Synthesis and study of new siderophore analog-ciprofloxacin conjugates with antibiotic activities against Pseudomonas aeruginosa and Burkholderia spp.

Antibacterial resistance is a healthcare burden. Among Gram-negative bacteria, Pseudomonas aeruginosa belongs to the first list of antibiotic-resistant "priority pathogens" described by the World Health Organization. Formerly Pseudomonas pseudomallei, Burkholderia pseudomallei, responsible for melioidosis, is considered as a potential bioterrorist weapon by the Centers of Diseases Control and Prevention. We are interested in the development of new ways to combat these bacteria, targeted due to their high level of resistance to antibiotics via a lack of membrane permeability or efflux. Using iron transport systems is a promising strategy to bypass the bacteria cell membrane and restore the activity of conventional antibiotics such as ciprofloxacin. Specific outer membrane receptors are necessary to most microbes as they allow iron uptake, essential for their survival through siderophore-dependent mechanisms. These systems may allow the introduction of antibacterial agents, chemically coupled to a natural or synthetic siderophore molecule to form siderophore-antibiotic conjugates. In this work, we describe the synthesis of six new siderophore analog-ciprofloxacin conjugates including cleavable linker or not. The siderophore analogs correspond to a mono-catechol or a hydroxypyridinone moiety recognized by both Pseudomonas and Burkholderia species. Physico-chemical studies showed that (i) conjugates were unable to interact or cross the membrane by passive diffusion and (ii) conjugates with cleavable linker are stable in physiologic environment. Biological evaluations have highlighted a promising compound 2d, bearing an hydroxypyridinone moiety with a cleavable linker, active on a large panel of strains of Pseudomonas aeruginosa, Burkholderia pseudomallei and Burkholderia thailandensis without toxicity observed in vitro.

Role of GPI-anchored enzyme in liposome detergent-resistance.

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.

Insertion of a glycosylphosphatidylinositol-anchored enzyme into liposomes.

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of (3)H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance-about 2.5 nm-between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein.