No abstract

We analysed the porphyrin-induced photodynamic inactivation of the membrane functions of bacteria through the in situ monitoring of changes in respiration rates, membrane permeability and membrane potential, using electrochemical sensors, such as oxygen, K(+) and tetraphenylphosphonium (TPP(+)) electrodes. We used two cationic porphyrins, tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TTMAPP) and tetrakis(4-N-methylpyridinium)porphyrin (TMPyP), along with an anionic porphyrin, tetrakis(4-sulfonatophenyl)porphyrin (TSPP), as a negative control. TTMAPP and TMPyP inhibited the respiration of bacteria within minutes of photo-irradiation at a concentration of 1 μM, where the survival of bacteria decreased, while TSPP did not affect the bacteria. The respiration of Staphylococcus aureus cells (Gram-positive bacterium) was more strongly inhibited than that of Escherichia coli cells (Gram-negative bacterium). Increasing the concentration of porphyrin strengthened the respiratory inhibition. Although TTMAPP increased the permeability to K(+) of the cytoplasmic membranes of bacteria, the change was relatively slow. Cationic porphyrins, showing the strong respiratory inhibition of S. aureus cells, induced the dissipation of membrane potential within minutes of photo-irradiation, in accord with the time traces of respiratory inhibition. Such a correlation strongly supported that porphyrin-induced photo-inactivation of bacteria involved rapid damage to the energy-producing system of bacteria induced by inhibition of the respiratory chain, leading to a dissipation of membrane potential. These results are discussed in connection with the ability of porphyrins to generate singlet oxygen and bind to the bacterial cell envelope.

We analyzed the photoinactivation of the membrane functions of bacteria and erythrocytes induced by xanthene dyes. The dyes tested were rose bengal, phloxine B, erythrosine B and eosin B. These dyes induced the leakage of K(+) from Staphylococcus aureus cells within minutes of photoirradiation, in the order of rose bengal > phloxine B > erythrosine B > eosin B. The ability of dyes to inhibit respiration was weak, except for rose bengal, and the dyes dissipated the membrane potential in similar time traces with changes in K(+) permeability. The xanthene dyes also induced the leakage of K(+) from bovine erythrocytes upon photoirradiation in the same order as that observed with bacteria. Furthermore, we found that the ability to cause the leakage of K(+) from erythrocytes was associated with dye-induced morphological changes, forming a crenated form from the normal discoid. These results are discussed in connection with the ability of xanthene dyes to generate singlet oxygen and bind to bacterial cells, and further compared with the actions of cationic porphyrins, which induced photoinactivation of bacteria through respiratory inhibition.

The synthesis and biological activity of a novel cyclic beta-sheet-type antimicrobial dehydropeptide based on gramicidin S (GS) is described. The GS analogue, containing two (Z)-(beta-3-pyridyl)-alpha,beta-dehydroalanine (DeltaZ3Pal) residues at the 4 and 4' positions (2), was synthesized by solution-phase methodologies using Boc-Leu-DeltaZ3Pal azlactone. Analogue 2 exhibited high antimicrobial activity against Gram-positive bacteria and had much lower hemolytic activity than wild-type GS and the corresponding (Z)-alpha,beta-dehydrophenylalanine (DeltaZPhe) analogue (1).