In vitro performance and in vivo fertility of antibiotic-free preserved boar semen stored at 5 °C

Background: Hypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility. Objectives: To examine chilling injury and the number and type of bacteria in boar semen stored at 5 °C in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions. Material and methods: Boar ejaculates were extended with AndroStar® Premium, stored at 17 °C with and at 5 °C without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study. Results: Lethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher (P 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 °C and semen stored at 17 °C with antibiotics. Conclusion: Despite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable

Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology

Untersuchung des Wirkmechanismus eines synthetischen zyklischen antimikrobiellen Peptids

Antimicrobial peptides represent an ancient class of molecules which is found throughout the animal and plant kingdom. In higher organisms they are part of the innate immune system and serve as first line defence against infectious pathogens. In contrast to conventional antibiotics, resistance development against antimicrobial peptides is rather low which has attracted a great deal of attention in the field of antibiotic research. Precise knowledge of the mechanism of antimicrobial action provides the basis for the development of novel, highly efficient antibiotic agents which selectively kill microbial pathogens. This study is focused on elucidating the antimicrobial mechanism of action of the synthetic cyclic hexapeptide cWFW. Unlike the majority of antimicrobial peptides, this small molecule does not kill bacteria by permeabilisation of the membrane. In order to analyse alternative mechanisms of action, i.e. peptide translocation into the cytoplasm and demixing of membrane lipids, different peptide derivatives were synthesised to fulfil the technical requirements for the spectrum of methods applied. Peptide modification involved labelling with different fluorescent reporter groups as well as amino acid substitution. Minor changes in the primary structure were found not only to reduce the antimicrobial activity but also to change the non-permeabilising mechanism of action of the parent peptide. However, two derivatives could be identified which conserved the properties of cWFW. Using HPLC analysis and fluorescence microscopy, the bacterial cytoplasmic membrane could be confirmed as target structure. Moreover, we observed pronounced accumulation of the antimicrobial peptide at distinct sites of the lipid matrix. Peptide translocation into the cells, however, could not be detected. In order to characterise peptide interactions with different membrane phospholipids, we performed live cell imaging and isothermal titration calorimetry. We found that cWFW preferentially integrates into membrane regions with high curvature strain such as the division septum and the cell poles. In addition, the cyclic hexapeptide strongly reduced the fluidity of the bacterial membrane and induced a demixing of phospholipids into large domains. The immense impact of peptide-induced phase separation was manifested in the delocalisation of membrane-associated proteins which is suggested to influence vital processes such as cell growth and division. The results obtained in the present study point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is considered of low risk to induce the development of bacterial resistance.Antimikrobielle Peptide gehören evolutionär betrachtet zu einer sehr alten Klasse von Molekülen und kommen in zahlreichen Tier- und Pflanzenarten vor. In höher entwickelten Organismen stellen sie einen Teil des Immunsystems dar und erfüllen eine essentielle Funktion bei der unspezifischen Abwehr von pathogenen Mikroorganismen. Im Gegensatz zu konventionellen Antibiotika ist die Gefahr der Resistenzentwicklung gegen antimikrobielle Peptide relativ gering, was ihnen eine zentrale Bedeutung für die Erforschung neuartiger Substanzklassen verleiht. Die Untersuchung des antimikrobiellen Wirkmechanismus trägt dabei entscheidend zur Entwicklung innovativer, hoch- spezifischer Antibiotika bei. Ziel der vorliegenden Arbeit ist die Aufklärung des antimikrobiellen Wirkmechanismus des synthetischen, zyklischen Hexapeptids cWFW. Es besitzt eine sehr hohe Aktivität gegen Gram positive und Gram negative Bakterienspezies, allerdings beruht die Wirkung des kleinen Moleküls nicht auf Membran-Permeabilisierung, wie es für die meisten anderen antimikrobiellen Peptide gezeigt wurde. Alternative Wirkmechanismen basieren entweder auf der Translokation von Peptiden ins Zytoplasma der Zellen oder auf direkter Interaktion mit bestimmten Membrankomponenten. Für die Untersuchung dieser Prozesse wurden verschiedene Derivate des Zyklopeptids synthetisiert. Wir konnten zeigen, dass minimale strukturelle Veränderungen, wie der Einbau von Fluorophoren oder Aminosäuresubstitution, generell zu einer Verringerung der antimikrobiellen Aktivität führen und zum Teil eine Permeabilisierung der bakteriellen Membran bewirkten. Dennoch konnten zwei Peptid-Derivate identifiziert werden, bei denen die nativen Eigenschaften von cWFW erhalten waren und die für weitergehende Versuche verwendet wurden. Mittels Fluoreszenzmikroskopie und HPLC-Analyse konnten wir starke Wechselwirkungen des Zyklopeptids mit der bakteriellen Membran und Akkumulation in bestimmten Bereichen der Lipid-Matrix beobachten. Eine Translokation ins Zytoplasma hingegen wurde nicht festgestellt. Um die Interaktion mit bestimmten Phospholipiden genauer zu charakterisieren, wurden Bindungsstudien an lebenden Zellen und, unter Anwendung von isothermaler Titrationskalorimetrie, auf Model-Membran-Level durchgeführt. Es konnte gezeigt werde, dass sich das Zyklopeptid bevorzugt in Lipidbereiche einlagert, die eine hohe Krümmungsspannung aufweisen, wie an den Polen und der Zellteilungsebene. Darüber hinaus reduziert cWFW erheblich die Fluidität der Bakterienmembran und bewirkt eine starke Entmischung von Phospholipiden, was durch die Bildung großflächiger Membrandomänen nachgewiesen werden konnte. Diese Phasenseparation hatte große Auswirkungen auf die Lokalisation und Funktionalität membran-ständiger Proteine, die eine wesentliche Rolle während des Zellwachstums und der Teilung spielen. Die Ergebnisse unserer Untersuchungen deuten darauf hin, dass die hohe bakterizide Aktivität des synthetischen Zyklopeptids cWFW auf einem neuartigen, bisher unbeschriebenen antimikrobiellen Wirkmechanismus beruht, welcher kaum Risiken für eine Resistenzentwicklung birgt

