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The study aimed to investigate the antibacterial activity, cytotoxicity, and mechanism of action of the non-ionic, cyclic lipopeptide, serrawettin W2-FL10 against Staphylococcus aureus . W2-FL10 exhibited potent activity against the Gram-positive bacteria S. aureus , Enterococcus faecalis , Enterococcus faecium , Listeria monocytogenes, and Bacillus subtilis , with minimum inhibitory concentration (MIC) values ranging from 6.3 to 31.3 μg/mL, while no activity was observed against Gram-negative bacteria. Broth microdilution assays showed that W2-FL10 interacted with key cell membrane components, such as lipid phosphatidyl glycerol and lipoteichoic acid of S. aureus . Upon membrane interaction, W2-FL10 dissipated membrane potential within 12 min and increased S. aureus membrane permeability within 28-40 min, albeit at slower rates and higher concentrations than the lytic peptide melittin. The observed membrane permeability, as detected with propidium iodide (PI), may be attributed to transmembrane pores/lesions, possibly dependent on dimer-driven lipopeptide oligomerization in the membrane. Scanning electron microscopy (SEM) imaging also visually confirmed the formation of lesions in the cell wall of one of the S. aureus strains, and cell damage within 1 h of exposure to W2-FL10, corroborating the rapid time-kill kinetics of the S. aureus strains. This bactericidal action against the S. aureus strains corresponded to membrane permeabilization by W2-FL10, indicating that self-promoted uptake into the cytosol may be part of the mode of action. Finally, this lipopeptide exhibited low to moderate cytotoxicity to the Chinese hamster ovarian (CHO) cell line in comparison to the control (emetine) with an optimal lipophilicity range (log D value of 2.5), signifying its potential as an antibiotic candidate., Importance: Antimicrobial resistance is a major public health concern, urgently requiring antibacterial compounds exhibiting low adverse health effects. In this study, a novel antibacterial lipopeptide analog is described, serrawettin W2-FL10 (derived from Serratia marcescens ), with potent activity displayed against Staphylococcus aureus . Mechanistic studies revealed that W2-FL10 targets the cell membrane of S. aureus , causing depolarization and permeabilization because of transmembrane lesions/pores, resulting in the leakage of intracellular components, possible cytosolic uptake of W2-FL10, and ultimately cell death. This study provides the first insight into the mode of action of a non-ionic lipopeptide. The low to moderate cytotoxicity of W2-FL10 also highlights its application as a promising therapeutic agent for the treatment of bacterial infections., Competing Interests: The authors declare no conflict of interest.

We report on the structure of Gramicidin S (GS) in a model membrane mimetic environment represented by the amphipathic solvent 1-octanol using one-dimensional (1D) and two-dimensional (2D) IR spectroscopy. To explore potential structural changes of GS, we also performed a series of spectroscopic measurements at differing temperatures. By analyzing the amide I band and using 2D-IR spectral changes, results could be associated to the disruption of aggregates/oligomers, as well as structural and conformational changes happening in the concentrated solution of GS. The ability of 2D-IR to enable differentiation in melting transitions of oligomerized GS structures is attributed to the sensitivity of the technique to vibrational coupling. Two melting transition temperatures were identified; at T m1 in the range 41-47 °C where the GS aggregates/oligomers disassemble and at T m2 = 57 ± 2 °C where there is significant change involving GS β-sheet-type hydrogen bonds, whereby it is proposed that there is loss of interpeptide hydrogen bonds and we are left with mainly intrapeptide β-sheet and β-turn hydrogen bonds of the smaller oligomers. Further analysis with quantum mechanical/molecular mechanics (QM/MM) simulations and second derivative results highlighted the participation of active GS side chains. Ultimately, this work contributes toward understanding the GS structure and the formulation of GS analogues with improved bioactivity.

Secondary metabolic profiling, using UPLC-MS E and molecular networking, revealed the secondary metabolites produced by Serratia marcescens NP10. The NP10 strain co-produced cyclic and open-ring stephensiolides (i.e., fatty acyl chain linked to Thr-Ser-Ser-Ile/Leu-Ile/Leu/Val) and glucosamine derivatives (i.e., fatty acyl chain linked to Val-glucose-butyric/oxo-hexanoic acid), with the structures of sixteen new stephensiolides (L-Y) and three new glucosamine derivatives (L-N) proposed. Genome mining identified sphA (stephensiolides) and gcd (glucosamine derivatives) gene clusters within Serratia genomes available on NBCI using antiSMASH, revealing specificity scores of the adenylation-domains within each module that corroborates MS E data. Of the nine RP-HPLC fractions, two stephensiolides and two glucosamine derivatives exhibited activity against Staphylococcus aureus (IC 50 of 25-79 µg/mL). 1 H NMR analysis confirmed the structure of the four active compounds as stephensiolide K, a novel analogue stephensiolide U, and glucosamine derivatives A and C. Stephensiolides K and U were found to cause membrane depolarisation and affect the membrane permeability of S. aureus, while glucosamine derivatives A and C primarily caused membrane depolarisation. New members of the stephensiolide and glucosamine derivative families were thus identified, and results obtained shed light on their antibacterial properties and mode of membrane activity., (© 2023. The Author(s).)

Tryptocidine C (TpcC), a Trp-rich cyclodecapeptide is a minor constituent in the antibiotic tyrothricin complex from Brevibacillus parabrevis. TpcC possesses a high tendency to oligomerise in aqueous solutions and dried TpcC forms distinct self-assembled nanoparticles. High-resolution scanning electron microscopy revealed the influence of different ethanol:water solvent systems on TpcC self-assembly, with the TpcC, dried from a high concentration in 15% ethanol, primarily assembling into small nanospheres with 24.3 nm diameter and 0.05 polydispersity. TpcC at 16 μM, near its CMC, formed a variety of structures such as small nanospheres, large dense nanospheroids and facetted 3-D-crystals, as well as sheets and coarse carpet-like structures which depended on ethanol concentration. Drying 16 μM TpcC from 75% ethanol resulted in highly facetted 3-D crystals, as well as small nanospheres, while those in 10% ethanol preparation had less defined facets. Drying from 20 to 50% ethanol led to polymorphic architectures with a few defined nanospheroids and various small nanoparticles, imbedded in carpet- and sheet-like structures. These polymorphic surface morphologies correlated with maintenance of fluorescence properties and the surface-derived antibacterial activity against Staphylococcus aureus over time, while there was a significant change in fluorescence and loss in activity in the 10% and 75% preparations where 3-D crystals were observed. This indicated that TpcC oligomerisation in solutions with 20-50% ethanol leads to metastable structures with a high propensity for release of antimicrobial moieties, while those leading to crystallisation limit active moieties release. TpcC nano-assemblies can find application in antimicrobial coatings, surface disinfectants, food packaging and wound healing materials., Competing Interests: Declaration of competing interest The authors declare that there is no reasonable conflict of interest and that this was an independent study without any interference, guidance, or regulation by commercial entities., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The ever-increasing global threat of common infections developing resistance to current therapeutics is rapidly accelerating the onset of a primitive post-antibiotic era in medicine. The prevention of further antimicrobial resistance development is unlikely due to the continued misuse of antibiotics, augmented by the lack of discovery of novel antibiotics. Screening large libraries of synthetic compounds have yet to offer effective replacements for current antibiotics. Due to historical successes, discovery from large and diverse natural sources and, more specifically, environmental bacteria, may still yield novel alternative antibiotics. However, the process of antibiotic discovery from natural sources is laborious and time-consuming as a result of outdated methodologies. Therefore, we have developed a simple and rapid preliminary screening assay to identify antibacterial-producing bacteria from natural sources. In brief, the assay utilizes the presence or absence of luminescence in bioluminescent reporter bacteria and test bacterium co-cultures in a 96-well plate format to determine the absence or presence of antibacterial compound production. Our assay, called the bioluminescent simultaneous antagonism (BSLA) assay, can accurately distinguish between known antibacterial-producing and non-producing test bacteria. The BSLA assay was validated by screening 264 unknown soil isolates which resulted in the identification of 10 antibacterial-producing isolates, effectively decreasing the pool of isolates for downstream analysis by 96%. By design, the assay is simple and requires only general laboratory equipment; however, we have shown that the assay can be scaled to automated high-throughput screening systems. Taken together, the BSLA assay allows for the rapid pre-screening of unknown bacterial isolates which, when coupled with innovative downstream dereplication and identification technologies, can effectively fast-track antimicrobial discovery.

