Membrane-Interactive Compounds from Pistacia lentiscus L. Thwart Pseudomonas aeruginosa Virulence

ABSTRACTPseudomonas aeruginosa is capable to deploy a collection of virulence factors that are not only essential for host infection and persistence, but also to escape from the host immune system and to become more resistant to drug therapies. Thus, developing anti-virulence agents that may directly counteract with specific virulence factors or disturb higher regulatory pathways controlling the production of virulence armories are urgently needed. In this regard, this study reports that Pistacia lentiscus L. fruit cyclohexane extract (PLFE1) thwarts P. aeruginosa virulence by targeting mainly the pyocyanin pigment production by interfering with 4-hydroxy-2-alkylquinolines molecules production. Importantly, the anti-virulence activity of PLFE1 appears to be associated with membrane homeostasis alteration through the modulation of SigX, an extracytoplasmic function sigma factor involved in cell wall stress response. A thorough chemical analysis of PLFE1 allowed us to identify the ginkgolic acid (C17:1) and hydroginkgolic acid (C15:0) as the main bioactive membrane-interactive compounds responsible for the observed increased membrane stiffness and anti-virulence activity against P. aeruginosa. This study delivers a promising perspective for the potential future use of PLFE1 or ginkgolic acid molecules as an adjuvant therapy to fight against P. aeruginosa infections.

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate

ABSTRACTThe virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the “late” 135S particles have detectable levels of the VP1 N-terminus trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.AUTHOR SUMMARYNonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membrane. This process remains poorly understood. For enterovirus such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normal internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded but has not yet externalized the membrane interactive peptides, the clear identification of a unique exit site VP1 N-terminus, the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion, direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes, and the observation that a significant portion of VP4 remains inside the particle and accounts for a feature that had been previously ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.

Biophysical investigation into the antibacterial action of modelin-5-NH2

Neutron diffraction studies showed that modelin-5-CONH2has potent antibacterial activity which involves membrane interactive, tilted α-helical structure.

Fluorescent Ion Efflux Screening Assay for Determining Membrane-Active Peptides

A major global health threat is the emergence of antibiotic-resistant microbes. Coupled with a lack of development of modified antibiotics, there is a need to develop new antimicrobial molecules and screening assays for them. In this study, we provide proof of concept that a large unilamellar vesicle (LUV) method used to study chloride ion efflux facilitated by ionophores and surfactant-like molecules that disrupt membrane integrity can be adapted to identify membrane-interactive antimicrobial peptides (AMPs) and to screen relative activity of AMPs. Lucigenin was encapsulated in LUVs in the presence of Cl– ion (NaCl), which quenches fluorescence, and then incubated with AMPs in 100 mM NaNO3 buffer. Upon AMP membrane interaction or disruption, the Cl– ion is exchanged with the NO3– ion, and the resultant lucigenin fluorescence is indicative of relative AMP activity. Seven AMPs were synthesized by solid-phase peptide chemistry and incubated with LUVs of different phospholipid compositions. Each AMP resulted in lucigenin fluorescence, which was dose dependent, and the relative fluorescence correlated with the minimum inhibitory concentration and minimum bactericidal concentration values for the corresponding peptide. Furthermore, using mammalian model phospholipid LUVs, lucigenin-induced fluorescence also correlated with the AMP cytotoxicity half-maximal inhibitory concentration values. The proline-rich AMP, Chex1-Arg20, which is non-lytic but interacts with the bacterial membrane resulted in lucigenin fluorescence of bacterial membrane model LUVs but not of mammalian membrane model LUVs. The fluorescent ion efflux assay developed here should have applicability for most AMPs and could be tailored to target particular bacterial species membrane composition, potentially leading to the identification of novel membrane-interactive AMPs. The rapid high-throughput method also allows for screening of relative AMP activity and toxicity before biological testing.

Functionalising the azobenzene motif delivers a light-responsive membrane-interactive compound with the potential for photodynamic therapy applications

A light-responsive azobenzene derivative provides a membrane-interactive compound with the potential for photodynamic therapy applications.

Discovery of novel membrane binding structures and functions

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome.