Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

Combinatorial Screening of Cationic Lipidoids Reveals How Molecular Conformation Affects Membrane-Targeting Antimicrobial Activity.

The search for next-generation antibacterial compounds that overcome the development of resistance can be facilitated by identifying how to target the cell membrane of bacteria. Understanding the key molecular features that enable interactions with lipids and lead to membrane disruption is therefore crucial. Here, we employ a library of lipid-like compounds (lipidoids) comprising modular structures with tunable hydrophobic and hydrophilic architecture to shed light on how the chemical functionality and molecular shape of synthetic amphiphilic compounds determine their activity against bacterial membranes. Synthesized from combinations of 8 different polyamines as headgroups and 13 acrylates as tails, 104 different lipidoids are tested for activity against a model Gram-positive bacterial strain (Bacillus subtilis). Results from the combinatorial screening assay show that lipidoids with the most potent antimicrobial properties (down to 2 µM) have intermediate tail hydrophobicity (i.e., c log P values between 3 and 4) and lower headgroup charge density (i.e., longer spacers between charged amines). However, the most important factor appeared to be the ability of a lipidoid to self-assemble into an inverse hexagonal liquid crystalline phase, as observed by small-angle X-ray scattering (SAXS) analysis. The lipidoids active at lowest concentrations, which induced the most significant membrane damage during propidium iodide (PI) permeabilization assays, were those that aggregated into highly curved inverse hexagonal liquid crystal phases. These observations suggest that the introduction of strong curvature stress into the membrane is one way to maximize membrane disruption and lipidoid antimicrobial activity. Lipidoids that demonstrated the ability to furnish this phase consisted of either (i) branched or linear headgroups with shorter linear tails or (ii) cyclic headgroups with 4 bulky nonlinear tails. On the contrary, lipidoids previously observed to adopt disc-like conformations that pack into bicontinuous cubic phases were significantly less effective against B. subtilis. The discovery of these structure-property relationships demonstrates that it is not simply a balance of hydrophobic and hydrophilic moieties that govern membrane-active antibacterial activity, but also their intrinsic curvature and collective behavior.

Bactericidal Activity to Escherichia coli: Different Modes of Action of Two 24-Mer Peptides SAAP-148 and OP-145, Both Derived from Human Cathelicidine LL-37.

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 Escherichia coli, 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 E. coli 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 E. coli CM, depending on the smooth or rough/deep-rough lipopolysaccharide (LPS) phenotypes of E. coli 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 E. coli 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.

Magainin 2 and PGLa in bacterial membrane mimics IV: Membrane curvature and partitioning.

We previously reported that the synergistically enhanced antimicrobial activity of magainin 2 (MG2a) and PGLa is related to membrane adhesion and fusion. Here, we demonstrate that equimolar mixtures of MG2a and L18W-PGLa induce positive monolayer curvature stress and sense, at the same time, positive mean and Gaussian bilayer curvatures already at low amounts of bound peptide. The combination of both abilities-membrane curvature sensing and inducing-is most likely the base for the synergistically enhanced peptide activity. In addition, our coarse-grained simulations suggest that fusion stalks are promoted by decreasing the free-energy barrier for their formation rather than by stabilizing their shape. We also interrogated peptide partitioning as a function of lipid and peptide concentration using tryptophan fluorescence spectroscopy and peptide-induced leakage of dyes from lipid vesicles. In agreement with a previous report, we find increased membrane partitioning of L18W-PGLa in the presence of MG2a. However, this effect does not prevail to lipid concentrations higher than 1 mM, above which all peptides associate with the lipid bilayers. This implies that synergistic effects of MG2a and L18W-PGLa in previously reported experiments with lipid concentrations >1 mM are due to peptide-induced membrane remodeling and not their specific membrane partitioning.

Where Electrostatics Matter: Bacterial Surface Neutralization and Membrane Disruption by Antimicrobial Peptides SAAP-148 and OP-145.

