780 research outputs found

    Rombocin, a Short Stable Natural Nisin Variant, Displays Selective Antimicrobial Activity against Listeria monocytogenes and Employs a Dual Mode of Action to Kill Target Bacterial Strains

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    Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31–35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes

    Cesin, a short natural variant of nisin, displays potent antimicrobial activity against major pathogens despite lacking two C-terminal macrocycles

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    Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound. </p

    Lipidated variants of the antimicrobial peptide nisin produced via incorporation of methionine analogs for click chemistry show improved bioactivity

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    The increase in antibiotic resistance calls for accelerated molecular engineering strategies to diversify natural products for drug discovery. Incorporation of non-canonical amino acids (ncAAs) is an elegant strategy for this purpose, offering a diverse pool of building blocks to introduce desired properties into antimicrobial lanthipeptides. We here report an expression system using Lactococcus lactis as host, for non-canonical amino acid incorporation with high efficiency and yield. We show that incorporating the more hydrophobic analog ethionine (instead of methionine) into nisin improves its bioactivity against several Gram-positive strains we tested. New-to-nature variants were further created by click chemistry. By azidohomoalanine (Aha) incorporation and subsequent click chemistry, we obtained lipidated variants at different positions in nisin or in truncated nisin variants. Some of them show improved bioactivity and specificity against several pathogenic bacterial strains. These results highlight the ability of this methodology for lanthipeptide multi-site lipidation, to create new-to-nature antimicrobial products with diverse features, and extending the toolbox for (lanthi)peptide drug improvement and discovery.</p

    Synthesis and Antimicrobial Activity of Aminoalkyl Resveratrol Derivatives Inspired by Cationic Peptides

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    Antimicrobial resistance is a global concern, far from being resolved. The need of new drugs against new targets is imminent. In this work, we present a family of aminoalkyl resveratrol derivatives with antibacterial activity inspired by the properties of cationic amphipathic antimicrobial peptides. Surprisingly, the newly designed molecules display modest activity against aerobically growing bacteria but show surprisingly good antimicrobial activity against anaerobic bacteria (Gram-negative and Gram-positive) suggesting specificity towards this bacterial group. Preliminary studies into the action mechanism suggest that activity takes place at the membrane level, while no cross-resistance with traditional antibiotics is observed. Actually, some good synergistic relations with existing antibiotics were found against Gram-negative pathogens. However, some cytotoxicity was observed, despite their low haemolytic activity. Our results show the importance of the balance between positively charged moieties and hydrophobicity to improve antimicrobial activity, setting the stage for the design of new drugs based on these molecules.Junta de Andalucía FQM-7316Dutch Research Council NWA.1228.191.006National Institute of Allergy and Infectious Diseases CL03T12C3

    Cell-to-cell non-conjugative plasmid transfer between Bacillus subtilis and lactic acid bacteria

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    Bacillus subtilis is a soil-dwelling bacterium that can interact with a plethora of other microorganisms in its natural habitat. Due to the versatile interactions and its ability to form nanotubes, i.e., recently described membrane structures that trade cytoplasmic content between neighbouring cells, we investigated the potential of HGT from B. subtilis to industrially-relevant members of lactic acid bacteria (LAB). To explore the interspecies HGT events, we developed a co-culturing protocol and provided proof of transfer of a small high copy non-conjugative plasmid from B. subtilis to LABs. Interestingly, the plasmid transfer did not involve conjugation nor activation of the competent state by B. subtilis. Moreover, our study shows for the first time non-conjugative cell-to-cell intraspecies plasmid transfer for non-competent Lactococcus lactis sp. cremoris strains. Our study indicates that cell-to-cell transformation is a ubiquitous form of HGT and can be potentially utilized as an alternative tool for natural (non-GMO) strain improvement

    Emulating nonribosomal peptides with ribosomal biosynthetic strategies

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    Peptide natural products are important lead structures for human drugs and many nonribosomal peptides possess antibiotic activity. This makes them interesting targets for engineering approaches to generate peptide analogues with, for example, increased bioactivities. Nonribosomal peptides are produced by huge mega-enzyme complexes in an assembly-line like manner, and hence, these biosynthetic pathways are challenging to engineer. In the past decade, more and more structural features thought to be unique to nonribosomal peptides were found in ribosomally synthesised and posttranslationally modified peptides as well. These streamlined ribosomal pathways with modifying enzymes that are often promiscuous and with gene-encoded precursor proteins that can be modified easily, offer several advantages to produce designer peptides. This review aims to provide an overview of recent progress in this emerging research area by comparing structural features common to both nonribosomal and ribosomally synthesised and posttranslationally modified peptides in the first part and highlighting synthetic biology strategies for emulating nonribosomal peptides by ribosomal pathway engineering in the second part.ISSN:2633-067

    Mutational Studies of the Mersacidin Leader Reveal the Function of Its Unique Two-Step Leader Processing Mechanism

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    The class II lanthipeptide mersacidin, a ribosomally synthesized and post-translationally modified peptide (RiPP), displays unique intramolecular structures, including a very small lanthionine ring. When applied in the growing field of RiPP engineering, these can add unique features to new-to-nature compounds with novel properties. Recently, a heterologous expression system for mersacidin in Escherichia coli was developed to add its modification enzymes to the RiPP engineering toolbox and further explore mersacidin biosynthesis and leader-processing. The dedicated mersacidin transporter and leader protease MrsT was shown to cleave the leader peptide only partially upon export, transporting GDMEAA-mersacidin out of the cell. The extracellular Bacillus amyloliquefaciens protease AprE was shown to release active mersacidin in a second leader-processing step after transport. The conserved LanT cleavage site in the mersacidin leader is present in many other class II lanthipeptides. In contrast to mersacidin, the leader of these peptides is fully processed in one step. This difference with mersacidin leader-processing raises fundamentally interesting questions about the specifics of mersacidin modification and processing, which is also crucial for its application in RiPP engineering. Here, mutational studies of the mersacidin leader-core interface were performed to answer these questions. Results showed the GDMEAA sequence is crucial for both mersacidin modification and leader processing, revealing a unique leader layout in which a LanM recognition site is positioned downstream of the conserved leader-protease LanT cleavage site. Moreover, by identifying residues and regions that are crucial for mersacidin-type modifications, the wider application of mersacidin modifications in RiPP engineering has been enabled

    Analysis of cross-functionality within LanBTC synthetase complexes from different bacterial sources with respect to production of fully modified lanthipeptides

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    Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), a biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven very low. Here, the possibility of the cross-functionality by LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis. Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis, but not in B. subtilis. The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis. In contrast, such a complex was not formed in B. subtilis. Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pull-down assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications, and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro, independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis, the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which secretion of premature peptide is prevented
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