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens -mediated transient tobacco plant ( Nicotiana benthamiana ) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log 2 , for at least four months before dropping to approximately 5-6 log 2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by Brevibacillus parabrevis , showed potent activity against chloroquine sensitive (CQS) Plasmodium falciparum . The identity of the aromatic residues in the aromatic dipeptide unit in cyclo-(D-Phe 1 -Pro 2 -(Phe 3 /Trp 3 )-D-Phe 4 /D-Trp 4 )-Asn 5 -Gln 6 -(Tyr 7 /Phe 7 /Trp 7 )-Val 8 -(Orn 9 /Lys 9 )-Leu 10 was proposed to have an important role in activity. CQS and resistant (CQR) P. falciparum strains were challenged with three representative cyclodecapeptides. Our results confirmed that cyclodecapeptides from tyrothricin had significantly higher antiplasmodial activity than the analogous gramicidin S, rivaling that of CQ. However, the previously hypothesized size and hydrophobicity dependent activity for these peptides did not hold true for P. falciparum strains, other than for the CQS 3D7 strain. The Tyr 7 in tyrocidine A (TrcA) with Phe 3 -D-Phe 4 seem to be related with loss in activity correlating with CQ antagonism and resistance, indicating a shared target and/or resistance mechanism in which the phenolic groups play a role. Phe 7 in phenycidine A, the second peptide containing Phe 3 -D-Phe 4 , also showed CQ antagonism. Conversely, Trp 7 in tryptocidine C (TpcC) with Trp 3 -D-Trp 4 showed improved peptide selectivity and activity towards the more resistant strains, without overt antagonism towards CQ. However, TpcC lead to similar parasite stage inhibition and parasite morphology changes than previously observed for TrcA. The disorganization of chromatin packing and neutral lipid structures, combined with amorphous hemozoin crystals, could account for halted growth in late trophozoite/early schizont stage and the nanomolar non-lytic activity of these peptides. These targets related to CQ antagonism, changes in neural lipid distribution, leading to hemozoin malformation, indicate that the tyrothricin cyclodecapeptides and CQ share a target in the malaria parasite. The differing activities of these cyclic peptides towards CQS and CQR P. falciparum strains could be due to variable target interaction in multiple modes of activity. This indicated that the cyclodecapeptide activity and parasite resistance response depended on the aromatic residues in positions 3, 4 and 7. This new insight on these natural cyclic decapeptides could also benefit the design of unique small peptidomimetics in which activity and resistance can be modulated.

A metabolomics/peptidomics and genomics approach, using UPLC-MS E , molecular networking, and genome mining, was used to describe the serrawettin W2 lipopeptide family produced by Serratia marcescens NP2. Seven known serrawettin W2 analogues were structurally elucidated along with 17 new analogues, which varied based on the first (fatty acyl length of C 8 , C 10 , C 12 , or C 12:1 ), fifth (Phe, Tyr, Trp, or Leu/Ile), and sixth (Leu, Ile, or Val) residues. Tandem MS results suggested that the previously classified serrawettin W3 may be an analogue of serrawettin W2, with a putative structure of cyclo(C 10 H 18 O 2 -Leu-Ser-Thr-Leu/Ile-Val). Chiral phase amino acid analysis enabled the distinction between l/d-Leu and l-Ile residues within nine purified compounds. 1 H and 13 C NMR analyses confirmed the structures of four purified new analogues. Additionally, genome mining was conducted using Serratia genome sequences available on the NCBI database to identify the swrA gene using the antiSMASH software. NRPSpredictor2 predicted the specificity score of the adenylation-domain within swrA with 100% for the first, second, and third modules (Leu-Ser-Thr), 60-70% for the fourth module (Phe/Trp/Tyr/Val), and 70% for the fifth module (Val/Leu/Ile), confirming MS E data. Finally, antibacterial activity was observed for compounds 6 and 11 against a clinical Enterococcus faecium strain.

