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Abstract
The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library® of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca2+ channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.
Highlights
Cryptococcus neoformans is an opportunistic fungal pathogen that can cause fatal infections in immunocompromised individuals
To identify small molecules that inhibit the intracellular growth of C. neoformans in macrophages, a high-throughput fluorescencebased automated screen was designed to score replication of a GFPexpressing C. neoformans in macrophages (Fig. 1)
Macrophages were infected with strain H99-Green fluorescence protein (GFP) in 96-well plates and the intracellular proliferation rate (IPR) of the yeast was quantified by GFP fluorescence after 18 h following phagocytosis and drug addition
Summary
Cryptococcus neoformans is an opportunistic fungal pathogen that can cause fatal infections in immunocompromised individuals. Despite the advent of highly active antiretroviral therapy, CM-related mortality remains prevalent among HIV/AIDS patients in developing regions [4,5]. The exact mechanisms facilitating intracellular survival of the pathogen within phagosomes are not fully understood, but recent data suggest that the process of phagosomal maturation is subtly perturbed leading to reduced antimicrobial activity in this compartment [9] and phagolysosomal damage [10]. Continued replication of the fungus eventually leads to lysis of the host macrophage or nonlytic release of the pathogen by a process termed vomocytosis [11,12]
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