Louic S. Vermeer

Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments.

Order parameters from deuterium NMR are often used to validate or calibrate molecular dynamics simulations. This paper gives a short overview of the literature in which experimental order parameters from 2H NMR are compared to those calculated from MD simulations. The different ways in which order parameters from experiment are used to calibrate and validate simulations are reviewed. In the second part of this review, a case study of cholesterol in a DMPC bilayer is presented. It is concluded that the agreement between experimental data and simulation is favorable in the hydrophobic region of the membrane, for both the phospholipids and cholesterol. In the interfacial region the agreement is less satisfactory, probably because of the high polarity of this region which makes the correct computation of the electrostatics more complex.

Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience

Background Culture-independent analysis of the respiratory secretions of people with cystic fibrosis (CF) has identified many bacterial species not previously detected using culture in this context. However, little is known about their clinical significance or persistence in CF airways. Methods The authors characterised the viable bacterial communities in the sputum collected from 14 patients at monthly intervals over 1 year using a molecular community profiling technique—terminal restriction fragment length polymorphism. Clinical characteristics were also collected, including lung function and medications. Ecological community measures were determined for each sample. Microbial community change over time within subjects was defined using ecological analytical tools, and these measures were compared between subjects and to clinical features. Results Bacterial communities were stable within subjects over time but varied between subjects, despite similarities in clinical course. Antibiotic therapy temporarily perturbed these communities which generally returned to pretreatment configurations within 1 month. Species usually considered CF pathogens and those not previously regarded as such exhibited similar patterns of persistence. Less diverse sputum bacterial communities were correlated to lung disease severity and relative abundance of Pseudomonas aeruginosa. Conclusion Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

Background: Antimicrobial peptides (AMPs) have the potential to act against multiple pathogenic targets. Results: AMPs that maintain conformational flexibility are more potent against multiple pathogens and less hemolytic. Conclusion: Antimicrobial action and hemolysis proceed via differing mechanisms. Significance: The potency, selectivity, and ability of AMPs to reach intracellular pathogens can be modulated using general principles. We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

NMR metabolomics of MTLn3E breast cancer cells identifies a role for CXCR4 in lipid and choline regulation.

The alpha chemokine receptor CXCR4 is up-regulated in certain types of breast cancer. Truncation of the C-terminus of this receptor alters cell morphology and increases invasiveness and metastatic potential. Here, to better understand the effects of CXCR4 expression and truncation in breast cancer cells, we have used high resolution magic angle spinning (HR-MAS) NMR studies of rat breast carcinoma MtLn3E cells to characterize the metabolite complement of cells heterologously expressing human CXCR4 or its C-terminal truncation mutant, Δ34-CXCR4. Notable reductions in choline levels were detected when either cells expressing wild-type CXCR4 or Δ34-CXCR4 were compared with cells containing an empty expression vector. Cells expressing CXCR4-Δ34 had reduced lipid content when compared with either the wild-type CXCR4 expressing cells or those containing the empty expression vector. Taken together, our results show that distinct effects on the metabolite complement can be linked to either CXCR4 expression or CXCR4 regulation. The metabolite markers for these two effects identified in the present study can, in turn, be used to further investigate the role of CXCR4 in metastasis.

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.

Incorporation of 2,3-Diaminopropionic Acid into Linear Cationic Amphipathic Peptides Produces pH-Sensitive Vectors

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pKa of the β‐NH2 in 2,3‐diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pKa of around 6.3 is stabilized by the increased electron‐withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large‐scale conformational changes are observed in Dap‐rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene‐delivery capability on designed cationic amphipathic peptides that incorporate Dap.

Combined Systems Approaches Reveal Highly Plastic Responses to Antimicrobial Peptide Challenge in Escherichia coli

Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies.

A relationship between Pseudomonal growth behaviour and cystic fibrosis patient lung function identified in a metabolomic investigation.

Chronic polymicrobial lung infections in adult cystic fibrosis patients are typically dominated by high levels of Pseudomonas aeruginosa. Determining the impact of P. aeruginosa growth on airway secretion composition is fundamental to understanding both the behaviour of this pathogen in vivo, and its relationship with other potential colonising species. We hypothesised that the marked differences in the phenotypes of clinical isolates would be reflected in the metabolite composition of spent culture media. 1H NMR spectroscopy was used to characterise the impact of P. aeruginosa growth on a synthetic medium as part of an in vitro CF lower airways model system. Comparisons of 15 CF clinical isolates were made and four distinct metabolomic clusters identified. Highly significant relationships between P. aeruginosa isolate cluster membership and both patient lung function (FEV1) and spent culture pH were identified. This link between clinical isolate growth behaviour and FEV1 indicates characterisation of P. aeruginosa growth may find application in predicting patient lung function while the significant divergence in metabolite production and consumption observed between CF clinical isolates suggests dominant isolate characteristics have the potential to play both a selective role in microbiota composition and influence pseudomonal behaviour in vivo.

Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity.

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.

Structural properties of a peptide derived from H+-V-ATPase subunit a

The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H(+)-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously suggested based on NMR studies of a similar peptide in DMSO. The pKa of both histidine residues that are important for proton transport was measured in water and in SDS. The differences that are found demonstrate that the histidine residues interact with the SDS polar heads. In detergent, circular dichroism data indicate that the secondary structure of the peptide depends on the pH and the type of detergent used. Using solid-state NMR, it is shown that the peptide is immobile in phospholipid bilayers, which means that it is probably not a single transmembrane helix in these samples. The environment is important for the structure of TM7, so in subunit a it is probably held in place by the other transmembrane helices of this subunit.