Modified antimicrobial and antifouling materials and surfaces can be used to limit the propagation of microorganisms on various surfaces and minimise the occurrence of infection, transfer, and spoilage. Increased demand for 'green' solutions for material treatment has pushed the focus towards to naturally produced antimicrobials. Tyrocidines, cyclo-decapeptides naturally produced by a soil bacterium Brevibacillus parabrevis , have a broad spectrum of activity against Gram-positive and Gram-negative bacteria, filamentous fungi, and yeasts. Continual losses in tyrocidine production highlighted the possible association of peptides to surfaces. It was found in this study that tyrocidines readily associates with many materials, with a selectivity towards polysaccharide-type materials, such as cellulose. Peptide-treated cellulose was found to remain active after exposure to a broad pH range, various temperatures, salt solutions, water washes, and organic solvents, with the sterilising activity only affected by 1% SDS and 70% acetonitrile. Furthermore, a comparison to other antimicrobial peptides showed the association between tyrocidines and cellulose to be unique in terms of antimicrobial activity. The robust association between the tyrocidines and various materials holds great promise in applications focused on preventing surface contamination and creating self-sterilising materials.

Surface colonization by microorganisms, combined with the rise in antibiotic resistance, is the main cause of production failures in various industries. Self-sterilising materials are deemed the best prevention of surface colonization. However, current screening methods for these sterilising materials are laborious and time-consuming. The disk diffusion antimicrobial assay and the Japanese industrial standard method for antimicrobial activity on solid surfaces, JIS Z 2801, were compared to our modified solid surface antimicrobial assay in terms of detecting the activity of antibiotic-containing cellulose disks against four bacterial pathogens. Our novel assay circumvents the long incubation times by utilising the metabolic active dye, resazurin, to shorten the time in which antibacterial results are obtained to less than 4 h. This assay allows for increased screening to identify novel sterilising materials for combatting surface colonisation.•Disk diffusion assay could only detect the activity of small compounds that leached from the material over 20-24 h.•JIS Z 2801 was also able to detect the surface activity of non-polar compounds, thought to be inactive based on the disk diffusion results.•The resazurin solid surface antimicrobial assay could obtain the same results as the JIS Z 2801, within a shorter time and in a high-throughput 96-well plate setup., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

Candida species are highly adaptable to environmental changes with their phenotypic flexibility allowing for the evasion of most host defence mechanisms. Moreover, increasing resistance of human pathogenic Candida strains has been reported against all four classes of available antifungal drugs, which highlights the need for combinational therapies. Tyrocidines are cyclic antimicrobial peptides that have shown synergistic activity with antifungal drugs such as caspofungin and amphotericin B. However, these cyclodecapeptides have haemolytic activity and cytotoxicity, but they have been used for decades in the clinic for topical applications. The tyrocidines tend to form higher-order structures in aqueous solutions and excessive aggregation can result in variable or diminished activity. Previous studies have shown that the tyrocidines prefer ordered association to celluloses. Therefore, a formulation with soluble cellulose was used to control the oligomer stability and size, thereby increasing the activity against Candida spp. Of the formulants tested, it was found that commercial hydroxy-propyl-methyl cellulose, E10M, yielded the best results with increased stability, increased anti- Candida activity, and improved selectivity. This formulation holds promise in topical applications against Candida spp. infections.