The need for alternative treatment of multi-drug-resistant bacteria led to the increased design of antimicrobial peptides (AMPs). AMPs exhibit a broad antimicrobial spectrum without a distinct preference for a specific species. Thus, their mechanism, disruption of fundamental barrier function by permeabilization of the bacterial cytoplasmic membrane is considered to be rather general and less likely related to antimicrobial resistance. Of all physico-chemical properties of AMPs, their positive charge seems to be crucial for their interaction with negatively charged bacterial membranes. Therefore, we elucidate the role of electrostatic interaction on bacterial surface neutralization and on membrane disruption potential of two potent antimicrobial peptides, namely, OP-145 and SAAP-148. Experiments were performed on Escherichia coli, a Gram-negative bacterium, and Enterococcus hirae, a Gram-positive bacterium, as well as on their model membranes. Zeta potential measurements demonstrated that both peptides neutralized the surface charge of E. coli immediately after their exposure, but not of E. hirae. Second, peptides neutralized all model membranes, but failed to efficiently disrupt model membranes mimicking Gram-negative bacteria. This was further confirmed by flow cytometry showing reduced membrane permeability for SAAP-148 and the lack of OP-145 to permeabilize the E. coli membrane. As neutralization of E. coli surface charges was achieved before the cells were killed, we conclude that electrostatic forces are more important for actions on the surface of Gram-negative bacteria than on their cytoplasmic membranes.

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell.

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

Membrane Activity of LL-37 Derived Antimicrobial Peptides against Enterococcus hirae: Superiority of SAAP-148 over OP-145.

The development of antimicrobial agents against multidrug-resistant bacteria is an important medical challenge. Antimicrobial peptides (AMPs), human cathelicidin LL-37 and its derivative OP-145, possess a potent antimicrobial activity and were under consideration for clinical trials. In order to overcome some of the challenges to their therapeutic potential, a very promising AMP, SAAP-148 was designed. Here, we studied the mode of action of highly cationic SAAP-148 in comparison with OP-145 on membranes of Enterococcus hirae at both cellular and molecular levels using model membranes composed of major constituents of enterococcal membranes, that is, anionic phosphatidylglycerol (PG) and cardiolipin (CL). In all assays used, SAAP-148 was consistently more efficient than OP-145, but both peptides displayed pronounced time and concentration dependences in killing bacteria and performing at the membrane. At cellular level, Nile Red-staining of enterococcal membranes showed abnormalities and cell shrinkage, which is also reflected in depolarization and permeabilization of E. hirae membranes. At the molecular level, both peptides abolished the thermotropic phase transition and induced disruption of PG/CL. Interestingly, the membrane was disrupted before the peptides neutralized the negative surface charge of PG/CL. Our results demonstrate that SAAP-148, which kills bacteria at a significantly lower concentration than OP-145, shows stronger effects on membranes at the cellular and molecular levels.

Disruption of the Cytoplasmic Membrane Structure and Barrier Function Underlies the Potent Antiseptic Activity of Octenidine in Gram-Positive Bacteria.

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 E. hirae and 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. IMPORTANCE 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.

Magainin 2 and PGLa in bacterial membrane mimics III: Membrane fusion and disruption.

We previously speculated that the synergistically enhanced antimicrobial activity of Magainin 2 and PGLa is related to membrane adhesion, fusion, and further membrane remodeling. Here we combined computer simulations with time-resolved in vitro fluorescence microscopy, cryoelectron microscopy, and small-angle X-ray scattering to interrogate such morphological and topological changes of vesicles at nanoscopic and microscopic length scales in real time. Coarse-grained simulations revealed formation of an elongated and bent fusion zone between vesicles in the presence of equimolar peptide mixtures. Vesicle adhesion and fusion were observed to occur within a few seconds by cryoelectron microscopy and corroborated by small-angle X-ray scattering measurements. The latter experiments indicated continued and time-extended structural remodeling for individual peptides or chemically linked peptide heterodimers but with different kinetics. Fluorescence microscopy further captured peptide-dependent adhesion, fusion, and occasional bursting of giant unilamellar vesicles a few seconds after peptide addition. The synergistic interactions between the peptides shorten the time response of vesicles and enhance membrane fusogenic and disruption properties of the equimolar mixture compared with the individual peptides.

