast, levels of ZEN, α-zearalenol and ß-zearalenol were not affected by the ensiling process. Based on these findings, ensiling is not a practical method for reducing the total amount of Fusarium mycotoxins present at harvest.
Assuntos
Fusarium/química , Silagem/análise , Tricotecenos/análise , Zea mays/metabolismo , Zearalenona/análise , Cromatografia Líquida , Silagem/microbiologia , Espectrometria de Massas em Tandem , Zea mays/microbiologiaRESUMO
The selective and sensitive analysis of mycotoxins in highly complex feed matrices is a great challenge. In this study, the suitability of OrbitrapTM-based high-resolution mass spectrometry (HRMS) for routine mycotoxin analysis in complex feeds was demonstrated by the successful validation of a full MS/data-dependent MS/MS acquisition method for the quantitative determination of eight Fusarium mycotoxins in forage maize and maize silage according to the Commission Decision 2002/657/EC. The required resolving power for accurate mass assignments (<5 ppm) was determined as 35,000 full width at half maximum (FWHM) and 70,000 FWHM for forage maize and maize silage, respectively. The recovery (RA), intra-day precision (RSDr), and inter-day precision (RSDR) of measurements were in the range of 94 to 108%, 2 to 16%, and 2 to 12%, whereas the decision limit (CCα) and the detection capability (CCß) varied from 11 to 88 µg/kg and 20 to 141 µg/kg, respectively. A set of naturally contaminated forage maize and maize silage samples collected in northern Germany in 2017 was analyzed to confirm the applicability of the HRMS method to real samples. At least four Fusarium mycotoxins were quantified in each sample, highlighting the frequent co-occurrence of mycotoxins in feed.
Assuntos
Contaminação de Alimentos/análise , Micotoxinas/análise , Silagem/análise , Zea mays/química , Cromatografia Líquida , Espectrometria de Massas/métodosRESUMO
As Streptomyces have shown an outstanding capacity for drug production, different campaigns in geographically distant locations currently aim to isolate new antibiotic producers. However, many of these newly isolated Streptomyces strains are classified as identical to already described species. Nevertheless, as discrepancies in terms of secondary metabolites and morphology are possible, we compared two Streptomyces strains with identical 16S rRNA gene sequences but geographically distant origins. Chosen were an Easter Island Streptomyces isolate (Streptomyces sp. SN25_8.1) and the next related type strain, which is Streptomyces griseus subsp. griseus DSM 40236T isolated from Russian garden soil. Compared traits included phylogenetic relatedness based on 16S rRNA gene sequences, macro and microscopic morphology, antibiotic activity and secondary metabolite profiles. Both Streptomyces strains shared several common features, such as morphology and core secondary metabolite production. They revealed differences in pigmentation and in the production of accessory secondary metabolites which appear to be strain-specific. In conclusion, despite identical 16S rRNA classification Streptomyces strains can present different secondary metabolite profiles and may well be valuable for consideration in processes for drug discovery.
RESUMO
The presence of two known anthraquinones, Lupinacidin A and Galvaquinone B, which have antitumor activity, has been identified in the sea anemone (Gyractis sesere) from Easter Island. So far, these anthraquinones have been characterized from terrestrial and marine Actinobacteria only. In order to identify the anthraquinones producer, we isolated Actinobacteria associated with the sea anemone and obtained representatives of seven actinobacterial genera. Studies of cultures of these bacteria by HPLC, NMR, and HRLCMS analyses showed that the producer of Lupinacidin A and Galvaquinone B indeed was one of the isolated Actinobacteria. The producer strain, SN26_14.1, was identified as a representative of the genus Verrucosispora. Genome analysis supported the biosynthetic potential to the production of these compounds by this strain. This study adds Verrucosispora as a new genus to the anthraquinone producers, in addition to well-known species of Streptomyces and Micromonospora. By a cultivation-based approach, the responsibility of symbionts of a marine invertebrate for the production of complex natural products found within the animal's extracts could be demonstrated. This finding re-opens the debate about the producers of secondary metabolites in sea animals. Finally, it provides valuable information about the chemistry of bacteria harbored in the geographically-isolated and almost unstudied, Easter Island.
Assuntos
Actinobacteria/metabolismo , Antraquinonas/isolamento & purificação , Antibióticos Antineoplásicos/isolamento & purificação , Anêmonas-do-Mar/microbiologia , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Animais , Antraquinonas/metabolismo , Antibióticos Antineoplásicos/metabolismo , Genoma Bacteriano/genética , Polinésia , Anêmonas-do-Mar/metabolismo , SimbioseRESUMO
Mechanochemical cycloreversion of cyclobutane is known from ultrasound experiments. It is, however, not clear which forces are required to induce the cycloreversion. In atomic force microscopy (AFM) experiments, on the other hand, it is notoriously difficult to assign the ruptured bond. We have solved this problem through the synthesis of tailored macrocycles, in which the cyclobutane mechanophore is bypassed by an ethylene glycol chain of specific length. This macrocycle is covalently anchored between a glass substrate and an AFM cantilever by polyethylene glycol linkers. Upon mechanical stretching of the macrocycle, cycloreversion occurs, which is identified by a defined length increase of the stretched polymer. The measured length change agrees with the value calculated with the external force explicitly included (EFEI) method. By using two different lengths for the ethylene glycol safety line, the assignment becomes unambiguous. Mechanochemical cycloreversion of cyclobutane is observed at forces above 1.7â nN.