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Browsing by Author "Rautenbach, Marina"

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Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self-assembly
(2012) Naidoo V.B.; Rautenbach, Marina
We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water-soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N′-suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self-assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing-directed self-assembly rather than non-specific aggregation or precipitation. The progress of suberoyldiglycylglycine self-assembly was observed to be via the formation of oligomers in the solution, which then self-assembled to form layered β-sheet type macrostructures. Within 24h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5-7days. © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Cell-surface alterations in class IIa bacteriocin resistant Listeria monocytogenes strains
(MAIK NAUKA/INTERPERIODICA, C/O KLUWER ACADEMIC-PLENUM PUBLISHERS, 233 SPRINGST, NEW YORK, USA, NY, 10013-1578, 2004) Vadyvaloo, Viveka; Arous S; Gravesen A; H�chard Y; Chauhan-Haubrock R; Hastings JW; Rautenbach, Marina
Characterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant
(SpringerOpen, 2017) Ndlovu, Thando; Rautenbach, Marina; Vosloo, Johann Arnold; Khan, Sehaam; Khan, Wesaal
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 C13–16 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–C10–C10 and Rha–C10–C10, Rha–Rha–C8–C10/Rha–Rha–C10–C8 and Rha–C8–C10/Rha–C10–C8, as well as Rha–Rha–C12–C10/Rha–Rha–C10–C12 and Rha–C12–C10/Rha–C10–C12. 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.
Direct surfactin-gramicidin S antagonism supports detoxification in mixed producer cultures of Bacillus subtilis and Aneurinibacillus migulanux
(MAIK NAUKA/INTERPERIODICA, C/O KLUWER ACADEMIC-PLENUM PUBLISHERS, 233 SPRINGST, NEW YORK, USA, NY, 10013-1578, 2012) Rautenbach, Marina; Ey�gh�-Bikong HA; Vlok NM; Stander MA; De Beer A
Impact of Different Wort Boiling Temperatures on the Beer Foam Stabilizing Properties of Lipid Transfer Protein 1
(2004) Van Nierop S.N.E.; Evans D.E.; Axcell B.C.; Cantrell I.C.; Rautenbach, Marina
Beer consumers demand satisfactory and consistent foam stability; thus, it is a high priority for brewers. Beer foam is stabilized by the interaction between certain beer proteins, including lipid transfer protein 1 (LTP1), and isomerized hop α-acids, but destabilized by lipids. In this study it was shown that the wort boiling temperature during the brewing process was critical in determining the final beer LTP1 content and conformation. LTP1 levels during brewing were measured by an LTP1 ELISA, using antinative barley LTP1 polyclonal antibodies. It was observed that the higher wort boiling temperatures (∼102 °C), resulting from low altitude at sea level, reduced the final beer LTP1 level to 2-3 μg/mL, whereas the lower wort boiling temperatures (∼96 °C), resulting from higher altitudes (1800 m), produced LTP1 levels between 17 and 35 μg/mL. Low levels of LTP1 in combination with elevated levels of free fatty acids (FFA) resulted in poor foam stability, whereas beer produced with low levels of LTP1 and FFA had satisfactory foam stability. Previous studies indicated the need for LTP1 denaturing to improve its foam stabilizing properties. However, the results presented here show that LTP1 denaturation reduces its ability to act as a binding protein for foam-damaging FFA. These investigations suggest that wort boiling temperature is an important factor in determining the level and conformation of LTP1, thereby favoring satisfactory beer foam stability.
Membranes of class IIa bacteriocin-resistant Listeria monocytogenes cells contain increased levels of desaturated and short-acyl-chain phosphatidylglycerols
(2002) Vadyvaloo, Viveka; Hastings J.W.; Van der Merwe M.J.; Rautenbach, Marina
A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target Listeria strains. We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant L. monocytogenes strains. Two wild-type strains, L. monocytogenes B73 and 412, two spontaneous mutants of L. monocytogenes B73 with intermediate resistance to leucocin A (±2.4 and ±4 times the 50% inhibitory concentrations [IC50] for sensitive strains), and two highly resistant mutants of each of the wild-type strains (<500 times the IC50 for sensitive strains) were analyzed. Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short- to long-acyl-chain species of PG in all the resistant L. monocytogenes strains in our study, although their sensitivities to leucocin A were significantly different. This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane. The presence of this phenomenon in a strain (L. monocytogenes 412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some L. monocytogenes strains. Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains. There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains. It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present.
