Search

The fungus Lasiodiplodia theobromae is one of the main causal agents of trunk canker and dieback of grapevine. The objective of this work was to evaluate the efficiency of photodynamic inactivation (PDI) of L. theobromae with synthetic and natural photosensitizers and irradiation with either sunlight or artificial photosynthetically active radiation. Although the growth of the mycelium could not be completely prevented with natural sunlight irradiation, phenothiazine dyes (methylene blue, MB; toluidine blue O, TBO), riboflavin and a cationic porphyrin (Tetra-Py + -Me) caused complete inhibition under continuous irradiation with artificial light. Free radicals were the main cytotoxic agents in the PDI with MB, indicating the predominance of the type I mechanism. PDI with MB or Tetra-Py + -Me may represent a promising approach for the sanitation of vine material in greenhouse nurseries, in order to reduce the risk of infection upon grafting.

Oxidative stress induced by photodynamic treatment of microbial cells causes irreversible damages to vital cellular components such as proteins. Photodynamic inactivation (PDI) of bacteria, a promising therapeutic approach for the treatment of superficial and localized skin and oral infections, can be achieved by exciting a photosensitizing agent with visible light in an oxygenated environment. Although some studies have addressed the oxidative alterations of PDI in bacterial proteins, the present study is the first to compare the electrophoretic profiles of proteins of Gram-positive and Gram-negative bacteria, having two structurally different porphyrins, with different kinetics of photoinactivation. The cationic porphyrins 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py(+)-Me-PF) and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py(+)-Me) were used to photosensitize Escherichia coli and Staphylococcus warneri upon white light irradiation at an irradiance of 4.0 mW cm(-2). After different photosensitization periods, proteins were extracted from bacteria and analyzed using one-dimensional SDS-PAGE. Apparent molecular weights and band intensities were determined after an irradiation period corresponding to a reduction of 4 log10 in cell viability. After photodynamic treatment, there was a general loss of bacterial proteins, assigned to large-scale protein degradation. Protein loss was more pronounced after PDI with Tri-Py(+)-Me-PF in both bacteria. There was also an increase in the concentration of some proteins as well as an increase in the molecular weight of other proteins. We show that proteins of E. coli and S. warneri are important targets of PDI. Although there is an attempt of cellular response to the PDI-induced damage by overexpression of a limited number of proteins, the damage is lethal. Our results show that changes occurring in the protein pattern during photodynamic treatment are different with the two photosensitizers, which helps to explain the different inactivation kinetics of the two bacteria. SDS-PAGE is a rational approach to assign the type of cellular response to stress that is being induced in the cells.

Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way., (Copyright © 2014 Elsevier B.V. All rights reserved.)