OP-145 and SAAP-148, two 24-mer antimicrobial peptides derived from human cathelicidin LL-37, exhibit killing efficacy against both Gram-positive and Gram-negative bacteria at comparable peptide ...concentrations. However, when it comes to the killing activity against
, the extent of membrane permeabilization does not align with the observed bactericidal activity. This is the case in living bacteria as well as in model membranes mimicking the
cytoplasmic membrane (CM). In order to understand the killing activity of both peptides on a molecular basis, here we studied their mode of action, employing a combination of microbiological and biophysical techniques including differential scanning calorimetry (DSC), zeta potential measurements, and spectroscopic analyses. Various membrane dyes were utilized to monitor the impact of the peptides on bacterial and model membranes. Our findings unveiled distinct binding patterns of the peptides to the bacterial surface and differential permeabilization of the
CM, depending on the smooth or rough/deep-rough lipopolysaccharide (LPS) phenotypes of
strains. Interestingly, the antimicrobial activity and membrane depolarization were not significantly different in the different LPS phenotypes investigated, suggesting a general mechanism that is independent of LPS. Although the peptides exhibited limited permeabilization of
membranes, DSC studies conducted on a mixture of synthetic phosphatidylglycerol/phosphatidylethanolamine/cardiolipin, which mimics the CM of Gram-negative bacteria, clearly demonstrated disruption of lipid chain packing. From these experiments, we conclude that depolarization of the CM and alterations in lipid packing plays a crucial role in the peptides' bactericidal activity.
With its broad antimicrobial spectrum and non-specific mode of action via membrane disruption, any resistance to octenidine (OCT) seems unlikely and has not been observed in clinical settings so far. ...In this study, we aimed to investigate the efficacy of OCT against Escherichia coli and mutants lacking specific lipid head groups which, due to altered membrane properties, might be the root cause for resistance development of membrane-active compounds. Furthermore, we aimed to test its efficacy under different experimental conditions including different solvents for OCT, bacterial concentration and methods for analysis. Our primary goal was to estimate how many OCT molecules are needed to kill one bacterium. We performed susceptibility assays by observing bacterial growth behavior, using a Bioscreen in an analogous manner for every condition. The growth curves were recorded for 20 h at 420–580 nm in presence of different OCT concentrations and were used to assess the inhibitory concentrations (IC100%) for OCT. Bacterial concentrations given in cell numbers were determined, followed by Bioscreen measurement by manual colony counting on agar plates and QUANTOMTM cell staining. This indicated a significant variance between both methods, which influenced IC100% of OCT, especially when used at low doses. The binding capacity of OCT to E. coli was investigated by measuring UV-absorbance of OCT exposed to bacteria and a common thermodynamic framework based on Bioscreen measurements. Results showed that OCT’s antimicrobial activity in E. coli is not affected by changes at the membrane level but strongly dependent on experimental settings in respect to solvents and applied bacterial counts. More OCT was required when the active was dissolved in phosphate or Hepes buffers instead of water and when higher bacterial concentration was used. Furthermore, binding studies revealed that 107–108 OCT molecules bind to bacteria, which is necessary for the saturation of the bacterial surface to initiate the killing cascade. Our results clearly demonstrate that in vitro data, depending on the applied materials and the methods for determination of IC100%, can easily be misinterpreted as reduced bacterial susceptibility towards OCT.
•Octenidine (OCT) kills Escherichia coli via membrane disruption.•OCT acts in a detergent-like manner.•On a cellular level, OCT depolarises and changes the fluidity of the cell membrane.•On a ...molecular level, OCT induces a complete loss of the packing order of bacterial phospholipids.
Octenidine (OCT) is a widely used antiseptic molecule with an antimicrobial spectrum covering a broad range of bacteria and fungi. However, the detailed molecular mechanism of killing has not yet been elucidated. The objective of our study was to investigate the mode of action of OCT's potent effect on Gram-negative bacteria using Escherichia coli as a model organism as well as corresponding model membranes. The effects of OCT on cellular morphology were observed by electron microscopy, changes affecting membrane integrity (surface charge, fluidity, permeabilisation and depolarisation) by zeta potential, fluorescence microscopy and spectroscopy. Specific interactions of OCT with membrane phospholipids were addressed using differential scanning calorimetry, X-ray scattering and fluorescence techniques. OCT neutralises the surface charge of E. coli leading to disruption of the outer membrane and dramatic loss of the cell wall and further penetrates through the periplasmic space reaching the inner membrane. Model membranes showed that OCT inserts into the hydrophobic fatty acyl chain region of the bilayer, inducing complete lipid disorder. The loss of membrane integrity is also reflected by membrane depolarisation and changes in membrane fluidity as shown by electron microscopy. Insertion of OCT into the outer and inner membrane of E. coli results in a chaotic lipid arrangement that leads to rapid disruption of the cell envelope. We propose that this unspecific mode of action based on purely physical interactions is the basis of the very broad antimicrobial profile and makes it unlikely that resistance to OCT will develop.