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.
Physiological implications of class IIa bacteriocin resistance in Listeria monocytogenes strains
(MAIK NAUKA/INTERPERIODICA, C/O KLUWER ACADEMIC-PLENUM PUBLISHERS, 233 SPRINGST, NEW YORK, USA, NY, 10013-1578, 2004) Vadyvaloo, Viveka; Snoep JL; Hastings JW; Rautenbach, Marina
Profiling the production of antimicrobial secondary metabolites by xenorhabdus khoisanae J194 under different culturing conditions
(Frontiers Media, 2020-03-30) Booysen, Elzaan; Rautenbach, Marina; Stander, Marietjie A.; Dicks, Leon M. T.
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.
Selective lysis of erythrocytes infected with the trophozoite stage of Plasmodium falciparum by polyene macrolide antibiotics
(2006) Wiehart U.I.M.; Rautenbach, Marina; Hoppe H.C.
The continuous increase in strains of the human malaria parasite Plasmodium falciparum resistant to most front-line antimalarial compounds is reason for grave clinical concern. The search for new drugs led us to investigate a number of membrane active polyene macrolide antibiotics, such as amphotericin B, nystatin, filipin and natamycin. The interaction of these compounds with sterols in bilayer cell membranes can lead to cell damage and ultimately cell lysis. The malaria parasite modifies the host erythrocyte membrane by changing the protein and lipid composition and thus the infected cell could be a selective target for membrane active compounds. We found that erythrocytes infected with the trophozoite stage of P. falciparum were particularly susceptible to lysis by amphotericin B (Fungizone™) and, to a lesser extent, nystatin, as determined by ELISA and various microscopy assays. Liposomal amphotericin B (AmBisome™) displayed a similar specificity for parasitised erythrocytes, but complete lysis required a longer incubation period. In contrast, filipin and natamycin did not distinguish between normal and parasite-infected erythrocytes, but lysed both at similar concentrations. In addition, when added to ring-stage cultures, the amphotericin B preparations and nystatin produced a marked disruption in parasite morphology in less than 2 h without an accompanying permeabilisation of the infected host cell, suggesting a second plasmodicidal mode of action. The results imply that selected polyene macrolide antibiotics or their derivatives could find application in the treatment of severe malaria caused by of P. falciparum. © 2006 Elsevier Inc. All rights reserved.
The synthesis and characterisation of analogues of the antimicrobial peptide iturin A₂
(Stellenbosch : Stellenbosch University, 1998-12) Rautenbach, Marina; Hofmeyr, J.-H. S.; Swart, P.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry
ENGLISH ABSTRACT: Iturin A, an antifungal lipopeptide, is produced by Bacillus subtilis. This cyclic peptide consists of seven D- and L-amino acid residues (L-Asn2-D-Tyr3-D-Asn4-L-Gln5-L-Pro6-DAsn7- L-Ser8) and a beta-amino fatty acid residue. Eight analogues of iturin A2 were synthesised and purified by high performance chromatography (HPLC). Electrospray ionisation mass spectrometry (ESI-MS), amino acid analysis and HPLC confirmed high chemical purity of the synthetic products. The influence of primary structure on conformation, hydrophobicity, interaction with alkali metal ions and bioactivity was investigated using the purified peptides. Two low energy in vacuo structures of a linear iturin A2 analogue (8-Beta), one with a distorted W-backbone structure and one with a twisted S-backbone structure, were predicted with HyperChem®4.5. Nuclear magnetic resonance spectrometry confirmed the existence of two slow interconverting conformations, possibly a W<->S equilibrium. The predicted S-structure of 8-Beta includes two turns that approximate beta-turns. In natural iturin A, the same two peptide moieties, beta-aminotetradecanoyl-L-Asn2-D-Tyr3-D-Asn4 and L-Gln5-L-Pro6-DAsn7- L-Ser8, each adopt a type II beta-turn conformation. ESI-MS fragmentation patterns of sodiated 8-Beta indicated that the sodium interacts with the majority of the amide bond oxygens in the predicted