Dissecting the Organization of a ß-Barrel Assembly Machinery (BAM) Complex by Neutron Reflectometry.

The ß-barrel assembly machinery (BAM) complex in Gram-negative bacteria facilitates the assembly of ß-barrel proteins into the outer membrane. Understanding the protein-protein interactions within this complex is essential for unravelling its functional mechanisms. Here, we present the use of neutron reflectometry for investigating the organization of ß-barrel membrane protein complexes in the membrane environment. The spatial organization, protein positioning, protein-lipid interactions, and conformational changes within the complex can be elucidated by this method.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'ß-signal' imprinted in the final ß-strand of the OMP engages the ß-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the ß-signal are repeated in other, internal ß-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the ß-signal, arranging several ß-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Coassembled Nitric Oxide-Releasing Nanoparticles with Potent Antimicrobial Efficacy against Methicillin-Resistant Staphylococcus aureus (MRSA) Strains.

Nitric oxide (NO)-releasing nanoparticles are effective nanomedicines with diverse therapeutic advantages compared with small molecule-based NO donors. Here, we report a new class of furoxan-based NO-releasing nanoparticles using a simple, creative yet facile coassembly approach. This is the first time we demonstrated that the coassembled NO-releasing nanoparticles with poly(ethylene glycol)101-block-poly(propylene glycol)56-block-poly(ethylene glycol)101 (Pluronic F127) had potent antimicrobial efficacies against methicillin-resistant Staphylococcus aureus (MRSA) strains. Nanoparticles obtained from the coassembly of either 4-(1-(3-methylpentan-5-ol)oxyl)(3-phenylsulfonyl) furoxan (compound 1) or 4-methoxy(3-phenylsulfonyl) furoxan (compound 2) with Pluronic F127 exhibit 4-fold improved antimicrobial activities compared to their self-assembled counterparts without Pluronic F127. 5(6)-Carboxylfluorescein (CF) leakage experiments further reveal that both coassembled NO-releasing nanoparticles show stronger interactions with lipid bilayers than those self-assembled alone. Subsequently, their strong plasma membrane-damaging capabilities are confirmed under both high-resolution optical microscopy and scanning electron microscopy characterizations. This coassembly approach could be readily applied to other small molecule-based antimicrobials, providing new solutions and important insights to further antimicrobial recipe design.

A polytherapy based approach to combat antimicrobial resistance using cubosomes.

A depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative 'superbugs' has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of "superbugs".

Phytantriol-Based Cubosome Formulation as an Antimicrobial against Lipopolysaccharide-Deficient Gram-Negative Bacteria.

Treatment of multidrug-resistant (MDR) bacterial infections increasingly relies on last-line antibiotics, such as polymyxins, with the urgent need for discovery of new antimicrobials. Nanotechnology-based antimicrobials have gained significant importance to prevent the catastrophic emergence of MDR over the past decade. In this study, phytantriol-based nanoparticles, named cubosomes, were prepared and examined in vitro by minimum inhibitory concentration (MIC) and time-kill assays against Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Phytantriol-based cubosomes were highly bactericidal against polymyxin-resistant, lipopolysaccharide (LPS)-deficient A. baumannii strains. Small-angle neutron scattering (SANS) was employed to understand the structural changes in biomimetic membranes that replicate the composition of these LPS-deficient strains upon treatment with cubosomes. Additionally, to further understand the membrane-cubosome interface, neutron reflectivity (NR) was used to investigate the interaction of cubosomes with model bacterial membranes on a solid support. These results reveal that cubosomes might be a new strategy for combating LPS-deficient Gram-negative pathogens.

Synthesis of Chromeno[4,3-b]pyrrol-4(1H)-ones through a Multicomponent Reaction and Cyclization Strategy.

A novel three-component reaction of 2-oxo-2H-chromene-3-carbaldehydes with isocyanides and anilines was developed for one-pot assembly of biologically intriguing chromeno[4,3-b]pyrrol-4(1H)-ones, which can also be transferred into diverse polycyclic fused scaffolds through further synthetic manipulations. The described method tolerates a broad substrate scope and proceeds in moderate to good yield via a sequential multicomponent reaction and intramolecular Michael cyclization.