A small diffusion pore in the outer membrane of Pseudomonas aeruginosa

Abstract

The permeability properties of the outer membrane of Pseudomonas aeruginosa were re-examined, since the reported conclusions are conflicting [Decad, M. G. and Nikaido, H. (1976) J. Bacteriol. 128, 325-336; Caulcott, C. A., Brown, M. R. W. and Gonda, I. (1984) FEMS Microbiol. Lett. 21, 119-1231, On the basis of the experimental evidence to be described below we conclude that the exclusion limit of the outer membrane of P. aeruginosa is smaller than the size of uncharged disaccharides but larger than the size of hexose. This conclusion is based on the following evidence. (a) Penetration of monosaccharides into the expanded periplasm was large and that of disaccharides was small, after the cells were plasmolyzed with 600 mosM NaCl. (b) A significant amount of protein was released after osmotic down-shock of cells treated with the hypertonic monosaccharides but not of cells treated with the hypertonic saccharides larger than disaccharides. (c) Centrifuged pellets of cells treated with hypertonic di, tri and tetrasaccharides weighed about 15 -20% less than that of cells treated with the isotonic monosaccharide, suggesting that the osmotic pressure was exerted on the outer membrane causing dehydration and shrinking of the cells. By contrast, cells treated with the hypertonic pentose and hexoses weighed about 0.1% and 6% less, respectively, than cells treated with the isotonic saccharide, suggesting that pentose diffused through the outer membrane freely.The outer membrane of gram-negative bacteria prevents the access of hydrophobic and large hydrophilic compounds into the cells [l -31. This membrane contains a set of poreforming proteins (porins) through which small hydrophilic nutrients and metabolic wastes diffuse [4, 51. The sieve property of the porin pores in various gram-negatives may, in terms of pore size, be roughly divided into two groups. (a) The porins having small pores showing an apparent exclusion limit close to the size of uncharged saccharides of M , about 700. These are the porins from Escherichia coli [5], Salmonella typhimurium [4], Proteus mirubilis [6], Enterobacter [6] and Yersinia pestis [7]. (b) The porins having large pores with an exclusion limit similar to the size of uncharged polysaccharides with M , of several thousands. This group includes the porins from Pseudomonas ueruginosa [2, 81, Paracoccus denitrificans [9], Anabaena variabilis [lo] and Chlamydia trachomatis [ll].The clinical relevance of P. aeruginosa is the high incidence of opportunistic infections and its resistance to a wide range of antibiotics [12]. This intrinsic drug resistance of P. aeruginosa is mainly due to the diffusion barrier of the outer membrane [13 -151. However, the pore in the outer membrane of P. aeruginosa was reported to be so large that it allows the diffusion of saccharides with Mr of several thousands [2, 81. The presence of the large pore in the membrane was confirmed by in vitro reconstitution experiments [8, 16, 171. To explain this apparent discrepancy it was proposed [13,15,16,18] that the major...

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane

“…This antibiotic resistance has been though to be due mostly to low antibiotic permeability across the outer membrane (1). The cause of this low outer membrane permeability has been assumed to be the presence of small diffusion pores that allow the diffusion of uncharged saccharides of Mr less than about 350 to 400 (4,40,41). However, it was reported earlier that protein F of the outer membrane of P. aeruginosa forms the large pore that allows the diffusion of uncharged saccharides with Mrs of several thousands (2,10,11,30).…”

“…Among the opportunistic pathogens to which immunocompromised patients are susceptible, P. aeruginosa persists longest during extensive antibiotic treatments (41). This antibiotic resistance has been though to be due mostly to low antibiotic permeability across the outer membrane (1).…”

“…On the other hand, Caulcott et al (4) and Yoneyama et al (40,41) recently showed that the outer membrane pore(s) in P. aeruginosa excluded di-or trisaccharides as determined by measuring the equilibrium of the test solutes across the intact outer membrane. If these recently reported results are correct, one can explain the intrinsic drug resistance of P. aeruginosa to structurally unrelated antibiotics without difficulty.…”

Pseudomonas aeruginosa Porin OprF Exists in Two Different Conformations

“…Although Yoneyama and Nakae (11) reported that glucose, but not disaccharides, can permeate through the outer membrane, this experiment was done with intact cells, and the contribution of sugar-specific channel OprB (46,47), for example, cannot be excluded. In addition, OprF, according to Yoshihara and Nakae (10), does not form channels of any size.…”

Use of Fluorescence Probes to Monitor Function of the Subunit Proteins of the MexA-MexB-OprM Drug Extrusion Machinery inPseudomonas aeruginosa

“…1 and 2). In Pseudomonas aeruginosa, active extrusion (3,4) combined with a tight diffusion barrier at the outer membrane (5,6) are the main reasons for low specific resistance against diverse antibiotics and other toxic elements (7).…”