Search Results
An Outer Membrane Vesicle-Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability
When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways.
Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections
In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
Bacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor
Liposomes have been studied for decades as nanoparticulate drug delivery systems for cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work, we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound (HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored antimicrobial target. The liposomes were composed of DOPG (18:1 (Δ9-cis) phosphatidylglycerol) and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 °C. Loading of the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm. Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug-resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably hydrophobic, antibiotics.