Environment-responsive two-fluorophore reporter system: A potential tool to monitor particulate vomocytosis

Statement of Purpose: Pathogens have evolved over time to evade the host immune system in various ways. For instance, the fungal species Cryptococcus neoformans, following engulfment by phagocytes, has been observed to stay alive within the acidic phagolysosome and escape through a process called vomocytosis1. Using this phenomenon, C. neoformans utilizes host immune cells to disseminate intection throughout the body. One condition due to infection of this microbe is cryptococcal meningitis (CM). Primarily affecting immunocompromised individuals, including an estimated 220,000 HIV/AIDS patients, CM causes -181,000 deaths/ year worldwide2.Greater understanding of vomocytosis could lead to the development of new CM treatments for patients, as well as novel biomaterial particulate vaccines. However, the underlying mechanisms of this phenomenon are unknown.To study vomocytosis, a method for quantifying phagocytosis and expulsion rates is required. Current studies use manual counting of vomocytic events3 (Fig. 1) or multi-step flow cytometry staining4 with limited success. This work characterizes a novel dual fluorescent reporter system for precise monitoring of phagocytic entry and vomocytic expulsion. The molecular signaling system is composed of tethered Enzyme-Labeled Fluorescence (ELF-97) and Fluorogen Activating Peptide (FAP). ELF-97 is a molecule that fluorescently activates upon cleavage by lysosomal phosphatases, indicating successful phagosomal uptake.The second indicator, FAP, is a protein that activates a cell- impermeable fluorogen (either aRED-np or MG Beta Tau) while outside of the cytoplasm and will be used to confirm escape from phagocytes. Here, poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) are used to demonstrate proof-of-concept for the transfer of the reporter system to C. neoformans and particulates.