Evidence for a Novel Mechanism of Antimicrobial Action of a Cyclic R-,W-Rich HexapeptideEvidence for a Novel Mechanism of Antimicrobial Action of a Cyclic R-,W-Rich Hexapeptide

The development of antimicrobial peptides as new class of antibiotic agents requires structural characterisation and understanding of their diverse mechanisms of action. As the cyclic hexapeptide cWFW (cyclo(RRRWFW)) does not exert its rapid cell killing activity by membrane permeabilisation, in this study we investigated alternative mechanisms of action, such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Using fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into the cells we could confirm the cytoplasmic membrane as the major peptide target. However, unexpectedly we observed accumulation of cWFW at distinct sites of the membrane. Further characterisation of peptide-membrane interaction involved live cell imaging to visualise the distribution of the lipid cardiolipin (CL) and isothermal titration calorimetry to determine the binding affinity to model membranes with different bacterial lipid compositions. Our results demonstrate a distribution of the cyclic peptide similar to that of cardiolipin within the membrane and highly preferred affinity of cWFW for CL-rich phosphatidylethanolamine (POPE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is neither based on membrane permeabilisation nor translocation into the cytoplasm but rather on preferred partitioning into particular lipid domains. As the phospholipids POPE/CL play a key role in the dynamic organisation of bacterial membranes we discuss the consequences of this peptide-lipid-interaction and outline the impact on antimicrobial peptide research

Carney Okla. Enterprise

Weekly newspaper from Carney, Oklahoma that includes local, state, and national news along with advertising

Relevance of Leptospira in boar and for the development of alternative antimicrobial concepts in boar semen preservation

Leptospirosis is a zoonotic disease of importance to public health and in livestock productions. It causes significant economic losses in pig breeding farms worldwide. However, actual transmission cycles and disease epidemiology in the pig population remain largely unknown. Despite the fact that the potential risk of venereal transmission of pathogenic Leptospira serovars in pigs has been a topic of discussion since the 1970s, reliable data are still lacking compared to other livestock species. Consequently, antibiotics are added to semen extenders to reduce bacterial contamination including pathogens like Leptospira. In view of the global threat of antimicrobial resistances, the routine use of antibiotics in porcine semen extenders is now under debate. Information about the prevalence of Leptospira infections in boar used for artificial insemination is needed for the development of novel antimicrobial concepts in pig insemination

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides

Influence of cWFW on cardiolipin distribution.

<p>Cardiolipin staining in <i>B</i>. <i>subtilis</i> 168 membranes was performed with NAO in untreated control cells and after peptide incubation with 12 μM cWFW. (A) Bright-field, (B) NAO fluorescence at 525 nm (binding to negative phospholipids) and (C) NAO fluorescence at 640 nm (specific interaction with CL). Untreated cells show distinct staining of cardiolipin at the septa and poles. However, a strong decrease in fluorescence intensity was observed in the presence of the antimicrobial peptide cWFW at both wavelengths, insets showing images processed with identical intensity settings as applied for control cells. At 640 nm NAO fluorescence was virtually abolished. Experiments were performed in duplicate, representative images of two independent experiments are shown.</p

Thermodynamic characterisation of cWFW binding to model membranes of different lipid compositions.

<p>LUVs (5 mM POPE/CL, 10 mM POPE/POPG and <i>E</i>. <i>coli</i> lipid extract) were titrated into 40 μM cWFW solution at 25°C. (A) Representative thermograms of the corresponding ITC titration experiments: differential heating power, <i>Δp</i>, versus time, POPE/CL (87.5/12.5 mol%, top) and POPE/POPG (75/25 mol%, bottom). (B) Isotherms of peptide adsorption to negatively charged POPE-based liposomes: integrated and normalised heat of reaction versus lipid/peptide molar ratio, POPE/CL (circles, left ordinate), <i>E</i>. <i>coli</i> extract (triangles, left ordinate) and POPE/POPG (squares, right ordinate). Solid lines represent best fits to experimental data in terms of a surface partition equilibrium modulated by electrostatic effects [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125056#pone.0125056.ref033" target="_blank">33</a>] assuming a lipid accessibility factor of γ = 0.5 for POPE/POPG, POPC/CL, γ = 0.6 for <i>E</i>.<i>coli</i> extract and γ = 1 for POPE/CL vesicles (see text for details).</p