Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa

Abstract

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular ⌬ 1 -dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N,N-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI 23 , chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl 2 . These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…In contrast, lipid A from N. gonorrhoeae is acylated in a symmetrical manner, with both sugar residues carrying three hydrocarbon chains (645). This difference in the positions of the acyl chains, however, cannot be the cause of the presumed high permeability of the neisserial OM, because P. aeruginosa, whose OM bilayer is at least as impermeable as that of Salmonella (503), produces lipid A that also has the symmetrical pattern of acylation (735) (Fig. 14B).…”

“…This apparent impermeability later turned out to be the result of slow influx across the OM counteracted by active efflux (456). Nevertheless, when the diffusion rates of very hydrophobic test solutes, i.e., steroids, were measured experimentally by coupling their influx with oxidation in the cytoplasm (503,504), their diffusion across the OM was found to be about 2 orders of magnitude slower than the diffusion through typical cell membranes containing phospholipid bilayers, showing that, indeed, the lipid bilayer domain of OM is an effective barrier.…”

Alteration of the Lipopolysaccharide Structure Affects the Functioning of the Xcp Secretory System in Pseudomonas aeruginosa

“…Indeed, enhanced sensitivity to antibiotics, detergents, and EDTA and leakage of periplasmic enzymes have been observed in deep rough mutants of Escherichia coli and Salmonella enterica serovar Typhimurium (19). In P. aeruginosa, the permeation of steroids has been shown to be increased up to 5.5-fold when the outer membrane contained defective LPS (38). Furthermore, both in vivo and in vitro studies have demonstrated that LPS plays a role in protein assembly (10,28,45).…”

Substrate Uptake and Subcellular Compartmentation of Anoxic Cholesterol Catabolism in Sterolibacterium denitrificans

“…Accumulating evidence indicates that the OM of Gram-negative bacteria serves as a selective permeability barrier, because its asymmetric structure with lipopolysaccharides results in an unusually slow influx of hydrophobic compounds such as steroids (49). Previous investigations have revealed that the diffusion of steroids across the OM of nonsteroid-degrading Gramnegative bacteria was approximately 2 orders of magnitude slower than diffusion through a cytoplasmic membrane containing phospholipid bilayers (17,49).…”

“…Previous investigations have revealed that the diffusion of steroids across the OM of nonsteroid-degrading Gramnegative bacteria was approximately 2 orders of magnitude slower than diffusion through a cytoplasmic membrane containing phospholipid bilayers (17,49). Although steroids could passively diffuse through the OM bilayer of S. denitrificans, our current data disproved the crucial role of passive diffusion in the cholesterol uptake of S. denitrificans because of the following: (i) the cholesterol uptake efficiency of cholesterol-grown S. denitrificans cells was significantly higher than that of palmitate-grown S. denitrificans cells and other tested proteobacterial species; (ii) the steroid uptake pattern of cholesterol-grown S. denitrificans cells exhibited high substrate selectivity; and (iii) the cholesterol uptake of S. denitrificans is not enhanced by preincubation with EDTA.…”