turns. The linear peptides associated with either one or two alkali metal ions, while the cyclic analogues associated only with one ion. The alkali metal ion selectivity sequence of all the lipopeptides was Na+>K+>Rb+, indicating a size limitation in interaction cavities. Iturin A possibly has a direct interaction with alkali metal ions and it is proposed that these ions are chelated by the carbonyl oxygens in either one of the two beta-turns of natural iturin A. It was found that the more hydrophobic the iturin A2 analogue, the better it interacted with lipid membranes and octadecanoylsilane matrices (HPLC retention), except if it had a high tendency to aggregate in solution. Aggregation in the membrane is part of iturin A’s mechanism of action. It is proposed that solution-phase aggregates are not the active form of iturin A as the lipopeptide preparations, which self-aggregated in solution, lost their antibacterial activity. Circular dichroism (CD) spectra of the peptides in liposomes revealed the possibility of type II b-turns in all the octalipopeptides. There is, however, a marked difference between the overall cyclic and linear structures in membranes, although diastereomers, differing in configuration of b-aminotetradecanoic acid (b-NC14) residue, had similar structures. The possibility of self-assembly of synthetic iturin A2 in antiparallel beta-sheets was also indicated by CD. Haemolytic activity of the iturin A2 analogues depended on cyclisation, inclusion of L-Asn2 and b-NC14 configuration. This activity is possibly stereoselective as synthetic iturin A2 and its linear analogue were the most haemolytic. Growth inhibition of Micrococcus luteus mainly depended on hydrophobic interaction and not on cyclisation or configuration of the beta-NC14 residue, therefore this activity differs in mechanism of action from that of haemolysis. Lysis of M. luteus protoplasts, however, decreased with decrease in peptide length: 8-Beta>7-Beta>6- Beta. The activity against Botrytis cinerea depended mainly on cyclisation. The hydrophobic hub, formed by the invariant Tyr residue and the beta-NC14 residue, is a possible key to antifungal activity. This hub is absent in the predicted S-structure of 8-Beta and may be influenced in cyclic 8-Beta and shorter analogues by the configuration of the beta-NC14 residue, resulting in good overall bioactivity of only the synthetic iturin A2.
The synthesis and characterisation of analogues of the antimicrobial peptide iturin A2
(Stellenbosch : Stellenbosch University, 1999) Rautenbach, Marina; Stellenbosch University. Faculty of . Dept. of .
Variants of lipopeptides and glycolipids produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa cultured in different carbon substrates
(SpringerOpen, 2017) Ndlovu, Thando; Rautenbach, Marina; Khan, Sehaam; Khan, Wesaal
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 C13–C15 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–C10–C10 and Rha–C10–C10 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.
What goes around comes around-a comparative study of the influence of chemical modifications on the antimicrobial properties of small cyclic peptides
(MDPI, 2013) Scheinpflug, Kathi; Nikolenko, Heike; Komarov, Igor V.; Rautenbach, Marina; Dathe, Margitta
Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells
Xenorhabdus khoisanae SB10 produces Lys-rich PAX lipopeptides and a Xenocoumacin in its antimicrobial complex
(BMC (part of Springer Nature), 2019-06-13) Dreyer, J.; Rautenbach, Marina; Booysen, E.; Van Staden, A. D.; Deane, S. M.; Dicks, L. M. T.
Background: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. Results: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1’ and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1’ were also detected. A small antibiotic compound, yellow in colour and λmax of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. Conclusion: With UPLC-MS and MSe analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1’ peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.

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