Species from the genus Xenorhabdus, endosymbiotic bacteria of Steinernema nematodes, produce several antibacterial and antifungal compounds, some of which are anti-parasitic. In this study, we report on the effect growth conditions have on the production of antimicrobial compounds produced by Xenorhabdus khoisanae J194. The strain was cultured in aerated and non-aerated broth, respectively, and on solid media. Production of antimicrobial compounds was detected after 24 h of growth in liquid media, with highest levels recorded after 96 h. Highest antimicrobial activity was obtained from cells cultured on solid media. By using ultraperformance liquid chromatography linked to mass spectrometry and HPLC, a plethora of known Xenorhabdus compounds were identified. These compounds are the PAX lipopeptides (PAX 1', PAX 3', PAX 5, and PAX 7E), xenocoumacins and xenoamicins. Differences observed in the MS-MS fractionation patterns collected in this study, when compared to previous studies indicated that this strain produces novel xenoamicins. Three novel antimicrobial compounds, khoicin, xenopep and rhabdin, were identified and structurally characterized based on MS-MS fractionation patterns, amino acid analysis and whole genome analysis. The various compounds produced under the three different conditions indicates that the secondary metabolism of X. khoisanae J194 may be regulated by oxygen, water activity or both. Based on these findings X. khoisanae J194 produce a variety of antimicrobial compounds that may have application in disease control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Booysen, Rautenbach, Stander and Dicks.)

An integrated approach that combines reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry, untargeted ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS E ) and molecular networking (using the Global Natural Products Social molecular network platform) was used to elucidate the metabolic profiles and chemical structures of the secondary metabolites produced by pigmented (P1) and non-pigmented (NP1) Serratia marcescens ( S. marcescens ) strains. Tandem mass spectrometry-based molecular networking guided the structural elucidation of 18 compounds for the P1 strain (including 6 serratamolides, 10 glucosamine derivatives, prodigiosin and serratiochelin A) and 15 compounds for the NP1 strain (including 8 serratamolides, 6 glucosamine derivatives and serratiochelin A) using the MS E fragmentation profiles. The serratamolide homologues were comprised of a peptide moiety of two L-serine residues (cyclic or open-ring) linked to two fatty acid chains (lengths of C 10 , C 12 , or C 12:1 ). Moreover, the putative structure of a novel open-ring serratamolide homologue was described. The glucosamine derivative homologues (i.e., N -butylglucosamine ester derivatives) consisted of four residues, including glucose/hexose, valine, a fatty acid chain (lengths of C 13 - C 17 and varying from saturated to unsaturated) and butyric acid. The putative structures of seven novel glucosamine derivative homologues and one glucosamine derivative congener (containing an oxo-hexanoic acid residue instead of a butyric acid residue) were described. Moreover, seven fractions collected during RP-HPLC, with major molecular ions corresponding to prodigiosin, serratamolides (A, B, and C), and glucosamine derivatives (A, C, and E), displayed antimicrobial activity against a clinical Enterococcus faecalis S1 strain using the disc diffusion assay. The minimum inhibitory and bactericidal concentration assays however, revealed that prodigiosin exhibited the greatest antimicrobial potency, followed by glucosamine derivative A and then the serratamolides (A, B, and C). These results provide crucial insight into the secondary metabolic profiles of pigmented and non-pigmented S. marcescens strains and confirms that S. marcescens strains are a promising natural source of novel antimicrobial metabolites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Clements, Rautenbach, Ndlovu, Khan and Khan.)

