Cryptococcus neoformans capsule protects cell from oxygen reactive species generated by antimicrobial photodynamic inactivation

Abstract

ABSTRACT Antimicrobial photodynamic inactivation (APDI) is based on the utilization of substances that can photosensitize biological tissues and are capable of being activated in the presence of light. Cryptococcus neoformans is an yeast surrounded by a capsule composed primarily of glucoronoxylomannan that plays an important role in its virulence. This yeast causes infection on skin, lungs and brain that can be associated with neurological sequelae and neurosurgical interventions, and its conventional treatment requires prolonged antifungal therapy, which presents important adverse effects. The aim of this study was to evaluate the protective effect of Cryptococcus neoformans capsule against reactive oxygen species generated by APDI. Cryptococcus neoformans KN99 D , which is a strain able to produce capsule, and CAP59 that does not present capsule production were submitted to APDI using methylene blue (MB), rose bengal (RB), and pL-ce6 as photosensitizers (PS). Then microbial inactivation was evaluated by counting colony form units following APDI and confocal laser scanni ng microscopy (CLSM) illustrated localization as well as the preferential accumulation of PS into the fungal cells. C. neoformans KN99 D was more resistant to APDI than CAP59 for all PSs tested. CLSM showed incorporation of MB and RB into the cytoplasm and a preferential uptake in mitochondria. A nuclear accumulation of MB was also observed. Contrarily, pL-ce6 appears accumulated in cell wall and cell membrane and minimal florescence was observed inside the fungal cells. In conclusion, the ability of C. neoformans to form capsule enhances survival following APDI . Keywords: Antimicrobial photodynamic chemotherapy; Conjugate Chlorine; Methylene Blue; Cryptococcus neoformans ; Photodynamic therapy; Photosensitizer; Rose Bengal.