Deep Plasma Proteome Profiling by Modulating Single Nanoparticle Protein Corona with Small Molecules.

The protein corona, a dynamic biomolecular layer that forms on nanoparticle (NP) surfaces upon exposure to biological fluids is emerging as a valuable diagnostic tool for improving plasma proteome coverage analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Here, we show that spiking small molecules, including metabolites, lipids, vitamins, and nutrients, into plasma can induce diverse protein corona patterns on otherwise identical NPs, significantly enhancing the depth of plasma proteome profiling. The protein coronas on polystyrene NPs when exposed to plasma treated with an array of small molecules (n=10) allowed for detection of 1793 proteins marking an 8.25-fold increase in the number of quantified proteins compared to plasma alone (218 proteins) and a 2.63-fold increase relative to the untreated protein corona (681 proteins). Furthermore, we discovered that adding 1000 µg/ml phosphatidylcholine could singularly increase the number of unique proteins within the protein corona (897 proteins). This specific concentration of phosphatidylcholine selectively depleted the four most abundant plasma proteins, including albumin, thus reducing concentration dynamic range of plasma proteome and boosting LC-MS/MS sensitivity for detection of proteins with lower abundance. By employing an optimized data-independent acquisition (DIA) approach, the inclusion of phosphatidylcholine led to the detection of 1436 proteins in plasma. This significant achievement is made utilizing only a single NP type and one small molecule to analyze a single plasma sample, setting a new standard in proteomic depth of the plasma sample. Given the critical role of plasma proteomics in biomarker discovery and disease monitoring, we anticipate widespread adoption of this methodology for identification and clinical translation of proteomic biomarkers into FDA approved diagnostics.

Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane.

Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS-polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials.

Computational identification of a systemic antibiotic for gram-negative bacteria.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its ß-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.

Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs.

There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class Gammaproteobacteria. Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates. IMPORTANCE Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens.

Detergent Titration as an Efficient Method for NMR Resonance Assignments of Membrane Proteins in Lipid-Bilayer Nanodiscs.

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded ß-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer.

Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.