Tryptocidine C (TpcC, cyclo[D-Phe 1 -Pro 2 -Trp 3 -D-Trp 4 -Asn 5 -Gln 6 -Trp 7 -Val 8 -Orn 9 -Leu 10 ]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp 7 replaced by Tyr 7 ). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 μM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 μM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

The concepts of polymer-peptide conjugation and self-assembly were applied to antimicrobial peptides (AMPs) in the development of a targeted antimalaria drug delivery construct. This study describes the synthesis of α-acetal, ω-xanthate heterotelechelic poly( N -vinylpyrrolidone) (PVP) via reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization, followed by postpolymerization deprotection to yield α-aldehyde, ω-thiol heterotelechelic PVP. A specific targeting peptide, GSRSKGT, for Plasmodium falciparum -infected erythrocytes was used to sparsely decorate the α-chain ends via reductive amination while cyclic decapeptides from the tyrocidine group were conjugated to the ω-chain end via thiol-ene Michael addition. The resultant constructs were self-assembled into micellar nanoaggregates whose sizes and morphologies were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro activity and selectivity of the conjugates were evaluated against intraerythrocytic P. falciparum parasites.

The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe 1 -L-Pro 2 -L-(Phe 3 /Trp 3 )-D-(Phe 4 /Trp 4 )-L-Asn 5 -L-Gln 6 -L-(Tyr 7 /Phe 7 /Trp 7 )-L-Val 8 -L-(Orn 9 /Lys 9 )-L-Leu 10 ], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A "peptidomics" approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe 3 -D-Phe 4 aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp 3 -D-Trp 4 . For the other variable aromatic residue position, Tyr 7 was preferentially incorporated above Trp 7 , with a minor incorporation of Phe 7 over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn 9 to Lys 9 incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery., Competing Interests: Declaration of competing interest The authors declare they have no conflict of interest in the publication of this research., (Copyright © 2020. Published by Elsevier B.V.)

The authors would like to correct the errors in the publication of the original article.

Bacteria isolated from different segments of the gastro-intestinal tract (GIT) of healthy free-range broilers were screened for probiotic properties. Six strains were selected and identified as Lactobacillus gallinarum, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus crispatus, Enterococcus faecalis and Bacillus amyloliquefaciens based on 16S rRNA, gyrB and recA gene sequence analyses. All six strains produced exopolysaccharides (EPS) and formed biofilms under conditions simulating the broiler GIT. Lactobacillus johnsonii DPN184 and L. salivarius DPN181 produced hydrogen peroxide, and L. crispatus DPN167 and E. faecalis DPN94 produced bile salt hydrolase (BSH) and phytase. Bacillus amyloliquefaciens DPN123 produced phytase, amylase, surfactin and iturin A1. No abnormalities were observed when broilers were fed the multi-strain combination, suggesting that it could be used as a probiotic.

Tyrocidines are a family of cyclic decapeptides produced by the soil bacterium, Brevibacillus parabrevis. These antibiotic peptides can be used to prevent infections in agriculture and food industry but also to prepare antimicrobial lozenges, creams, and dressings for medical applications. It has been observed that the tyrocidines interact with saccharides such as cellulose from their soil environment, as well as sugars in culture media and glycans in fungal cell walls. Here, we investigated the interactions of tyrocidines with glucose, sucrose, and cellotetraose (as cellulose model) in a quantitative fashion utilising CD and NMR spectroscopy. The CD and NMR spectra of tyrocidine A (TrcA) were analysed as a function of solvent composition, and the spectral properties agree with the formation of oligomeric structures that are governed by β-sheet secondary structures once the acetonitrile content of the solvent is increased. Saccharides seem to also induce TrcA spectral changes reverting those induced by organic solvents. The CD spectral changes of TrcA in the presence of glucose agree with new ordered H-bonding, possibly β-sheet structures. The amides involved in intramolecular H-bonding remained largely unaffected by the environmental changes. In contrast, amides exposed to the exterior and/or involved in TrcA intermolecular association show the largest 1 H chemical shift changes. CD and NMR spectroscopic investigations correlated well with TrcA-glucose interactions characterized by a dissociation constant around 200 μM. Interestingly, the association of cellotetraose corresponds closely to the additive effect from four glucose moieties, while a much higher dissociation constant was observed for sucrose. Similar trends to TrcA for binding to the three saccharides were observed for the analogous tyrocidines, tyrocidine B, and tyrocidine C. These results therefore indicate that the tyrocidine interactions with the glucose monosaccharide unit are fairly specific and reversible., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent. IMPORTANCE Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them., (Copyright © 2018 Wenzel et al.)