Antimicrobial peptide activity in asymmetric bacterial membrane mimics.

We report on the response of asymmetric lipid membranes composed of palmitoyl oleoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylglycerol, to interactions with the frog peptides L18W-PGLa and magainin 2 (MG2a), as well as the lactoferricin derivative LF11-215. In particular we determined the peptide-induced lipid flip-flop, as well as membrane partitioning of L18W-PGLa and LF11-215, and vesicle dye-leakage induced by L18W-PGLa. The ability of L18W-PGLa and MG2a to translocate through the membrane appears to correlate with the observed lipid flip-flop, which occurred at the fastest rate for L18W-PGLa. The higher structural flexibility of LF11-215 in turn allows this peptide to insert into the bilayers without detectable changes of membrane asymmetry. The increased vulnerability of asymmetric membranes to L18W-PGLa in terms of permeability, appears to be a consequence of tension differences between the compositionally distinct leaflets, but not due to increased peptide partitioning.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Evolution of the analytical scattering model of live Escherichia coli.

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

Bridging the Antimicrobial Activity of Two Lactoferricin Derivatives in E. coli and Lipid-Only Membranes.

We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1-5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

Octenidine: Novel insights into the detailed killing mechanism of Gram-negative bacteria at a cellular and molecular level.

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.

Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation.

We studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding article of this series, we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in the membranes. Here, we focus on the structures of the peptide-lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic, and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles with a collapsed interbilayer spacing resulting from peptide-induced adhesion. Interestingly, the adhesion does not lead to a peptide-induced lipid separation of charged and charge-neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle x-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.

Blood Compatibility-An Important but Often Forgotten Aspect of the Characterization of Antimicrobial Peptides for Clinical Application.

Acylation of antimicrobial peptides mimics the structure of the natural lipopeptide polymyxin B, and increases antimicrobial and endotoxin-neutralizing activities. In this study, the antimicrobial properties of lactoferrin-based LF11 peptides as well as blood compatibility as a function of acyl chain length were investigated. Beyond the classical hemolysis test, the biocompatibility was determined with human leukocytes and platelets, and the influence of antimicrobial peptides (AMPs) on the plasmatic coagulation and the complement system was investigated. The results of this study show that the acylation of cationic peptides significantly reduces blood tolerance. With increasing acyl chain length, the cytotoxicity of LF11 peptides to human blood cells also increased. This study also shows that acylated cationic antimicrobial peptides are inactivated by the presence of heparin. In addition, it could be shown that the immobilization of LF11 peptides leads to a loss of their antimicrobial properties.

Magainin 2 and PGLa in Bacterial Membrane Mimics I: Peptide-Peptide and Lipid-Peptide Interactions.

We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies, both peptides, as well as their equimolar mixture, were found to remain upon adsorption in a surface-aligned topology and to induce significant membrane perturbation, as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so-called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with the formation of parallel heterodimers at concentrations below a synergistic increase of dye leakage from vesicles. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup region as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.

Membrane Sphingolipids Regulate the Fitness and Antifungal Protein Susceptibility of Neurospora crassa.

