Accumulation of endogenous free radicals is required to induce titan-like cell formation in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an encapsulated yeast able to cause disease (mainly meningoencephalitis) among immunosuppressed patients, mostly HIV+. This yeast can form the so-called titan cells in vivo, which are cells of an abnormally larger size due to an increase in both the capsule and the cell body size (total size reaching between 50 70 μm). This phenomenon can be partially reproduced in vitro to obtain cells of an intermediate size (25 30 μm), which have been denominated titan-like cells. In this work, we have screened 1,520 compounds from the Prestwick Chemical Library and identifiedoff-patentdrugs that inhibited titan-like cell formation in vitro. We developed an automated fluorescence-basedmicroscopy assay and identified64 compounds as possible inhibitors of titan-like cells. We chose 10 of these compounds to perform dose-response curves and confirmedthem as inhibitors of titan-like cell formation. Several of the compounds identifiedhad antioxidant properties (i.e., retinoic acid), indicating a possible role of free radicals during titan cell formation. Using fluorescentprobes, we found that there was an endogenous accumulation of reactive oxygen species (ROS) during cell growth, which was inhibited in the presence of retinoic acid. Furthermore, we found that during titanization, there were significantchanges in the mitochondria, which is the main organelle where ROS are produced. We hypothesize that an intracellular increase of free radicals at the mitochondria might be a triggering signal to induce titanization. IMPORTANCE Cryptococcus neoformans is an excellent model to investigate fungal pathogenesis. This yeast can produce "titan cells,"which are cells of an abnormally larger size that contribute to the persistence of the yeast in the host. In this work, we have used a new approach to characterize them by identifying drugs that inhibit this process. We have used a repurposing off-patentdrug library, combined with an automatic method to image and analyze fungal cell size. In this way, we have identifiedmany compounds that inhibit this transition. Interestingly, several compounds were antioxidants, allowing us to confirmthat endogenous ROS and mitochondrial changes are important for titan cell formation. This work provides new evidence of the mechanisms required for titanization. Furthermore, the future characterization of the inhibitory mechanisms of the identifiedcompounds by the scientificcommunity will contribute to better understand the role of titan cells in virulence.