Browsing by Author "Laubscher, Wikus Ernst"

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    The multifaceted antibacterial mechanisms of the pioneering peptide antibiotics tyrocidine and gramicidin S
    (American Society for Microbiology, 2018-10-09) Wenzel, Michaela; Rautenbach, Marina; Vosloo, J. Arnold; Siersma, Tjalling; Aisenbrey, Christopher H. M.; Zaitseva, Ekaterina; Laubscher, Wikus Ernst; Van Rensburg, Wilma; Behrends, Jan C.; Bechinger, Burkhard; Hamoen, Leendert W.
    ENGLISH ABSTRACT: 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.
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    Production, characterisation and activity of selected and novel antibiotic peptides from soil bacteria
    (Stellenbosch : Stellenbosch University, 2016-12) Laubscher, Wikus Ernst; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The ever increasing development of pathogen resistance towards conventional antibiotics has necessitated the search for novel antimicrobial molecules. It has been suggested that antimicrobial peptides will form the foundation of a new generation of antibiotics. These small natural antibiotics possess rapid killing mechanisms against a broad spectrum of pathogens. They often disrupt multiple cellular targets resulting in decreased risk of resistance, making them ideal candidates as novel antimicrobials. Selection of a screening source is challenging as antimicrobial peptides are ubiquitously produced by most organisms. However, soil bacteria have historically been shown to produce a large variety of clinically significant antibiotics, including antimicrobial peptides. Furthermore, the soil biome possesses a vast bacterial diversity that has been to date largely unexplored, making it an ideal resource for the discovery of novel antimicrobial peptides. The overall objective of this study was therefore to isolate, identify and characterise novel antimicrobial peptides from soil bacteria. A commercial soil additive containing a lower bacterial diversity than natural soil environments was first used to validate a method for novel antimicrobial peptide discovery. With the goal of discovering novel antimicrobial peptides, this method included the isolation, identification and characterisation of antimicrobial producing bacteria and their active components. Only two of the isolates from the soil additive, denoted LB.4 and LB.5, were selected for further purification and characterisation of their antimicrobial compounds. The LB.4 and LB.5 isolates were determined to be strains of Brevibacillus laterosporus and Bacillus licheniformis respectively. In this dissertation they are referred to as Br. laterosporus LB.4 and B. licheniformis LB.5. The antimicrobial compound produced by B. licheniformis LB.5 was determined to be the well–known antimicrobial peptide, bacitracin A. It was found that Br. laterosporus LB.4 produces two potentially novel antimicrobial peptides termed LB.4-1223 and LB.4-1273. Although the amino acid composition was shown to be: F, L/I, M, N, P, V and Y, the amino acid sequences remain to be determined and their novelty could therefore not be confirmed. Brevibacillus parabrevis was used as a positive control as it is known to produce antimicrobial peptides from the tyrocidine group. The biological activity and mode of action data of the isolated peptides were therefore compared to that of tryptocidine C, a characterised analogue from the tyrocidine group of peptides. Antimicrobial dose response analysis revealed that LB.4-1223, LB.4-1273 and tyrocidine C possess antimicrobial and haemolytic activity, while bacitracin A only showed potent antimicrobial activity. Biophysical studies indicated that both bacitracin A and tryptocidine C disorientated lipid bilayers, however, only tryptocidine C resulted in the formation of transmembrane pores. This was the first study to show pore formation by tryptocidine C. It is known that bacitracin elicits its antimicrobial activity by inhibiting peptidoglycan synthesis, but this study provided the first insight into the interactions between bacitracin A and cellular membranes. However, whether these interactions results in microbial inhibition is still unknown. Screening of an environmental soil sample yielded three isolates with significant antimicrobial activity and suitable low molecular mass spectra to suggest antimicrobial peptide production. A literature study suggested that one of the isolates produces peptides from the bogorol group of antimicrobial peptides, while the other two isolates produce previously uncharacterised compounds. These compounds will be investigated in future studies. This dissertation describes an antimicrobial discovery and characterisation study that led to the discovery of novel antimicrobial peptides/compounds. Furthermore, it was shown that the laborious, time consuming nature of traditional microbiological screening methodology demands that a more effective, higher-throughput methodology be developed to meet the demand for novel antibiotics discovery.