Marina Rautenbach

Membranes of Class IIa Bacteriocin-Resistant Listeria monocytogenes Cells Contain Increased Levels of Desaturated and Short-Acyl-Chain Phosphatidylglycerols

ABSTRACT 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.

A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity.

We have developed a highly sensitive micro-gel well diffusion assay for the determination of antimicrobial activity. In essence, the normal radial diffusion type assay was adapted to perform it in a microtiter plate. We compared our micro-gel well diffusion assay to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus luteus as the indicator organism. The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method. Data analysis to calculate minimum inhibitory concentration, 50% microbial growth inhibition and maximum inhibitory concentration was refined by generating dose-response curves with the software package Prism 3.0 (Graphpad Software Inc.). The minimum inhibitory concentrations, determined by the three methods, were significantly different (P<0. 001), highlighting the limitations involved in comparing data obtained from different methods.

Short cationic antimicrobial peptides interact with ATP.

ABSTRACT The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. α-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP.

Antifungal peptides: To be or not to be membrane active.

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.

Synergistic activity of the tyrocidines, antimicrobial cyclodecapeptides from Bacillus aneurinolyticus, with amphotericin B and caspofungin against Candida albicans biofilms

ABSTRACT Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.

Optimised quantification of the antiyeast activity of different barley malts towards a lager brewing yeast strain.

The brewing of beer involves two major biological systems, namely malted barley (malt) and yeast. Both malt and yeast show natural variation and assessing the impact of differing malts on yeast performance is important in the optimisation of the brewing process. Currently, the brewing industry uses well-established tests to assess malt quality, but these frequently fail to predict malt-associated problem fermentations, such as incomplete fermentations, premature yeast flocculation (PYF) and gushing of the final beer product. Antimicrobial compounds, and in particular antiyeast compounds in malt, may be one of the unknown and unmeasured malt factors leading to problem fermentations. In this study, the adaptation of antimicrobial assays for the determination of antiyeast activity in malt is described. Our adapted assay was able to detect differing antiyeast activities in nine malt samples. For this sample set, malts associated with PYF during fermentation and gushing activity in beer showed high antiyeast activity. Both PYF and gushing are malt quality issues associated with fungal infection of barley in the field which may result in elevated antimicrobial activity in the barley grain. Also, two more malts that passed the normal quality control tests were also observed to have high antiyeast activity and such malts must be considered as suspect. Based on our results, this assay is a useful measure of malt quality as it quantifies the antiyeast activity in malt which may adversely impact on brewery fermentation.

Anti-listerial activity and structure–activity relationships of the six major tyrocidines, cyclic decapeptides from Bacillus aneurinolyticus

Six major tyrocidines, purified from the antibiotic tyrothricin complex produced by Bacillus aneurinolyticus, showed significant lytic and growth inhibitory activity towards the gram+ bacteria, Micrococcus luteus and Listeria monocytogenes, but not against the gram- bacterium, Escherichia coli. The isolated natural tyrocidines were in particular more active against the leucocin A (antimicrobial peptide) resistant strain, L. monocytogenes B73-MR1, than the sensitive L. monocytogenes B73 strain. Remarkably similar structure-activity trends toward the three gram+ bacteria were found between growth inhibition and different physicochemical parameters (solution amphipathicity, theoretical lipophilicity, side-chain surface area and mass-over-charge ratio).

β-sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus.

The structures of two major tyrocidines, antibiotic peptides from Bacillus aneurinolyticus, in an aqueous environment were studied using nuclear magnetic resonance spectroscopy, restrained molecular dynamics (MD), circular dichroism, and mass spectrometry. TrcA and TrcC formed β-structures in an aqueous environment. Hydrophobic and hydrophilic residues were not totally separated into nonpolar and polar faces of the peptides, indicating that tyrocidines have low amphipathicity. In all the β-structures, residues Trp(4)/Phe(4) and Orn(9) were on the same face. The ability of the peptides to form dimers in aqueous environment was studied by replica exchange MD simulations. Both peptides readily dimerize, and predominant complex structures were characterized through cluster analysis. The peptides formed dimers by either associating sideways or stacking on top of each other. Dimers formed through sideways association were mainly stabilized by hydrogen bonding, while the other dimers were stabilized by hydrophobic interactions. The ability of tyrocidine peptides to form different types of dimers with different orientations suggests that they can form larger aggregates, as well.

Construction and validation of a detailed kinetic model of glycolysis in Plasmodium falciparum.

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.

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.