Exploring the Membrane Mechanism of the Bioactive Peptaibol Ampullosporin A Using Lipid Monolayers and Supported Biomimetic Membranes

Ampullosporin A is an antimicrobial, neuroleptic peptaibol, the behavior of which was investigated in different membrane mimetic environments made of egg yolk L-α-phosphatidylcholine. In monolayers, the peptaibol adopted a mixed α/310-helical structure with an in-plane orientation. The binding step was followed by the peptide insertion into the lipid monolayer core. The relevance of the inner lipid leaflet nature was studied by comparing ampullosporin binding on a hybrid bilayer, in which this leaflet was a rigid alkane layer, and on supported fluid lipid bilayers. The membrane binding was examined by surface plasmon resonance spectroscopy and the effect on lipid dynamics was explored using fluorescence recovery after photobleaching. In the absence of voltage and at low concentration, ampullosporin A substantially adsorbed onto lipid surfaces and its interaction with biomimetic models was strongly modified depending on the inner leaflet structure. At high concentration, ampullosporin A addition led to the lipid bilayers disruption

Membrane permeabilisation and antimycoplasmic activity of the 18-residue peptaibols, trichorzins PA

AbstractThe membrane permeabilisation properties of six linear natural 18-residue peptaibols, termed trichorzins PA, have been assessed on liposomes and on mollicutes (trivial name, mycoplasmas), a class of parasitic bacteria characterized by a small genome, the lack of a cell wall, a minute cell size, and the incorporation in their plasma membrane of exogenously supplied cholesterol. The trichorzins PA used in this study (PA II, PA IV–VI, PA VIII, and PA IX) differ between them by amino acid or amino alcohol substitutions at positions 4, 7, and 18, and form slightly amphipathic α-helices. They proved bactericidal for mollicutes belonging to the genera Acholeplasma, Mycoplasma, and Spiroplasma, with minimal inhibitory concentrations (3.12≤MICs≤50 μM) generally 2 to 4 fold higher than those of alamethicin F50, a related 20-residue peptide (1.56≤MICs≤12.5 μM). Spiroplasma cells were apparently not protected by the presence of spiralin on their surface. The activities of the six trichorzins PA were not influenced by their sequence variations and no synergistic effect was observed. Consistent with the marginal effect of cholesterol on the incorporation of the trichorzins PA into liposome bilayers, the antibiotic activity was independent of the amount of cholesterol in the membranes of the different mollicutes. The trichorzins PA and alamethicin inhibited the motility of Spiroplasma melliferum, the helical cells being deformed and split into coccoid forms. Membrane potential measurements in Acholeplasma laidlawii and S. melliferum showed that trichorzin PA V and alamethicin F50 very efficiently depolarized the plasma membrane of mollicutes. This was consistent with fluorescence and 23Na NMR measurements on liposomes that revealed the permeabilisation of the lipid bilayer and the nonselective ionophoric activity of the trichorzins PA. These data suggest that the bactericidal activity exhibited by the trichorzins PA on mollicutes is due to the permeabilisation of the plasma membrane

The Rsr1/Bud1 GTPase Interacts with Itself and the Cdc42 GTPase during Bud-Site Selection and Polarity Establishment in Budding Yeast

Bimolecular fluorescence complementation assays allow the visualization of the homotypic and heterotypic GTPase interactions in vivo. The Rsr1 homotypic interaction involves its polybasic region and depends on its GDP-GTP exchange factor. Dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry

Specific Evolution of F1-Like ATPases in Mycoplasmas

F1F0 ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, β, γ and ε subunits of F1 ATPases and could form an F1-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F1-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F1-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F1-like structure is associated with a hypothetical X0 sector located in the membrane of mycoplasma cells

