Cell outer membrane

Abstract: Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

Abstract: Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species. For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments. For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P. fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems. Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P. fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport. For the three other strains (P. fluorescens ATCC 13525, P. chlororaphis ATCC 9446, and P. aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments. In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.

Abstract: 1. The potentials and resistances associated with the cell membranes of the rabbit corneal epithelium were studied with 3 M-KCl-filled micro-electrodes.2. In the isolated cornea, the transepithelial potential was identical in polarity and magnitude to the simultaneously measured total corneal potential. In contrast to previous findings, the stromal potential was positive to the tear side. Negative stromal potentials apparently derive from inadequate electrodes or method of penetration, and were not found to be a function of filling solution. Transepithelial potential was also identical to over-all corneal potential in the living rabbit eye.3. In the isolated preparation, the average potential profile occurred in three distinct steps across the epithelium. By means of iontophoretic dye injection it was shown that these steps occurred across the outer membrane of the squamous cell, the transition region between the wing and basal cell, and across the inner membrane of the basal cell.4. The transverse membrane resistance of the outer epithelial membrane accounted for 60% of total corneal resistance. As a result, short-circuit current, which depolarizes the cornea, led to a hyperpolarization of the outer membrane, while affecting deeper membrane potentials little or not at all.5. The spontaneous potential of the outer membrane varied inversely with corneal potential in both normal and chloride-free Ringer, while the potential of the inner membrane of the basal cell was relatively constant, approaching the theoretical Nernst potential for potassium. The potential of the outer membrane was at chloride equilibrium and was sensitive to extracellular shunts. A Thevenin equivalent drawn for the epithelium suggested that half of the outer membrane potential could be attributed to loop currents. The potential step between wing and basal cells could be accounted for in terms of loop currents driven by the corneal potential through the epithelium.6. The potential profile of the frog corneal epithelium was similar to that of the rabbit. However, the major resistance in the frog cornea was associated with the basal cell membrane rather than with the squamous cell outer membrane. Quasi-instantaneous rectification was found for both epithelia. In the rabbit chloride rectified inwardly.