Monitoring Organelle‐Specific Responses to Amphotericin B in Mammalian Cells and Candida albicans Biofilms

Abstract

The use of indwelling medical devices such as catheters or dentures in combination with immunosuppressive therapies increase the body's susceptibility to fungal infections as a pathway to life threatening systemic infections. In particular, Candida albicans, an opportunistic pathogen existing in the normal microbial flora can produce serious systemic infections. This species is unique in its ability to exist in two phases: a yeast form and hyphal biofilm. Once it advances into its biofilm formation, Candida albicans is resistant towards drug treatment, making it immensely difficult to treat in clinical settings. Confocal laser microscopy using organelle‐specific fluorescent probes provides an internal view of Candida albicans along the stages of development and drug response. We have developed and deployed a series of our own naphthalimide‐based fluorescent probes targeted to mitochondria, lysosomes, and the Golgi apparatus to monitor organelle vitality in real time. The brightness, chemical and photo‐stability of our organelle probes allows us to image live cells in their appropriate culture media to observe cellular responses to drug treatments. The resistance to photobleaching makes them useful for longer periods of laser exposure as well as 3D imaging. We have investigated the various liposomal as well as micellar formulations of Amphotericin B including Fungizone, Abelcet, and Ambisome. All formulations of Amphotericin B display some level of toxicity, but lipid formulations such as Abelcet and Ambisome have shown a great reduction in human toxicity without significantly sacrificing their antifungal activity. Using our organelle probes in vitro, we are able to monitor the changes in specific organelles in both model human cells and Candida albicans yeast and hyphal cells to pinpoint the intracellular responses to Amphotericin B in both the target and host. Thus far we have found that Amphotericin B, especially Fungizone, leads to depolarization of mitochondria in some human cell lines while having little acute effect on mitochondria in Candida albicans biofilms. Comparable Amphotericin B doses in the forms of Abelcet and Ambisome, on the other hand, seem to allow for the maintenance of mitochondrial membrane potential in human cells as seen by persistent polarization following multiple hours of incubation. We are currently investigating the time‐dependent relationship between organelle vitality and the differing Amphotericin B formulations with all our probes and cell lines to provide insight as to how Amphotericin B affects mammalian, yeast and biofilm cells differently. Gaining this intracellular perspective of cell health will open doors for the optimization of antifungal drugs through pinpointing their effective mechanisms against fungi while minimizing cellular casualties resulting from organelle decline.Support or Funding InformationOffice of Research and Sponsored Programs‐UW‐Eau Claire