Browsing by Author "Van Rensburg, Wilma"

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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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    The tyrocidines in the creation of antimicrobial cellulose and sterilizing materials
    (Stellenbosch : Stellenbosch University, 2020-04) Van Rensburg, Wilma; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The rising resistance of pathogenic bacteria is of great concern, especially since resistance has been reported for five of the six common hospital acquired infections. Furthermore, continual infections occurring in the food industry can become costly to the companies and negatively impact consumers. Modified antimicrobial and antifouling materials and surfaces can be used limit the propagation of microorganisms on various surfaces and minimise the occurrence of infection and spoilage. These materials can prevent pathogenic cell adhesion or kill cells with the release of active compounds or kill cells on contact. Active compounds used to functionalise materials can include silver nanoparticles, antimicrobially active polymers, antibiotics, enzymes and chemically synthesised peptides. Due to the increased demand for more environmentally friendly practises, naturally produced antimicrobial peptides are considered. Antimicrobial peptides have a broad spectrum of activity and resistance is less likely to develop due to their membranolytic mode of action. Tyrocidines and analogues (Trcs) are antimicrobial cyclodecapeptides with a potent broad spectrum of activity against Gram-positive bacteria, filamentous fungi, human pathogens Candida albicans and malaria parasite Plasmodium falciparum. The peptides have been shown to adsorb onto various surfaces while maintaining activity, with a selectivity towards cellulose. The goal for this study was therefore to determine the application in commercial materials, the robustness of the cellulose-peptide material and conditions that dictate the cellulose-peptide interaction, as well as the molecular descriptors in peptide interaction. In order to facilitate antimicrobial screening of Trcs treated materials, a high throughput solid surface assay was developed that gave comparable results to that observed with an industrial standard assay. The assay was developed for detection of antimicrobial activity within four hours, by utilising a cell viability or metabolic active dye, resazurin. A key factor that allowed for the fast detection was the use of ten times more cells per cm² as what is used in other solid surface assays, which allowed for the selection of only the best performing antimicrobial materials. Optimisation was confirmed with four model organisms Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa against materials that contained five different antimicrobial agents (gentamicin, bacitracin, ampicillin, gramicidin S and tetracycline). Screening of association of a Trc mixture containing 11 cyclodecapeptides (Trc mix) to laboratory and commercial materials showed that the peptides in this mixture maintained activity and showed full inhibition of L. monocytogenes by peptide treated cellulose-based materials and plastics. Trc mix treated cellulose proved to be robust in terms of cell exposure (killing up to 90% of 10⁷ cells/cm² within 10 minutes of contact time), temperature and solvents. The only solvents that resulted in decreased activity were 1% m/v SDS and 70% v/v acetonitrile. Comparison between cellulose treated with tyrocidine and other antimicrobial peptides, showed the tyrocidine-cellulose outperforming the other peptides in terms of inhibition of L. monocytogenes and E. coli. Study into the aggregation of Trcs using different percentages acetonitrile, as a denaturant of larger hydrophobic driven oligomers, showed the formation of two distinct types of oligomer groups: one group stabilised with hydrogen bonding formed at higher acetonitrile concentrations and one driven by hydrophobic interactions formed in aqueous solutions. The hydrophobic driven self-assembly structures were temperature stable and attributed to the temperature and solvent stability of the Trc-treated cellulose. It was also proposed to be a key factor in the association between Trc and cellulose, apart from the possible Maillard reaction that drive covalent linkages of some of the peptides in the initial seeding layer on the cellulose. Trcs showed a concentration dependent association linked to the formation of an optimal aggregate size that allows for association. Interaction with various cellulose derivates (glucose, cellobiose, hydropropyl cellulose) showed conformational changes of the peptide resulting in higher activity against L. monocytogenes and higher haemolytic activity. Studies into the molecular interaction, using FTIR and NMR, between cellulose and Trcs with the cellulose derivatives as cellulose models showed the amino acids exposed to the solvent environment to be most effected by the presence of the saccharides and therefore involved in the peptide:cellulose interaction. The hypothesis is that the peptide forms self-assembled structures driven by hydrophobic interactions in aqueous solutions allowing the formation of hydrogen bonds between the hydroxyl groups on cellulose and Orn⁹, Asn⁵, and Gln⁶ followed by Trp⁴ and Tyr⁷. Upon association the peptide oligomers would form a layered sheet like structure on the cellulose surface with above mentioned amino acids associated to the cellulose and exposed to the solvent environment. Thereby allowing for further self-assembly and/or antimicrobial activity as Orn⁹ and Trp⁴ are both key residues in the activity of Trcs. Furthermore, the peptide can arrange and re-arrange its conformation based on the environment to better suit association. The creation of functional materials by harnessing Trcs’ natural ability to associate to surfaces is more environmentally friendly but also creates an antimicrobial material that is robust in terms of activity and solvent/environment exposure. Therefore, materials functionalised with Trcs hold great promise in preventing surface colonization by resistant pathogens while still being “green” in its environmental footprint.