The membrane sphingolipid glucosylceramide (GlcCer) plays an important role in fungal fitness and adaptation to most diverse environments. Moreover, reported differences in the structure of GlcCer between fungi, plants and animals render this pathway a promising target for new generation therapeutics. Our knowledge about the GlcCer biosynthesis in fungi is mainly based on investigations of yeasts, whereas this pathway is less well characterized in molds. We therefore performed a detailed lipidomic profiling of GlcCer species present in Neurospora crassa and comprehensively show that the deletion of genes encoding enzymes involved in GlcCer biosynthesis affects growth, conidiation and stress response in this model fungus. Importantly, our study evidences that differences in the pathway intermediates and their functional role exist between N. crassa and other fungal species. We further investigated the role of GlcCer in the susceptibility of N. crassa toward two small cysteine-rich and cationic antimicrobial proteins (AMPs), PAF and PAFB, which originate from the filamentous ascomycete Penicillium chrysogenum. The interaction of these AMPs with the fungal plasma membrane is crucial for their antifungal toxicity. We found that GlcCer determines the susceptibility of N. crassa toward PAF, but not PAFB. A higher electrostatic affinity of PAFB than PAF to anionic membrane surfaces might explain the difference in their antifungal mode of action.

Interaction of two antitumor peptides with membrane lipids - Influence of phosphatidylserine and cholesterol on specificity for melanoma cells.

R-DIM-P-LF11-322 and DIM-LF11-318, derived from the cationic human host defense peptide lactoferricin show antitumor activity against human melanoma. While R-DIM-P-LF11-322 interacts specifically with cancer cells, the non-specific DIM-LF11-318 exhibits as well activity against non-neoplastic cells. Recently we have shown that cancer cells expose the negatively charged lipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane, while non-cancer cells just expose zwitterionic or neutral lipids, such as phosphatidylcholine (PC) or cholesterol. Calorimetric and zeta potential studies with R-DIM-P-LF11-322 and cancer-mimetic liposomes composed of PS, PC and cholesterol indicate that the cancer-specific peptide interacts specifically with PS. Cholesterol, however, reduces the effectiveness of the peptide. The non-specific DIM-LF11-318 interacts with PC and PS. Cholesterol does not affect its interaction. The dependence of activity of R-DIM-P-LF11-322 on the presence of exposed PS was also confirmed in vitro upon PS depletion of the outer leaflet of cancer cells by the enzyme PS-decarboxylase. Further corresponding to model studies, cholesterol depleted melanoma plasma membranes showed increased sensitivity to R-DIM-P-LF11-322, whereas activity of DIM-LF11-318 was unaffected. Microscopic studies using giant unilamellar vesicles and melanoma cells revealed strong changes in lateral distribution and domain formation of lipids upon addition of both peptides. Whereas R-DIM-P-LF11-322 enters the cancer cell specifically via PS and reaches an intracellular organelle, the Golgi, inducing mitochondrial swelling and apoptosis, DIM-LF11-318 kills rapidly and non-specifically by lysis of the plasma membrane. In conclusion, the specific interaction of R-DIM-P-LF11-322 with PS and sensitivity to cholesterol seem to modulate its specificity for cancer membranes.

Effect of Technically Relevant X-Ray Doses on the Structure and Function of Alcohol Dehydrogenase and Hen Egg-White Lysozyme.

PURPOSE: The effect of different irradiation doses on the structure and activity of lyophilized powders of Hen Egg-White Lysozyme (HEWL) and alcohol dehydrogenase (ADH) was investigated using these substances as models for robust and sensitive proteins, respectively. Three doses were selected to cover the ranges of radio-sterilization (25kGy), treatment of blood products (25Gy) and annual background radiation dose (approximately 2mGy). The results offer an initial screening of different irradiation doses and support the development of X-ray imaging methods as non-destructive process analytical technology (PAT) tools for detecting the visible particulate matters in such products. METHODS: HEWL and ADH were exposed to X-rays in the solid state. The effect of irradiation was determined directly after irradiation and after storage. Structural changes and degradation were investigated using SAXS, SDS-PAGE and HPLC-MS. Protein functionality was assessed via activity assays. RESULTS: Lower irradiation doses of 25Gy and 2mGy had no significant impact on the structure and enzyme activity. The dose of 25kGy caused a significant decrease in the enzyme activity and structural changes immediately after irradiation of ADH and after storage of irradiated HEWL at -20°C. CONCLUSION: The results emphasize the importance of careful selection of radiation doses for development of X-ray imaging methods as PAT tools inspection of solid biopharmaceutical products.