Biosurfactants are unique secondary metabolites, synthesised non-ribosomally by certain bacteria, fungi and yeast, with their most promising applications as antimicrobial agents and surfactants in the medical and food industries. Naturally produced glycolipids and lipopeptides are found as a mixture of congeners, which increases their antimicrobial potency. Sensitive analysis techniques, such as liquid chromatography coupled to mass spectrometry, enable the fingerprinting of different biosurfactant congeners within a naturally produced crude extract. Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, isolated from wastewater, were screened for biosurfactant production. Biosurfactant compounds were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Results indicated that B. amyloliquefaciens ST34 produced C 13-16 surfactin analogues and their identity were confirmed by high resolution ESI-MS and UPLC-MS. In the crude extract obtained from P. aeruginosa ST5, high resolution ESI-MS linked to UPLC-MS confirmed the presence of di- and monorhamnolipid congeners, specifically Rha-Rha-C 10 -C 10 and Rha-C 10 -C 10 , Rha-Rha-C 8 -C 10 /Rha-Rha-C 10 -C 8 and Rha-C 8 -C 10 /Rha-C 10 -C 8 , as well as Rha-Rha-C 12 -C 10 /Rha-Rha-C 10 -C 12 and Rha-C 12 -C 10 /Rha-C 10 -C 12 . The crude surfactin and rhamnolipid extracts also retained pronounced antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains and the pathogenic yeast Candida albicans. In addition, the rapid solvent extraction combined with UPLC-MS of the crude samples is a simple and powerful technique to provide fast, sensitive and highly specific data on the characterisation of biosurfactant compounds.

The quantitative and qualitative effect of water immiscible and miscible carbon-rich substrates on the production of biosurfactants, surfactin and rhamnolipids, by Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, respectively, was analysed. A small-scale high throughput 96 deep-well micro-culture method was utilised to cultivate the two strains in mineral salt medium (MSM) supplemented with the water miscible (glucose, glycerol, fructose and sucrose) and water immiscible carbon sources (diesel, kerosene and sunflower oil) under the same growth conditions. The biosurfactants produced by the two strains were isolated by acid precipitation followed by an organic solvent extraction. Ultra-performance liquid chromatography coupled to electrospray ionisation mass spectrometry was utilised to analyse yields and characterise the biosurfactant variants. For B. amyloliquefaciens ST34, maximum surfactin production was observed in the MSM supplemented with fructose (28 mg L -1 ). In addition, four surfactin analogues were produced by ST34 using the different substrates, however, the C 13 -C 15 surfactins were dominant in all extracts. For P. aeruginosa ST5, maximum rhamnolipid production was observed in the MSM supplemented with glucose (307 mg L -1 ). In addition, six rhamnolipid congeners were produced by ST5 using different substrates, however, Rha-Rha-C 10 -C 10 and Rha-C 10 -C 10 were the most abundant in all extracts. This study highlights that the carbon sources utilised influences the yield and analogues/congeners of surfactin and rhamnolipids produced by B. amyloliquefaciens and P. aeruginosa, respectively. Additionally, glucose and fructose were suitable substrates for rhamnolipid and surfactin, produced by P. aeruginosa ST5 and B. amyloliquefaciens ST34, which can be exploited for bioremediation or as antimicrobial agents.

It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~Srf↔GS+Srf correlated to apparent K d values of 6-9 μM in gas-phase and 1 μM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low μM K d app values of GS~Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na + nor Ca 2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca 2+ and Na + from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. Graphical Abstract ᅟ.

