Wujing Xian

Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin–lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin–lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin–lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

Liquid-crystalline ordering of antimicrobial peptide–DNA complexes controls TLR9 activation

Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs 1-5). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

Pentobra: A Potent Antibiotic with Multiple Layers of Selective Antimicrobial Mechanisms against Propionibacterium Acnes.

Although antibiotics are a common treatment for acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in skin are compounded by increasing resistance of Propionibacterium acnes clinical isolates. To address these problems, we engineered Pentobra, a peptide-aminoglycoside molecule which has multiple mechanisms of antibacterial action, and investigated whether it can be a potential candidate for the treatment of acne. Pentobra combines the potent ribosomal activity of aminoglycosides with the bacteria-selective membrane-permeabilizing abilities of antimicrobial peptides (AMPs). Pentobra demonstrated potent and selective killing of P. acnes, but not against human skin cells in vitro. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5–7 logs) against multiple P. acnes clinical strains. Moreover, EM studies showed that Pentobra had robust membrane activity, as treatment with Pentobra killed P. acnes cells and caused leakage of intracellular contents. Pentobra may also have potential anti-inflammatory effects as demonstrated by suppression of some P. acnes-induced chemokines. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in comedones extracts isolated from human donors. Our work demonstrates that equipping aminoglycosides with selective membrane activity is a viable approach for developing antibiotics against P. acnes that are effective in cutaneous environments.

Designing Hybrid Antibiotic Peptide Conjugates To Cross Bacterial Membranes

We design hybrid antibiotic peptide conjugates that can permeate membranes. Integration of multiple components with different functions into a single molecule is often problematic, due to competing chemical requirements for different functions and to mutual interference. By examining the structure of antimicrobial peptides (AMPs), we show that it is possible to design and synthesize membrane active antibiotic peptide conjugates (MAAPCs) that synergistically combine multiple forms of antimicrobial activity, resulting in unusually strong activity against persistent bacterial strains.

Molecular Motor Dnm1 Synergistically Induces Membrane Curvature To Facilitate Mitochondrial Fission

Dnm1 and Fis1 are prototypical proteins that regulate yeast mitochondrial morphology by controlling fission, the dysregulation of which can result in developmental disorders and neurodegenerative diseases in humans. Loss of Dnm1 blocks the formation of fission complexes and leads to elongated mitochondria in the form of interconnected networks, while overproduction of Dnm1 results in excessive mitochondrial fragmentation. In the current model, Dnm1 is essentially a GTP hydrolysis-driven molecular motor that self-assembles into ring-like oligomeric structures that encircle and pinch the outer mitochondrial membrane at sites of fission. In this work, we use machine learning and synchrotron small-angle X-ray scattering (SAXS) to investigate whether the motor Dnm1 can synergistically facilitate mitochondrial fission by membrane remodeling. A support vector machine (SVM)-based classifier trained to detect sequences with membrane-restructuring activity identifies a helical Dnm1 domain capable of generating negative Gaussian curvature (NGC), the type of saddle-shaped local surface curvature found on scission necks during fission events. Furthermore, this domain is highly conserved in Dnm1 homologues with fission activity. Synchrotron SAXS measurements reveal that Dnm1 restructures membranes into phases rich in NGC, and is capable of inducing a fission neck with a diameter of 12.6 nm. Through in silico mutational analysis, we find that the helical Dnm1 domain is locally optimized for membrane curvature generation, and phylogenetic analysis suggests that dynamin superfamily proteins that are close relatives of human dynamin Dyn1 have evolved the capacity to restructure membranes via the induction of curvature mitochondrial fission. In addition, we observe that Fis1, an adaptor protein, is able to inhibit the pro-fission membrane activity of Dnm1, which points to the antagonistic roles of the two proteins in the regulation of mitochondrial fission.

How to Teach Old Antibiotics New Tricks

Antimicrobial peptides (AMPs), or more generally host defense peptides, have broad-spectrum antimicrobial activity and use nonspecific interactions to target generic features common to the membranes of many pathogens. As a result, development of resistance to such natural defenses is inhibited compared to conventional antibiotics. The disadvantage of AMPs, however, is that they are often not very potent. In contrast, traditional antibiotics typically have strong potency, but due to a broad range of bacterial defense mechanisms, there are many examples of resistance. Here, we explore the possibility of combining these two classes of molecules. In the first half of this chapter, we review the fundamentals of membrane curvature generation and the various strategies recently used to mimic this membrane activity of AMPs using different classes of synthetic molecules. In the second half, we show that it is possible to impart membrane activity to molecules with no previous membrane activity, and summarize some of our recent works which aim to combine advantages of traditional antibiotics and AMPs into a single molecule with multiple mechanisms of killing as well as multiple mechanisms of specificity.