Fluorescent proteins as singlet oxygen photosensitizers: Mechanistic studies in photodynamic inactivation of bacteria

Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer, light and oxygen to produce reactive oxygen species (ROS), mainly singlet oxygen (¹O₂), to photo-oxidize important biomolecules and induce cell death. aPDT is a promising alternative to standard antimicrobial strategies, but its mechanisms of action are not well understood. One of the reasons for that is the lack of control of the photosensitizing drugs location. Here we report the use of geneticallyencoded fluorescent proteins that are also ¹O₂ photosensitizers to address the latter issue. First, we have chosen the red fluorescent protein TagRFP as a photosensitizer, which unlike other fluorescent proteins such as KillerRed, is able to produce ¹O₂ but not other ROS. TagRFP photosensitizes ¹O₂ with a small, but not negligible, quantum yield. In addition, we have used miniSOG, a more efficient ¹O₂ photosensitizing fluorescent flavoprotein that has been recently engineered from phototropin 2. We have genetically incorporated these two photosensitizers into the cytosol of E. coli and demonstrated that intracellular ¹O ₂ is sufficient to kill bacteria. Additional assays have provided further insight into the mechanism of cell death. Photodamage seems to occur primarily in the inner membrane, and extends to the outer membrane if the photosensitizer's efficiency is high enough. These observations are markedly different to those reported for external photosensitizers, suggesting that the site where ¹O₂ is primarily generated proves crucial for inflicting different types of cell damage.