Most antifungal peptides (AFPs), if not all, have membrane activity, while some also have alternative targets. Fungal membranes share many characteristics with mammalian membranes with only a few differences, such as differences in sphingolipids, phosphatidylinositol (PI) content and the main sterol is ergosterol. Fungal membranes are also more negative and a better target for cationic AFPs. Targeting just the fungal membrane lipids such as phosphatidylinositol and/or ergosterol by AFPs often translates into mammalian cell toxicity. Conversely, a specific AFP target in the fungal pathogen, such as glucosylceramide, mannosyldiinositol phosphorylceramide or a fungal protein target translates into high pathogen selectivity. However, a lower target concentration, absence or change in the specific fungal target can naturally lead to resistance, although such resistance in turn could result in reduced pathogen virulence. The question is then to be or not to be membrane active - what is the best choice for a successful AFP? In this review we deliberate on this question by focusing on the recent advances in our knowledge on how natural AFPs target fungi., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The tyrocidines and analogues are cyclic decapeptides produced by Brevibacillus parabrevis with a conserved sequence of cyclo(D-Phe 1 -Pro 2 -X 3 -x 4 -Asn 5 -Gln 6 -X 7 -Val 8 -X 9 -Leu 10 ) with Trp 3,4 /Phe 3,4 in the aromatic dipeptide unit, Lys 9 /Orn 9 as their cationic residue and Tyr (tyrocidines), Trp (tryptocidines) or Phe (phenicidines) in position 7. Previous studies indicated they have a broad antifungal spectrum with the peptides containing a Tyr residue in position 7 being more active than those with a Phe or Trp residue in this position. Detailed analysis of antifungal inhibition parameters revealed that Phe 3 -D-Phe 4 in the aromatic dipeptide unit lead to more consistent activity against the three filamentous fungi in this study. These peptides exhibited high membrane activity and fast leakage kinetics against model membranes emulating fungal membranes, with selectivity towards ergosterol containing membranes. More fluid membranes and doping of liposomes with the sphingolipid, glucosylceramide, led to a decreased permeabilising activity. Peptide-induced uptake of membrane impermeable dyes was observed in hyphae of both Fusarium solani and Botrytis cinerea, with uptake more pronounced at the hyphal growth tips that are known to contain ergosterol-sphigolipid rich lipid rafts. Tyrocidine interaction with these rafts may lead to the previously observed fungal hyperbranching. However, the leakage of model membranes and Bot. cinerea did not correlate directly with the antifungal inhibition parameters, indicating another target or mode of action. Proteinase K treatment of target fungi had a minimal influence or even improved the tyrocidine activity, ruling out a mannoprotein target in the fungal cell wall. β-glucanase treatment of Bot. cinerea did not significantly affect the tyrocidine activity, but there was a significant loss in activity towards the β-glucanase treated F. solani. This study showed the tyrocidine antifungal membrane activity is selective towards ergosterol and possibly lipid rafts, but also point to additional targets such as the cell wall β-glucans that could modulate their activity., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Unlabelled: The enzymes in the Embden-Meyerhof-Parnas pathway of Plasmodium falciparum trophozoites were kinetically characterized and their integrated activities analyzed in a mathematical model. For validation of the model, we compared model predictions for steady-state fluxes and metabolite concentrations of the hexose phosphates with experimental values for intact parasites. The model, which is completely based on kinetic parameters that were measured for the individual enzymes, gives an accurate prediction of the steady-state fluxes and intermediate concentrations. This is the first detailed kinetic model for glucose metabolism in P. falciparum, one of the most prolific malaria-causing protozoa, and the high predictive power of the model makes it a strong tool for future drug target identification studies. The modelling workflow is transparent and reproducible, and completely documented in the SEEK platform, where all experimental data and model files are available for download., Database: The mathematical models described in the present study have been submitted to the JWS Online Cellular Systems Modelling Database (http://jjj.bio.vu.nl/database/penkler). The investigation and complete experimental data set is available on SEEK (10.15490/seek.1., Investigation: 56)., (© 2015 FEBS.)