The search for novel compounds to combat multi-resistant bacterial infections includes exploring the potency of antimicrobial peptides and derivatives thereof. Complementary to high-throughput ...screening techniques, biophysical and biochemical studies of the biological activity of these compounds enable deep insight, which can be exploited in designing antimicrobial peptides with improved efficacy. This approach requires the combination of several techniques to study the effect of such peptides on both bacterial cells and simple mimics of their cell envelope, such as lipid-only vesicles. These efforts carry the challenge of bridging results across techniques and sample systems, including the proper choice of membrane mimics. This review describes some important concepts toward the development of potent antimicrobial peptides and how they translate to frequently applied experimental techniques, along with an outline of the biophysics pertaining to the killing mechanism of antimicrobial peptides.
Display omitted
•Collection of available experimental techniques to characterize the modes of action of antimicrobial peptides.•Fundamental features of membrane-active antimicrobial peptide interactions with live bacteria and lipid-only membrane mimics.•Discussion of diverse aspects contributing to the identification of compromised membrane integrity, including critical assessment of advantages and limitations of different techniques and corresponding data interpretation.•Discussion of concepts that apply the comparison between live cells and model membranes.
S-adenosyl-L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological processes: more than 50 AdoMet-dependent methyltransferases methylate a broad spectrum of ...cellular compounds including nucleic acids, proteins and lipids. Common to all AdoMet-dependent methyltransferase reactions is the release of the strong product inhibitor S-adenosyl-L-homocysteine (AdoHcy), as a by-product of the reaction. S-adenosyl-L-homocysteine hydrolase is the only eukaryotic enzyme capable of reversible AdoHcy hydrolysis to adenosine and homocysteine and, thus, relief from AdoHcy inhibition. Impaired S-adenosyl-L-homocysteine hydrolase activity in humans results in AdoHcy accumulation and severe pathological consequences. Hyperhomocysteinemia, which is characterized by elevated levels of homocysteine in blood, also exhibits a similar phenotype of AdoHcy accumulation due to the reversal of the direction of the S-adenosyl-L-homocysteine hydrolase reaction. Inhibition of S-adenosyl-L-homocysteine hydrolase is also linked to antiviral effects. In this review the advantages of yeast as an experimental system to understand pathologies associated with AdoHcy accumulation will be discussed.
The antimicrobial killing mechanism of octenidine (OCT), a well-known antiseptic is poorly understood. We recently reported its interaction with Gram-negative bacteria by insertion of OCT into the ...outer and cytoplasmic membrane of Escherichia coli, resulting in a chaotic lipid rearrangement and rapid disruption of the cell envelope. Its action primarily disturbs the packing order of the hydrophobic moiety of a lipid, which consequently might result in a cascade of multiple effects at a cellular level. Here, we investigated OCT's impact on two different Gram-positive bacteria, Enterococcus hirae and Bacillus subtilis, and their respective model membranes. In accordance with our previous results, OCT induced membrane disorder in all investigated model systems. Electron and fluorescence microscopy clearly demonstrated changes in cellular structure and membrane integrity. These changes were accompanied by neutralization of the surface charge in both
B. subtilis and membrane disturbances associated with permeabilization. Similar permeabilization and disordering of the lipid bilayer was also observed in model membranes. Furthermore, experiments performed on strongly versus partly anionic membranes showed that the lipid disordering effect induced by OCT is a result of maximized hydrophobic over electrostatic forces without distinct neutralization of the surface charge or discrimination between the lipid head groups. Indeed, mutants lacking specific lipid head groups were also susceptible to OCT to a similar extent as the wild type. The observed unspecific mode of action of OCT underlines its broad antimicrobial profile and renders the development of bacterial resistance to this molecule less likely.
OCT is a well-established antiseptic molecule routinely used in a large field of clinical applications. Since the spread of antimicrobial resistance has restricted the use of antibiotics worldwide, topically applied antiseptics like OCT, with a broad spectrum of antimicrobial activity and high safety profile, gain increasing importance for effective infection prevention and therapy. To eliminate a wide spectrum of disease-causing microorganisms, a compound's antiseptic activity should be unspecific or multitarget. Our results demonstrate an unspecific mechanism of action for OCT, which remained largely unknown for years. OCT disturbs the barrier function of a bacterial cell, a function that is absolutely fundamental for survival. Because OCT does not distinguish between lipids, the building blocks of bacterial membranes, its mode of action might be attributed to all bacteria, including (multi)drug-resistant isolates. Our results underpin OCT's potent antiseptic activity for successful patient outcome.
In eukaryotes, S-adenosyl-l-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-l-homocysteine, a product and potent competitive inhibitor of S-adenosyl-l-methionine ...(AdoMet)-dependent methyltransferases. De novophosphatidylcholine(PC)synthesisrequiresthreeAdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-l-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.
PDF