Exploring Spiroplasma Biology: Opportunities and Challenges

Spiroplasmas are cell-wall-deficient helical bacteria belonging to the class Mollicutes. Their ability to maintain a helical shape in the absence of cell wall and their motility in the absence of external appendages have attracted attention from the scientific community for a long time. In this review we compare and contrast motility, shape determination and cytokinesis mechanisms of Spiroplasma with those of other Mollicutes and cellwalled bacteria. The current models for rod-shape determination and cytokinesis in cell-walled bacteria propose a prominent role for the cell wall synthesis machinery. These models also involve the cooperation of the actin-like protein MreB and FtsZ, the bacterial homolog of tubulin. However the exact role of the cytoskeletal proteins is still under much debate. Spiroplasma possess MreBs, exhibit a rod-shape dependent helical morphology, and divide by an FtsZ-dependent mechanism. Hence, spiroplasmas represent model organisms for deciphering the roles of MreBs and FtsZ in fundamental mechanisms of non-spherical shape determination and cytokinesis in bacteria, in the absence of a cell wall. Identification of components implicated in these processes and deciphering their functions would require genetic experiments. Challenges in genetic manipulations in spiroplasmas are a major bottleneck in understanding their biology. We discuss advancements in genome sequencing, gene editing technologies, superresolution microscopy and electron cryomicroscopy and tomography, which can be employed for addressing long-standing questions related to Spiroplasma biology

Effects on mollicutes (wall-less bacteria) of synthetic peptides comprising a signal peptide or a membrane fusion peptide, and a nuclear localization sequence (NLS) – a comparison with melittin

AbstractIn order to investigate the effect of primary amphipathic peptides on mollicutes (wall-less bacteria), we have synthesised five molecules (P1, P2, P3, JM123, and JM133) comprising a 16 to 18-residue hydrophobic sequence and the nuclear localization sequence (NLS) PKKKRKV of simian virus 40 large-T antigen, C-terminated by a cysteamide group. The hydrophobic cluster was in P1 the signal sequence of the heavy chain of Caiman crocodilus immunoglobulin G and in JM123 the fusion peptide of human immunodeficiency virus 1 glycoprotein gp41 in which phenylalanine7 was replaced by a tryptophan residue. The homologues P2, P3, and JM133 were obtained by slight alterations of these sequences. Circular dichroism spectroscopy revealed that, in liposomes, P-series peptides were mainly under the form of β-sheets whereas JM-series peptides displayed a high proportion of turns. These peptides proved to be bactericidal for some mollicutes, notably Acholeplasma laidlawii, but were much less potent than melittin. Furthermore, their antibiotic activity was independent of the average thickness of the plasma membrane hydrophobic core whilst that of melittin was inversely related to the thickness. Melittin and the synthetic peptides abolished spiroplasma cell motility and helicity, but only melittin and P-series peptides split the cells into globular forms displaying an average diameter of ca. 1 μm. In contrast to melittin, the synthetic peptides agglutinated spiroplasmas, suggesting that their polycationic NLS was exposed on the cell surface. P-series peptides decreased, though less efficiently than melittin, A. laidlawii and Spiroplasma melliferum membrane potential (ΔΨ) and transmembrane pH gradient (ΔpH), at concentrations much lower than their minimal inhibitory concentrations whilst JM-series peptides had no effect on ΔΨ and ΔpH in the same conditions. Actually, the bactericidal activity of these peptides towards mollicutes was proportional to their ability to collapse the electrochemical transmembrane potential

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic ?-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic a helix with a + 5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic ?-sheeted peptides [(LK)[n]K], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/ water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/ depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold

Construction and purification of the truncated PGK proteins.

<p>(A) Schematic illustration of PGK protein showing positions of amino acids interacting with its two substrates in glycolysis. The N-terminal part of the PGK and more precisely, amino acids D23, N25, R38, H62 and R121 are implicated in 1,3-biphosphoglycerate binding. The C-terminal part of the PGK containing amino acids G236, G310, N334, P336, G338 to E341 and G371 to T373 are implicated in ADP binding. Hinge regions containing amino acids L198 to P201 and V389 to T391 connect the two others domains of PGK and are responsible for inducing conformational change during catalysis. (B) Schematic illustration of the PGK and deletion constructs. Full-length PGK protein (amino acids 1 to 412) was deleted from C-terminus and PGK-FL1 (amino acids 1 to 302), PGK-FL2 (amino acids 1 to 205), PGK-FL3 (amino acids 1 to 101), were produced. N-terminal deletion produced PGK-FL4 (amino acids 101 to 412). PGK-FL5 (amino acids 49 to 154) was constructed by deleting both amino terminal and carboxy-terminal ends of PGK. (C) All His₆-tagged proteins were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017357#s2" target="_blank">Materials and Methods</a>, and subjected to SDS-PAGE (lane 1). Parallel gels were transferred onto nitrocellulose membranes. Saturated blots were probed with anti-His monoclonal antibodies (lane 2).</p